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

The authors have nothing to disclose.

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 transparent, whereas the left atrium is usually darker and more red in color25,48. Furthermore, it is essential not to stretch the SAN tissue while working with it or mounting it onto the electrode grid, as the tissue is easily mechanically damaged51. A tip to verify healthy and properly dissected SAN tissue is to examine it in Complete Tyrode&#39;s solution under the microscope to verify that the tissue is beating. Once the technique is mastered, at least 90% of tissue preparations should be good for recording.
Several considerations can improve the likelihood of successful recordings and subsequent data analysis. To ensure the best recordings, solutions should be carefully prepared and tested on practice mouse samples prior to experimental recordings. We worked extensively to adapt and modify the recording solution to optimize the health of the SAN tissue. Additionally, ensure that the gas used for the recording is carbogen (i.e., 95% O2/5% CO2). Single cell recordings often use pure oxygen due to the specific chemical composition of the Tyrode&#39;s solution used for that application, but the solution used for recording on the MEA requires carbogen in order to maintain a stable pH. Using pure oxygen with the Tyrode&#39;s solution for the MEA recordings will cause fluctuations in the pH which can lead to rapid deterioration of the tissue. Assessing the amplitude maxima in the channels as previously described will help determine if the tissue is of good recording quality. Finally, to aid analysis after the recording, taking an image of the mounted SAN tissue on the MEA electrode array after the recording is finished is very helpful. The tissue can shift slightly during the initial set up of the MEA, and this provides the most accurate assessment of electrode placement for analysis.
We propose MEA recordings as a thorough and accurate way to characterize SAN firing rate. An advantage of the MEA technique is that it allows the experimenter to capture firing rates on par with single cell recordings without the need to have extensive electrophysiological expertise. The MEA technique also has the advantage of eliminating the potential confounding influences of neurohumoral and mechano-electric mechanisms, which are inherent in isolated heart recordings and in vivo autonomic blockade measurements21. Ventricular contraction and respiration are the main mechano-electric influences that could alter SAN firing, but they are eliminated in our tissue preparation 52,53. While our technique eliminates most of the autonomic influences on the SAN, a limited number of remaining ICNS projections in the right atria could potentially impact SAN firing, a theoretical limitation which should be kept in mind during the interpretation of results5,6,22,23. Another advantage of the MEA technique described here is that it can be adapted for many other types of cardiac studies. For example, although this protocol demonstrated the effects of 4-AP on SAN activity, future studies could look at an almost limitless number of pharmacological agents, as well as the effects of gene mutations on SAN intrinsic firing. For example, ivabradine, a specific blocker of the SAN-specific Hcn4 channel, could be used to study funny current contributions to the firing rate54. The MEA system can also be used to measure cardiac function in other regions of the heart, allowing for detailed, region-specific characterization. However, recording from other heart regions would require different dissection approaches and the possibility of thin tissue sectioning before recording. One potentially significant limitation of this technique is the high cost of purchasing an MEA system which can be prohibitive. Several MEA systems are available on the market with similar characteristics and functionality, but the high cost remains the same. However, once the initial equipment and software is acquired, the cost to maintain and use the MEA system is fairly low. Another limitation is that the MEA system only allows recording of extracellular field potentials which are not conducive to precise comparisons of action potential characteristics (e.g., amplitude and shape) between preparations such as can be attained with single cell intracellular recordings. In summary, this protocol provides an efficient workflow to measure and analyze intrinsic cardiac firing rates in mouse SAN tissue with the ability to study the effects of pharmacological intervention on firing rate in a highly specific manner.

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>4-Aminopyridine</td>
			<td>Sigma</td>
			<td>A78403-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>22 gauge syringe needle</td>
			<td>Fisher Scientific</td>
			<td>14-826-5A</td>
			<td>Used for dissection</td>
		</tr>
		<tr>
			<td>23 gauge syringe needle</td>
			<td>Fisher Scientific</td>
			<td>14-826-6C</td>
			<td>Used for dissection</td>
		</tr>
		<tr>
			<td>60mm Petri Dishes</td>
			<td>Genesee Scientific</td>
			<td>32-105G</td>
			<td></td>
		</tr>
		<tr>
			<td>500mL Pyrex Bottle</td>
			<td>Fisher Scientific</td>
			<td>06-414-1C</td>
			<td>Used to store solutions</td>
		</tr>
		<tr>
			<td>1000 mL Pyrex Bottle</td>
			<td>Fisher Scientific</td>
			<td>06-414-1D</td>
			<td>Used to store solutions</td>
		</tr>
		<tr>
			<td>Bone Forceps</td>
			<td>Fine Science Tools</td>
			<td>16060-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride dihydrate (CaCl2&#183;2H2O)</td>
			<td>Sigma-Aldrich</td>
			<td>C5080-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbogen (95% O2, 5% CO2)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Castroviejo Scissors, 4&#34;</td>
			<td>Fine Science Tools</td>
			<td>15024-10</td>
			<td></td>
		</tr>
		<tr>
			<td>D-(+)-Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G7021-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Data Acquisition PC</td>
			<td></td>
			<td></td>
			<td>CPU: Intel Xeon or Intel Core i7, Memory: 8GB, HDD: 1TB, Graphic Card: NVIDIA or On-board, Screen: 1920x1080</td>
		</tr>
		<tr>
			<td>Dissection Microscope</td>
			<td>Jenco</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting Pins</td>
			<td>Fine Science Tools</td>
			<td>26002-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #2 Laminectomy Forceps</td>
			<td>Fine Science Tools</td>
			<td>11223-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #55 Forceps</td>
			<td>Fine Science Tools</td>
			<td>11295-51</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Extra Fine Graefe Forceps</td>
			<td>Fine Science Tools</td>
			<td>11152-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Chamber</td>
			<td>Grainger</td>
			<td>49WF30</td>
			<td>Used for mouse euthanization</td>
		</tr>
		<tr>
			<td>Harp Anchor Kit</td>
			<td>Warner Instruments</td>
			<td>&#160;SHD-22CL/15 WI 64-0247</td>
			<td></td>
		</tr>
		<tr>
			<td>HCl</td>
			<td>Fisher Chemicals</td>
			<td>SA48-4</td>
			<td>Used for pH balancing</td>
		</tr>
		<tr>
			<td>Hemostat</td>
			<td>Fine Science Tools</td>
			<td>13013-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Aurobindo Pharma Limited IDA, Pashamylaram</td>
			<td>NDC 63739-953-25</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H3375-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted Microscope</td>
			<td>Motic</td>
			<td>AE2000</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Patterson Veterinary</td>
			<td>07-893-1389</td>
			<td></td>
		</tr>
		<tr>
			<td>Lab Tape</td>
			<td>Fisher Scientific</td>
			<td>15-950</td>
			<td></td>
		</tr>
		<tr>
			<td>Light for Dissection Microscope</td>
			<td>Dolan-Jenner</td>
			<td>MI150DG 660000391014</td>
			<td></td>
		</tr>
		<tr>
			<td>Magesium chloride (MgCl2)</td>
			<td>Sigma-Aldrich</td>
			<td>208337-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>MED64 Head Amplifier</td>
			<td>MED64</td>
			<td>MED-A64HE1S</td>
			<td></td>
		</tr>
		<tr>
			<td>MED64 Main Amplifier</td>
			<td>MED64</td>
			<td>MED-A64MD1A</td>
			<td></td>
		</tr>
		<tr>
			<td>MED64 Perfusion Cap</td>
			<td>MED64</td>
			<td>MED-KCAP01</td>
			<td></td>
		</tr>
		<tr>
			<td>MED64 Perfusion Pipe Holder Kit</td>
			<td>MED64</td>
			<td>MED-KPK02</td>
			<td></td>
		</tr>
		<tr>
			<td>MED64 ThermoConnector</td>
			<td>MED64</td>
			<td>MED-CP04</td>
			<td></td>
		</tr>
		<tr>
			<td>Mesh</td>
			<td>&#160;Warner Instruments</td>
			<td>640246</td>
			<td></td>
		</tr>
		<tr>
			<td>Microelectrode array (MEA)</td>
			<td>Alpha Med Scientific</td>
			<td>MED-R515A</td>
			<td></td>
		</tr>
		<tr>
			<td>Mobius Software</td>
			<td>WitWerx Inc.</td>
			<td></td>
			<td>Specific software for the MED64</td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Fisher Chemicals</td>
			<td>S320-500</td>
			<td>Used for pH balancing</td>
		</tr>
		<tr>
			<td>Normal Saline</td>
			<td>Ultigiene</td>
			<td>NDC 50989-885-17</td>
			<td></td>
		</tr>
		<tr>
			<td>Paint Brush</td>
			<td>Fisher Scientific</td>
			<td>NC1751733</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Genesee Scientific</td>
			<td>PM-996</td>
			<td></td>
		</tr>
		<tr>
			<td>Peristaltic Pump</td>
			<td>Gilson</td>
			<td>F155009</td>
			<td></td>
		</tr>
		<tr>
			<td>Peristaltic Pump Tubing</td>
			<td>Fisher Scientific</td>
			<td>14-171-298</td>
			<td>1/8&#39;&#39; Interior Diameter</td>
		</tr>
		<tr>
			<td>Polyethyleneimine</td>
			<td>Sigma</td>
			<td>P3143</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride (KCl)</td>
			<td>Sigma-Aldrich</td>
			<td>P9333-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic (KH2PO4)</td>
			<td>Sigma-Aldrich</td>
			<td>P5655-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>Sigma</td>
			<td>S6297</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Fisher Scientific</td>
			<td>S671-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Sylgruard Elastomer Kit</td>
			<td>Dow Corning</td>
			<td>184 SIL ELAST KIT 0.5KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Phosphate Monobasic</td>
			<td>Sigma</td>
			<td>S6566</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium tetraborate</td>
			<td>Sigma</td>
			<td>S9640</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Scissors</td>
			<td>Fine Science Tools</td>
			<td>14074-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfer Pipets (3mL graduated)</td>
			<td>Samco Scientific</td>
			<td>225</td>
			<td></td>
		</tr>
	</tbody>
</table>

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 individual heart beats that reflect intrinsic cardiac pacemaking activity. The interspike intervals should be the same for every channel even if they may not be perfectly aligned across channels due to small differences in their location relative to the initiation site of depolarization (Figure 8). Although the shape of the waveforms for a given channel should be consistent, the shape of the waveforms will vary across channels depending on the location of the electrode in the tissue (Figure 8). The degree of contact of the tissue with the electrode may also influence waveform characteristics, such as the amplitude. However, the amplitude maxima should be at least 0.5 mV for the majority of channels if the preparation is satisfactory. From the 10 recorded traces, the three consecutive channels that best meet the quality criteria described above were chosen for further analysis described below. Figure 10A shows a sample of stable beat frequency (top panel) and interspike interval (middle panel) for three consecutive traces. Tissue that does not meet these criteria should not be recorded as there is likely tissue damage that will hinder accurate data collection. Figure 11 shows examples of bad extracted spike patterns which are either absent (A), influenced by noise (B), or unstable (C).
The sample data displayed in the figures was collected from a 45-day old male wildtype Black Swiss (Tac:N:NIHS-BC) mouse. The analysis procedure depicted in Figure 9 and Figure 10 was used to extract the intrinsic firing rate and display baseline spikes that can be seen in Figure 12A. The firing rate is the average rate across 60,000 ms from each of the three traces, but the spike pattern in Figure 12A shows 5 s of representative spiking from a single trace. Using automated analysis software, the intrinsic firing rate (i.e., beat frequency) of the selected three traces across all 64 channels was found to be approximately 320 bpm in our sample data (Figure 12A). In general, we observe a range of values of about 290-340 bpm in our recordings for wildtype mice. The firing rate can also be used as a secondary method to assess preparation quality. Rates that are either unstable or significantly lower than 300 bpm are less likely to be good for analysis. These values are comparable to both isolated heart and single cell recordings which report intrinsic heart rates in the range of approximately 300-500 bpm25,48,49. Therefore, the MEA recording technique is capable of generating reliable and accurate measures of intrinsic heart rate.
An advantage of the MEA system is that it allows easy application of drug agents to test pharmacological effects. In the sample experiment, we tested the effects of 1 mM 4-AP on firing rate, which should slow SAN activity since blockade of voltage-gated K+ channels is known to impair action potential repolarization in SA cells24,50. Figure 12B shows that the introduction of 4-AP increased the interspike intervals as expected. This prolonged spike interval corresponded to a decrease in the beat frequency from 320 bpm to 210 bpm. This firing rate following 4-AP administration is similar to a previous study that examined the effects of 4-AP on SAN firing rate using single electrode recordings of isolated tissue. That study measured a firing rate of approximately 190 bpm in the presence of 4-AP50. Thus, the MEA system can be used as a convenient and valuable tool for testing pharmacological effects of drug interventions on intrinsic cardiac function.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62735/62735fig01.jpg" /> 
Figure 1: Coating the microelectrode array (MEA) prior to use. (A) The MEA is composed of a small plastic dish with a grid array of 64 microelectrodes in the center (as shown in the panel A1) and four reference electrodes around the periphery in a square pattern. (B)&#160;Addition of 1 mL of PEI buffer to coat the MEA. (C)&#160;Covering the MEA dish with thermoplastic film for incubation overnight at room temperature. (D) Aspirating the PEI buffer from the MEA dish, which is followed by at least four rinses with distilled water. (E) Storing the coated MEA probe under ultrapure water to prevent it from drying out. <a href="https://www.jove.com/files/ftp_upload/62735/62735fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62735/62735fig02.jpg" /> 
Figure 2: Tools used for sinoatrial node (SAN) dissection. The following tools are used during the dissection part of the protocol:&#160;(i) Petri dish with silicone elastomer and small dissection pins; (ii) Plastic transfer pipette; (iii) Castroviejo scissor, size 4&#34;; (iv) Surgical scissors (straight) for cutting procedures; (v) Dumont #2 Laminectomy forceps; (vi) Dumont #55 forceps; (vii) Extra fine Graefe forceps; (viii) Hemostats (curved). <a href="https://www.jove.com/files/ftp_upload/62735/62735fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62735/62735fig03.jpg" /> 
Figure 3: Removal of the heart. (A)&#160;Transverse incision in the skin just beneath the bottom of the rib cage from about the left costal arch to the right costal arch. (B)&#160;Peritoneal incision. (C,D)&#160;Incision of the diaphragm along the thorax to expose the thoracic cavity. (E)&#160;Removal of the heart following excision of the lungs. <a href="https://www.jove.com/files/ftp_upload/62735/62735fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62735/62735fig04.jpg" /> 
Figure 4: Dissection of the sinoatrial (SAN) node. (A) The appearance of the heart in the Petri dish following removal from the body. (B)&#160;Insertion of the syringe needle through the inferior vena cava (IVC) and superior vena cava (SVC) of the right atrium. The pin in the apex of the heart is also shown. (C) Excision of the apex (i.e., the bottom half) of the heart to release the blood. The pins in the atrial appendages are also shown. (D) The final appearance of the SAN region of the right atrium at the end of dissection. The boxed region corresponds to the approximate location of the SAN. The SAN artery can also be faintly seen coursing through the SAN in a vertical orientation. The Abbreviations: AO, aorta; CT, crista terminalis; IVC, inferior vena cava; LA, left atrium; RA, right atrium; RAA, right atrial appendage; SAN, sinoatrial node; SVC, superior vena cava. <a href="https://www.jove.com/files/ftp_upload/62735/62735fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/62735/62735fig05.jpg" /> 
Figure 5: Schematic of the microelectrode array (MEA) recording system setup. The following components comprise the system: (A)&#160;gas cylinder (carbogen: 95% O2/ 5% CO2); (B) conical flask with distilled water to humidify the gas; (C) recording Tyrode&#39;s solution bottle that provides inflow to the MEA dish; (D) peristaltic pump to pump solution to and from the MEA dish; (E) temperature regulator; (F) MEA connector plate which receives signals from the MEA dish; (G) amplifier; (H)&#160;computer; (I)&#160;collection bottle for used waste solution from the MEA dish. <a href="https://www.jove.com/files/ftp_upload/62735/62735fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/62735/62735fig06.jpg" /> 
Figure 6: Positioning of the SA nodal tissue on MEA. (A)&#160;Tools used in positioning the tissue: (i) Mesh with 1.5-mm grid size, (ii) harp anchor, (iii) bone forceps, (iv) paint brush. (B) Positioning of the tissue on the MEA. The yellow box indicates the approximate area of the sinoatrial node region under the mesh and anchor in the MEA dish. (C)&#160;Arrangement of the MEA dish with enclosed tissue on the connector plate for field potential recording: (i) inlet for the recording solution; (ii) inlet for gas (carbogen); (iii) outlet for the solution; (iv) microelectrode connector plate; (v) perfusion cap; (vi) reference electrode ring attached to the cap; (vii) tape to hold the cap. <a href="https://www.jove.com/files/ftp_upload/62735/62735fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/62735/62735fig07.jpg" /> 
Figure 7: Setting the data acquisition protocol in the software. (A) An example of recording template showing arrangement of all 64 channels. (B)&#160;An example of the software input properties for the recording conditions. (C) An example of the Annotations menu showing how to Add New Phase during the recording, such as for measuring drug effects. <a href="https://www.jove.com/files/ftp_upload/62735/62735fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/62735/62735fig08.jpg" /> 
Figure 8: Different regions of the tissues showing different activity waveforms. Example screen shot showing waveforms with different shapes and amplitudes in different channels. However, all channels show identical interspike intervals and firing frequencies. The channels within the red box correspond approximately to the electrodes placed within the SAN region of the tissue. <a href="https://www.jove.com/files/ftp_upload/62735/62735fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/62735/62735fig09.jpg" /> 
Figure 9: Beat frequency analysis template. Example template showing arrangement of all 64 channels in beat frequency analysis template. The Replay Raw Data File inset shows an example of the input properties of the analysis window. In this example, traces 5 to 7 have been selected for analysis and the duration of analysis for each trace has been designated as 60,000 ms. <a href="https://www.jove.com/files/ftp_upload/62735/62735fig09large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 10" class="xfigimg" src="/files/ftp_upload/62735/62735fig10.jpg" /> 
Figure 10: Defining analysis parameters for spike extraction. (A)&#160;Representative analysis template results for 3 selected traces of a single channel. The top panel displays beat frequency for the three selected traces (three defined groupings of data points), and each point represents a 10-s average for beat frequency during the specific trace. The middle panel displays inter-spike interval for the three selected traces (three defined groupings of data points), and each data point represents the inter-spike interval between two consecutive spikes. The lower left panel shows selected representative extracted spikes for the last 5 s of the third trace, whereas the lower right panel shows an extracted waveform derived from the 5-s group of extracted spikes in the lower left panel. (B) Expanded view of the analysis window showing parameters used in the analysis of beating frequency for the 3 traces. <a href="https://www.jove.com/files/ftp_upload/62735/62735fig10large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 11" class="xfigimg" src="/files/ftp_upload/62735/62735fig11.jpg" /> 
Figure 11: Representative figure showing good versus bad data extraction for a particular channel. Bad data extraction: (A) Absence of extracted spikes; (B) Extracted spikes with noise signals; (C)&#160;Unstable extracted spikes. (D) Good data showing stable extracted spikes without noise signals. <a href="https://www.jove.com/files/ftp_upload/62735/62735fig11large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 12" class="xfigimg" src="/files/ftp_upload/62735/62735fig12.jpg" /> 
Figure 12: Recordings at baseline and after administration of 1mM 4-Aminopyridine (4-AP). (A) Baseline recording from a single microelectrode shows waveforms with a stable firing frequency of 320 bpm in a WT heart. (B) Following administration of 4-AP, the firing frequency slows to a stable rate of 210 bpm. <a href="https://www.jove.com/files/ftp_upload/62735/62735fig12large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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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Nanyang Technological University

Research

JoVE Journal

Medicine

2.9K Views.  Southern Methodist University. 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.

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

A Strategy to Identify de Novo Mutations in Common Disorders such as Autism and Schizophrenia

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, the de novo mutation rate in humans is relatively high, so new mutations are generated at a high frequency in the population. However, de novo mutations have not been reported in most common diseases. Mutations in genes leading to severe diseases where there is a strong negative selection against the phenotype, such as lethality in embryonic stages or reduced reproductive fitness, will not be transmitted to multiple family members, and therefore will not be detected by linkage gene mapping or association studies. The observation of very high concordance in monozygotic twins and very low concordance in dizygotic twins also strongly supports the hypothesis that a significant fraction of cases may result from new mutations. Such is the case for diseases such as autism and schizophrenia. Second, despite reduced reproductive fitness1 and extremely variable environmental factors, the incidence of some diseases is maintained worldwide at a relatively high and constant rate. This is the case for autism and schizophrenia, with an incidence of approximately 1% worldwide. Mutational load can be thought of as a balance between selection for or against a deleterious mutation and its production by de novo mutation. Lower rates of reproduction constitute a negative selection factor that should reduce the number of mutant alleles in the population, ultimately leading to decreased disease prevalence. These selective pressures tend to be of different intensity in different environments. Nonetheless, these severe mental disorders have been maintained at a constant relatively high prevalence in the worldwide population across a wide range of cultures and countries despite a strong negative selection against them2. This is not what one would predict in diseases with reduced reproductive fitness, unless there was a high new mutation rate. Finally, the effects of paternal age: there is a significantly increased risk of the disease with increasing paternal age, which could result from the age related increase in paternal de novo mutations. This is the case for autism and schizophrenia3. The male-to-female ratio of mutation rate is estimated at about 4&#x2013;6:1, presumably due to a higher number of germ-cell divisions with age in males. Therefore, one would predict that de novo mutations would more frequently come from males, particularly older males4. A high rate of new mutations may in part explain why genetic studies have so far failed to identify many genes predisposing to complexes diseases genes, such as autism and schizophrenia, and why diseases have been identified for a mere 3% of genes in the human genome. Identification for de novo mutations as a cause of a disease requires a targeted molecular approach, which includes studying parents and affected subjects. The process for determining if the genetic basis of a disease may result in part from de novo mutations and the molecular approach to establish this link will be illustrated, using autism and schizophrenia as examples.

Molecular genetic strategy for finding de novo mutations causing common disorders such as autism and schizophrenia.

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, ...

Cardiac Stress Test Induced by Dobutamine and Monitored by Cardiac Catheterization in Mice

Dobutamine is a &beta;-adrenergic agonist with an affinity higher for receptor expressed in the heart (&beta;1) than for receptors expressed in the arteries (&beta;2). When systemically administered, it increases cardiac demand. Thus, dobutamine unmasks abnormal rhythm or ischemic areas potentially at risk of infarction. 
Monitoring of heart function during a cardiac stress test can be performed by either ecocardiography or cardiac catheterization. The latter is an invasive but more accurate and informative technique that the former.
Cardiac stress test induced by dobutamine and monitored by cardiac catheterization accomplished as described here allows, in a single experiment, the measurement of the following hemodynamic parameters: heart rate (HR), systolic pressure, diastolic pressure, end-diastolic pressure, maximal positive pressure development (dP/dtmax) and maximal negative pressure development (dP/dtmin), at baseline conditions and under increasing doses of dobutamine.
As expected, in normal mice we observed a dobutamine dose-related increase in HR, dP/dtmax and dP/dtmin. Moreover, at the highest dose tested (12 ng/g/min) the cardiac decompensation of high fat diet-induced obese mice was unmasked.

We describe the protocol to perform a cardiac stress test induced by dobutamine and monitored by cardiac catheterization in normal mice. Also we show its application to unmask subclinical cardiac disease in high fat diet-induced obese mice.

Dobutamine is a &beta;-adrenergic agonist with an affinity higher for receptor expressed in the heart (&beta;1) than for receptors expressed in the ...

A Method to Study the Impact of Chemically-induced Ovarian Failure on Exercise Capacity and Cardiac Adaptation in Mice

The risk of cardiovascular disease (CVD) increases in post-menopausal women, yet, the role of exercise, as a preventative measure for CVD risk in post-menopausal women has not been adequately studied. Accordingly, we investigated the impact of voluntary cage-wheel exercise and forced treadmill exercise on cardiac adaptation in menopausal mice. The most commonly used inducible model for mimicking menopause in women is the ovariectomized (OVX) rodent. However, the OVX model has a few dissimilarities from menopause in humans. In this study, we administered 4-vinylcyclohexene diepoxide (VCD) to female mice, which accelerates ovarian failure as an alternative menopause model to study the impact of exercise in menopausal mice. VCD selectively accelerates the loss of primary and primordial follicles resulting in an endocrine state that closely mimics the natural progression from pre- to peri- to post-menopause in humans. To determine the impact of exercise on exercise capacity and cardiac adaptation in VCD-treated female mice, two methods were used. First, we exposed a group of VCD-treated and untreated mice to a voluntary cage wheel. Second, we used forced treadmill exercise to determine exercise capacity in a separate group VCD-treated and untreated mice measured as a tolerance to exercise intensity and endurance.

Two exercise paradigms were tested on a newly developed chemically induced menopausal mouse model to examine the impact of menopause on exercise capacity and cardiac adaption to exercise.

The risk of cardiovascular disease (CVD) increases in post-menopausal women, yet, the role of exercise, as a preventative measure for CVD risk in ...

Multi-electrode Array Recordings of Human Epileptic Postoperative Cortical Tissue

Epilepsy, affecting about 1% of the population, comprises a group of neurological disorders characterized by the periodic occurrence of seizures, which disrupt normal brain function. Despite treatment with currently available antiepileptic drugs targeting neuronal functions, one third of patients with epilepsy are pharmacoresistant. In this condition, surgical resection of the brain area generating seizures remains the only alternative treatment. Studying human epileptic tissues has contributed to understand new epileptogenic mechanisms during the last 10 years. Indeed, these tissues generate spontaneous interictal epileptic discharges as well as pharmacologically-induced ictal events which can be recorded with classical electrophysiology techniques. Remarkably, multi-electrode arrays (MEAs), which are microfabricated devices embedding an array of spatially arranged microelectrodes, provide the unique opportunity to simultaneously stimulate and record field potentials, as well as action potentials of multiple neurons from different areas of the tissue. Thus MEAs recordings offer an excellent approach to study the spatio-temporal patterns of spontaneous interictal and evoked seizure-like events and the mechanisms underlying seizure onset and propagation. Here we describe how to prepare human cortical slices from surgically resected tissue and to record with MEAs interictal and ictal-like events ex vivo.

We here describe how to perform multi-electrode array recordings of human epileptic cortical tissue. Epileptic tissue resection, slice preparation and multi-electrode array recordings of interictal and ictal events are demonstrated in detail.

Epilepsy, affecting about 1% of the population, comprises a group of neurological disorders characterized by the periodic occurrence of seizures, which ...

Ultrasound Based Assessment of Coronary Artery Flow and Coronary Flow Reserve Using the Pressure Overload Model in Mice

Transthoracic Doppler echocardiography (TTDE) is a clinically useful, noninvasive tool for studying coronary artery flow velocity and coronary flow reserve (CFR) in humans. Reduced CFR is accompanied by marked intramyocardial and pericoronary fibrosis and is used as an indication of the severity of dysfunction. This study explores, step-by-step, the real-time changes measured in the coronary flow velocity, CFR and systolic to diastolic peak velocity (S/D) ratio in the setting of an aortic banding model in mice. By using a Doppler transthoracic imaging technique that yields reproducible and reliable data, the method assesses changes in flow in the septal coronary artery (SCA), for a period of over two weeks in mice, that previously either underwent aortic banding or thoracotomy. 
During imaging, hyperemia in all mice was induced by isoflurane, an anesthetic that increased coronary flow velocity when compared with resting flow. All images were acquired by a single imager. Two ratios, (1) CFR, the ratio between hyperemic and baseline flow velocities, and (2) systolic (S) to diastolic (D) flow were determined, using a proprietary software and by two independent observers. Importantly, the observed changes in coronary flow preceded LV dysfunction as evidenced by normal LV mass and fractional shortening (FS). 
The method was benchmarked against the current gold standard of coronary assessment, histopathology. The latter technique showed clear pathologic changes in the coronary artery in the form of peri-coronary fibrosis that correlated to the flow changes as assessed by echocardiography. 
The study underscores the value of using a non-invasive technique to monitor coronary circulation in mouse hearts. The method minimizes redundant use of research animals and demonstrates that advanced ultrasound-based indices, such as CFR and S/D ratios, can serve as viable diagnostic tools in a variety of investigational protocols including drug studies and the study of genetically modified strains.

Coronary flow reserve (CFR) is useful for assessment of myocardial oxygen demand and evaluation of cardiovascular risk. This study establishes a step-by-step transthoracic Doppler echocardiographic (TTDE) method for longitudinal monitoring of the changes in CFR, as measured from coronary artery in mice, under the experimental pressure overload of aortic banding.

Transthoracic Doppler echocardiography (TTDE) is a clinically useful, noninvasive tool for studying coronary artery flow velocity and coronary flow ...

Cardiac Catheterization in Mice to Measure the Pressure Volume Relationship: Investigating the Bowditch Effect

Animal models that mimic human cardiac disorders have been created to test potential therapeutic strategies. A key component to evaluating these strategies is to examine their effects on heart function. There are several techniques to measure in vivo cardiac mechanics (e.g., echocardiography, pressure/volume relations, etc.). Compared to echocardiography, real-time left ventricular (LV) pressure/volume analysis via catheterization is more precise and insightful in assessing LV function. Additionally, LV pressure/volume analysis provides the ability to instantaneously record changes during manipulations of contractility (e.g., &#946;-adrenergic stimulation) and pathological insults (e.g., ischemia/reperfusion injury). In addition to the maximum (+dP/dt) and minimum (-dP/dt) rate of pressure change in the LV, an accurate assessment of LV function via several load-independent indexes (e.g., end systolic pressure volume relationship and preload recruitable stroke work) can be attained. Heart rate has a significant effect on LV contractility such that an increase in the heart rate is the primary mechanism to increase cardiac output (i.e., Bowditch effect). Thus, when comparing hemodynamics between experimental groups, it is necessary to have similar heart rates. Furthermore, a hallmark of many cardiomyopathy models is a decrease in contractile reserve (i.e., decreased Bowditch effect). Consequently, vital information can be obtained by determining the effects of increasing heart rate on contractility. Our and others data has demonstrated that the neuronal nitric oxide synthase (NOS1) knockout mouse has decreased contractility. Here we describe the procedure of measuring LV pressure/volume with increasing heart rates using the NOS1 knockout mouse model.

This article describes the measurement of murine left ventricular function via pressure/volume analysis at different heart rates.

Animal models that mimic human cardiac disorders have been created to test potential therapeutic strategies. A key component to evaluating these ...

Myocardial Infarction in Neonatal Mice, A Model of Cardiac Regeneration

Myocardial infarction induced by coronary artery ligation has been used in many animal models as a tool to study the mechanisms of cardiac repair and regeneration, and to define new targets for therapeutics. For decades, models of complete heart regeneration existed in amphibians and fish, but a mammalian counterpart was not available. The recent discovery of a postnatal window during which mice possess regenerative capabilities has led to the establishment of a mammalian model of cardiac regeneration. A surgical model of mammalian cardiac regeneration in the neonatal mouse is presented herein. Briefly, postnatal day 1 (P1) mice are anesthetized by isoflurane and placed on an ice pad to induce hypothermia. After the chest is opened, and the left anterior descending coronary artery (LAD) is visualized, a suture is placed around the LAD to inflict myocardial ischemia in the left ventricle. The surgical procedure takes 10-15 min. Visualizing the coronary artery is crucial for accurate suture placement and reproducibility. Myocardial infarction and cardiac dysfunction are confirmed by triphenyl-tetrazolium chloride (TTC) staining and echocardiography, respectively. Complete regeneration 21 days post myocardial infarction is verified by histology. This protocol can be used to as a tool to elucidate mechanisms of mammalian cardiac regeneration after myocardial infarction.

This protocol describes a highly reproducible model of cardiac regeneration by surgical induction of myocardial infarction in the left ventricle of postnatal day 1 mice. The method involves induction of hypothermic anesthesia and ligation of the left anterior descending coronary artery.

Myocardial infarction induced by coronary artery ligation has been used in many animal models as a tool to study the mechanisms of cardiac repair and ...

A Closed-chest Model to Induce Transverse Aortic Constriction in Mice

Research on cardiac hypertrophy and heart failure is frequently based on pressure overload mouse models induced by TAC. The standard procedure is to perform a partial thoracotomy to visualize the transverse aortic arch. However, the surgical trauma caused by the thoracotomy in open-chest models changes the respiratory physiology as the ribs are dissected and left unattached after chest closure. To prevent this, we established a minimally invasive, closed chest approach via lateral thoracotomy. Herein we approach the aortic arch via the 2nd intercostal space without entering the chest cavities, leaving the mouse with a less traumatic injury to recover from. We perform this operation using standard laboratory settings for open chest TAC procedures with equal survival rates. Apart from maintaining physiological breathing patterns due to the closed chest approach, the mice seem to benefit by showing rapid recovery, as the less invasive technique appears to facilitate a fast healing process and to reduce immune response after trauma.

Here, we present a protocol of Transverse Aortic constriction (TAC) via a lateral thoracotomy. This technique is a minimally invasive, closed chest surgical procedure aiming to simulate pressure overload and heart failure in mice utilizing standard TAC laboratory settings.

Research on cardiac hypertrophy and heart failure is frequently based on pressure overload mouse models induced by TAC. The standard procedure is to ...

Adeno-Associated Virus-Mediated Delivery of CRISPR for Cardiac Gene Editing in Mice

The clustered, regularly interspaced, short, palindromic repeat (CRISPR) system has greatly facilitated genome engineering in both cultured cells and living organisms from a wide variety of species. The CRISPR technology has also been explored as novel therapeutics for a number of human diseases. Proof-of-concept data are highly encouraging as exemplified by recent studies that demonstrate the feasibility and efficacy of gene editing-based therapeutic approach for Duchenne muscular dystrophy (DMD) using a murine model. In particular, intravenous and intraperitoneal injection of the recombinant adeno-associated virus (rAAV) serotype rh.74 (rAAVrh.74) has enabled efficient cardiac delivery of the Staphylococcus aureus CRISPR-associated protein 9 (SaCas9) and two guide RNAs (gRNA) to delete a genomic region with a mutant codon in exon 23 of mouse Dmd gene. This same approach can also be used to knock out the gene-of-interest and study their cardiac function in postnatal mice when the gRNA is designed to target the coding region of the gene. In this protocol, we show in detail how to engineer rAAVrh.74-CRISPR vector and how to achieve highly efficient cardiac delivery in neonatal mice.

Here we provide a detailed protocol to carry out in vivo cardiac gene editing in mice using recombinant Adeno-Associated Virus(rAAV)-mediated delivery of CRISPR. This protocol offers a promising therapeutic strategy to treat dystrophic cardiomyopathy in Duchenne muscular dystrophy and can be used to generate cardiac-specific knockout in postnatal mice.

The clustered, regularly interspaced, short, palindromic repeat (CRISPR) system has greatly facilitated genome engineering in both cultured cells and ...

Transdermal Measurement of Glomerular Filtration Rate in Mice

Transdermal analysis of glomerular filtration rate (GFR) is an established technique that is used to assess renal function in mouse and rat models of acute kidney injury and chronic kidney disease. The measurement system consists of a miniaturized fluorescence detector that is directly attached to the skin on the back of conscious, freely moving animals, and measures the excretion kinetics of the exogenous GFR tracer, fluorescein-isothiocyanate (FITC) conjugated sinistrin (an inulin analog). This system has been described in detail in rats. However, because of their smaller size, measurement of transcutaneous GFR in mice presents additional technical challenges. In this paper we therefore provide the first detailed practical guide to the use of transdermal GFR monitors in mice based on the combined experience of three different investigators who have been performing this assay in mice over a number of years.

Here we describe a protocol to measure glomerular filtration rate (GFR) in conscious, freely moving mice using a transdermal GFR monitor.

Transdermal analysis of glomerular filtration rate (GFR) is an established technique that is used to assess renal function in mouse and rat models of ...