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

Summary

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.

Abstract

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.

Introduction

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 heartbeat^1,2. 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....

Protocol

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

1. Make 25 mM borate buffer.
   1. Dissolve 0.953 g of Na₂B₄O₇·10 H₂O in 80 mL of distilled water.
   2. Adjust the pH to 8.4 with HCl and then add distilled water to a final volume of 100 mL.
2. Make a 0.1% stock solution of polyethyleneimine (PEI).

Results

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 ≥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 he.......

Discussion

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.......

Materials

List of materials used in this article

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