Optical Mapping of Langendorff-perfused Rat Hearts

Summary

This article describes a high temporal and spatial resolution technique to optically image action potential movement on the surface of Langendorff-perfused rat hearts using a potentiometric dye (di-8-ANEPPS).

Abstract

Optical mapping of the cardiac surface with voltage-sensitive fluorescent dyes has become an important tool to investigate electrical excitation in experimental models that range in scale from cell cultures to whole-organs^{[1, 2]}. Using state-of-the-art optical imaging systems, generation and propagation of action potentials during normal cardiac rhythm or throughout initiation and maintenance of arrhythmias can be visualized almost instantly^[1]. The latest commercially-available systems can provide information at exceedingly high spatiotemporal resolutions and were based on custom-built equipment initially developed to overcome the obstacles imposed by more conventional electrophysiological methods^[1]. Advancements in high-resolution and high-speed complementary metal-oxide-semiconductor (CMOS) cameras and intensely-bright, light-emitting diodes (LEDs) as well as voltage-sensitive dyes, optics, and filters have begun to make electrical signal acquisition practical for cardiovascular cell biologists who are more accustomed to working with microscopes. Although the newest generation of CMOS cameras can acquire 10,000 frames per second on a 16,384 pixel array, depending on the type of sample preparation, long-established fluorescence acquisition technologies such as photodiode arrays, laser scanning systems, and cooled charged-coupled device (CCD) cameras still have some distinct advantages with respect to dynamic range, signal-to-noise ratio, and quantum efficiency^{[1, 3]}. In the present study, Lewis rat hearts were perfused ex vivo with a crystalloid perfusate (Krebs-Henseleit solution) at 37°C on a modified Langendorff apparatus. After a 20 minute stabilization period, the hearts were intermittently perfused with 11 mMol/L 2,3-butanedione monoxime to eliminate contraction-associated motion during image acquisition. For optical mapping, we loaded hearts with the fast-response potentiometric probe di-8-ANEPPS^[4] (5 μMol/L) and briefly illuminated the preparation with 475±15 nm excitation light. During a typical 2 second period of illumination, >605 nm light emitted from the cardiac preparation was imaged with a high-speed CMOS camera connected to a horizontal macroscope. For this demonstration, hearts were paced at 300 beats per minute with a coaxial electrode connected to an isolated electrical stimulation unit. Simultaneous bipolar electrographic recordings were acquired and analyzed along with the voltage signals using readily-available software. In this manner, action potentials on the surface of Langendorff-perfused rat hearts can be visualized and registered with electrographic signals.

Protocol

Part 1: Prepare solutions and the isolated perfused heart system

1. On the morning of the experiment, 4.0 L of Krebs-Henseleit solution is prepared as previously described^{[5, 6]}.
2. 11 mMol/L 2,3-butanedione monoxime (BDM) is dissolved in 1.0 L of Krebs-Henseleit solution decanted from the perfusate prepared in step 1.1.
3. An additional 150 mL of Krebs-Henseleit is removed from the perfusate prepared in step 1.1 and mixed with 5 Mol/L di-8-ANEPPS (diluted from a 10 mMol/L stock dissolved in dimethyl sulfoxide (DMSO)).
4. These 3 solutions are transferred to water-jacketed glass reservoirs (Radnoti) where they are pre-warmed to 41....

Discussion

Removal of the heart from anesthetized rats must be performed rapidly to avoid myocardial ischemia. If ischemia or insufficient coronary perfusion occurs, the heart will likely develop arrhythmias and may become infarcted. Additionally, these hearts will show insufficient fluorescence emission for informative recordings and subsequent analyses. Before loading the dye, myocardial cells need to be adequately perfused with Krebs-Henseleit solution to establish and maintain a physiologic electrolyte milieu for the stability .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
CardioCMOS-SM128f	Equipment	RedShirtImaging, LLC
CardioPlex Software	Equipment	RedShirtImaging, LLC
LUXEON LED Light Source 460-490 nm	Equipment	Lumileds Lighting, US, LLC, San Jose, CA 95131 USA	LXHL-PB02
ECG Amplifier Type 689 Hugo Sachs Elektronik	Equipment	Harvard Apparatus	730149
Dichroic Mirror 505 nm	Equipment	Semrock	FF505-SDi01-25x36
Emission Filter 605 nm Long Pass	Equipment	SciMedia
THT Sideways	Equipment	SciMedia	25 BM-8
Mini Ball Joint Holder	Equipment	Harvard Apparatus	BS4 73-0177
Small Stimulation Electrode Set	Equipment	Harvard Apparatus	BS4 73-0160
BM-6 Benchtop Vibration Isolation Platform	Equipment	Technology Inc., Inglewood, CA 90301	25 BM-6
Monopolar ECG Electrode	Equipment	Harvard Apparatus	BS4 73-0200
Roller Pump SCI 400	Equipment	Watson-Marlow Pumps Group	401U/D1
Roller Pump MasterFlex Easy Load II	Equipment	Cole-Parmer	Model 77201-60
Tubing Marprene #14	Equipment	Watson-Marlow Pumps Group	902.0016.016
MasterFlex Tubing	Equipment	PharMed, Westlake, OH 44145 USA	06485-25
S48 Square Pulse Stimulator	Equipment	Grass Technologies	Model S48
SIU5 RF TRANSFORMER ISOLATION UNIT	Equipment	Grass Technologies	Model SIU5
5 Liter Water Jacketed Reservoir	Equipment	Radnoti Glass Technology Inc.	120142-5
2 Liter Water Jacketed Reservoir	Equipment	Radnoti Glass Technology Inc.	120142-2
0.5 Liter Water Jacketed Reservoir	Equipment	Radnoti Glass Technology Inc.	120142-0
0.25 Liter Water Jacketed Reservoir	Equipment	Radnoti Glass Technology Inc.	120142-025
10 ml Heating Coil	Equipment	Radnoti Glass Technology Inc.	158822
Compliance Bubble Trap	Equipment	Radnoti Glass Technology Inc.	130149
Luer Disconnect Cannula	Equipment	Harvard Apparatus	72-1444
3-Way stopcock, FLL to MLT, No Port Covers	Equipment	Harvard Apparatus	BS4 72-2630
Thermocouple Thermometer	Equipment	Cole-Parmer	WU-91100-40
Ultra Fine IT-Series Flexible Microprobe	Equipment	PhysiTemp Instruments Inc., Clifton, NJ 07013 USA	IT-24P
Oscilloscope Tektronix TDS 1002	Equipment	Tektronix, Inc.	TDS 1002B
2,3-Butanedione monoxime	Reagent	Sigma-Aldrich	B0753
Ketamine HCl	Reagent	Hospira Inc.	RL-0065
Xylazine	Reagent	Lloyd, Inc.	LB15705A
E-TOXA-CLEAN®	Reagent	Sigma-Aldrich	E9029
Di-8-ANEPPS	Reagent	Invitrogen	D-3167