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).
Part 1: Prepare solutions and the isolated perfused heart system
Part 2: Harvest the rat heart and set-up Langendorff perfusion
Part 3: Load heart with potentiometric dye and acquire electrographic and optical signals
Part 4: Analyze acquisition information using RedShirt Imaging Software
Part 5: Representative results
If the perfused heart preparation was motionless during recording, the optical signals show one distinct peak for every pixel involved in a change of the emission intensity of di-8-ANEPPS. The corresponding movies (Figures 3 and 4) demonstrate an excitation wave front propagating across the epicardial surface of the heart as well as the simultaneously acquired electrographic recordings (Figure 2).
Figure 1. A photograph of a Langendorff-perfused heart preparation depicting the positions of the pacing electrode on the right atrium and the ECG leads as described in step 3.2.
Figure 2. Representative optical signals and electrographic recordings from a perfused Lewis rat heart. Panel A shows an image of the epicardial surface used for optical imaging. The position of the pixels selected to demonstrate the changes in fluorescence emission over time in Panel B are indicated by colored arrows. Electrographic signals are shown in Panel C with the red line indicating atrial activation and the light blue line corresponding to the ventricular signal. Please click here to see a larger version of this figure.
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 of electrical impulses. Accurate preparation of the perfusate is also necessary to maintain proper organ viability and function. Differences in the electrolyte concentration or insufficient filtering of the perfusate will probably lead to fatal myocardial dysfunction and heart rhythm disorders. For the optical recordings, the heart must be completely loaded with voltage-sensitive dye. This is especially important for the atrial myocardium as these chambers are not well-perfused by the coronary arteries. We have found that additional intra-cavitary perfusion of the atria will establish a good optical signal. Furthermore, acquisition of top-quality voltage tracings requires the perfused heart to be motionless; otherwise, changes in fluorescence emission cannot be reliably used to track changes in membrane potential with high fidelity due to artifacts caused by signal drift. This will result in multiple peaks for one pixel rather than a single peak. Other methods to eliminate motion artifacts from optical recordings include mechanical immobilization, treatment with other excitation-contraction uncouplers (e.g. cytochalasin D, blebbistatin), signal processing, and by mathematical modeling[1, 8]. Finally, the method described here merely provides information regarding action potential movement on the cardiac epicardial surface. Alternative tissue preparations and infrared potentiometric dyes may resolve the electrical propagation characteristics in other regions of the heart.
The authors have nothing to disclose.
This work is supported by research grants from the National Institutes of Health (HL068915; HL088206) and contributions to the Cardiac Conduction Fund at Children’s Hospital Boston.
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
CardioCMOS-SM128f | Equipment | RedShirt Imaging, Decatur, GA 30030 USA | ||
CardioPlex Software | Equipment | RedShirt Imaging, Decatur, GA 30030 USA | ||
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, Holliston, MA 01746 USA | 730149 | |
Dichroic Mirror 505 nm | Equipment | Semrock, Rochester, NY 14624 USA | FF505-SDi01-25×36 | |
Emission Filter 605 nm Long Pass | Equipment | SciMedia, Costa Mesa, CA 92626 USA | ||
THT Sideways | Equipment | SciMedia, Costa Mesa, CA 92626 USA | 25 BM-8 | |
Mini Ball Joint Holder | Equipment | Harvard Apparatus, Holliston, MA 01746 USA | BS4 73-0177 | |
Small Stimulation Electrode Set | Equipment | Harvard Apparatus, Holliston, MA 01746 USA | BS4 73-0160 | |
BM-6 Benchtop Vibration Isolation Platform | Equipment | Technology Inc., Inglewood, CA 90301 | 25 BM-6 | |
Monopolar ECG Electrode | Equipment | Harvard Apparatus, Holliston, MA 01746 USA | BS4 73-0200 | |
Roller Pump SCI 400 | Equipment | Watson-Marlow Bredel Inc., Wilmington, MA 01887 USA | 401U/D1 | |
Roller Pump MasterFlex Easy Load II | Equipment | Cole Parmer, Vernon Hills, Illinois 60061 USA | Model 77201-60 | |
Tubing Marprene #14 | Equipment | Watson-Marlow Bredel Inc., Wilmington, MA 01887 USA | 902.0016.016 | |
MasterFlex Tubing | Equipment | PharMed, Westlake, OH 44145 USA | 06485-25 | |
S48 Square Pulse Stimulator | Equipment | Grass Technologies, West Warwick, RI 02893 USA | Model S48 | |
SIU5 RF TRANSFORMER ISOLATION UNIT | Equipment | Grass Technologies, West Warwick, RI 02893 USA | Model SIU5 | |
5 Liter Water Jacketed Reservoir | Equipment | Radnoti Glass Technology Inc., Monrovia CA 91016 USA | 120142-5 | |
2 Liter Water Jacketed Reservoir | Equipment | Radnoti Glass Technology Inc., Monrovia CA 91016 USA | 120142-2 | |
0.5 Liter Water Jacketed Reservoir | Equipment | Radnoti Glass Technology Inc., Monrovia CA 91016 USA | 120142-0 | |
0.25 Liter Water Jacketed Reservoir | Equipment | Radnoti Glass Technology Inc., Monrovia CA 91016 USA | 120142-025 | |
10 ml Heating Coil | Equipment | Radnoti Glass Technology Inc., Monrovia CA 91016 USA | 158822 | |
Compliance Bubble Trap | Equipment | Radnoti Glass Technology Inc., Monrovia CA 91016 USA | 130149 | |
Luer Disconnect Cannula | Equipment | Harvard Apparatus, Holliston, MA 01746 USA | 72-1444 | |
3-Way stopcock, FLL to MLT, No Port Covers | Equipment | Harvard Apparatus, Holliston, MA 01746 USA | BS4 72-2630 | |
Thermocouple Thermometer | Equipment | Cole Parmer, Vernon Hills, Illinois 60061 USA | 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., Beaverton, OR 97005 USA | TDS 1002B | |
2,3-Butanedione monoxime | Reagent | Sigma, St. Louis, MO 63132 USA | B0753 | |
Ketamine HCl | Reagent | Hospira Inc., Lake Forest, IL 60045 USA | RL-0065 | |
Xylazine | Reagent | Lloyd Inc., Iowa 51601 USA | LB15705A | |
E-TOXA-CLEAN® | Reagent | Sigma, St. Louis, MO 63132 USA | E9029 | |
Di-8-ANEPPS | Reagent | Invitrogen, Carlsbad, CA 92008 USA | D-3167 |