Name: Optical Mapping of Langendorff-perfused Rat Hearts (Video) | JoVE Journal
Uploaded: 2009-08-11T19:00:00.000+00:00
Duration: 11 min 48 s
Description: Learn optical mapping of action potentials on Langendorff-perfused rat hearts using di-8-ANEPPS dye. This high-resolution technique enables detailed study of cardiac electrical activity and arrhythmia mechanisms.

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5 minutes

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.

Part 1: Prepare solutions and the isolated perfused heart system
<ol>
<li>On the morning of the experiment, 4.0 L of Krebs-Henseleit solution is prepared as previously described[5, 6].</li>
<li>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.</li>
<li>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)).</li>
<li>These 3 solutions are transferred to water-jacketed glass reservoirs (Radnoti) where they are pre-warmed to 41.0° C and oxygenated with a submerged bubbler (Radnoti) using 0.2 μm-filtered 95% O2; 5% CO2 gas. Solutions from the 3 stock reservoirs are pumped to the wall-mounted Langendorff system with MasterFlex™ L/S peristaltic pumps and low-absorption silicone tubing (Cole-Parmer). </li>
<li>Prior to assembly,the Langendorff system glassware (Radnoti) is meticulously washed with E-TOXA-CLEAN reagent (Sigma-Aldrich) and thoroughly rinsed with 0.1 M/L HCl, 100% ethanol, and distilled water.</li>
<li>The non-recirculating Langendorff apparatus was constructed to deliver 70 mm Hg constant-pressure from 3 separate oxygenated reservoirs, which are separated from the heart with water-jacketed heating coils and bubble traps (Radnoti)[7]. The 3 independent perfusion lines converge above the heart at two 3-way stopcocks, allowing us to precisely control delivery of the solutions prepared in steps 1.1 to 1.3. Water-jacketed glassware is connected in series with MasterFlex PharMed BPT tubing (Cole-Parmer) and warmed to 39° C with distilled water using two E100 circulators (Lauda). Heat loss through the platinum-cured silicone tubing (Cole-Parmer) that connects the pressure-head reservoirs to the heating coils and bubble traps resulted in a 37° C perfusate reaching the heart. </li>
</ol>
Part 2: Harvest the rat heart and set-up Langendorff perfusion
<ol>
<li>To induce deep general anesthesia in 200-250 g Lewis rats, 100 mg/kg Ketamine and 10 mg/kg Xylazine is injected into the intraperitoneal cavity. To this mixture, we add 500 U/kg Heparin to prevent blood coagulation and myocardial ischemia during the explantation procedure.</li>
<li>For easy access to the heart and great vessels, the anterior chest wall is removed. After that, surrounding tissue is carefully dissected and the pericardial sack opened.</li>
<li>Following identification of the inferior vena cava, this vessel is ligated with 5-0 silk (Ethicon) and the entire heart-lung block is explanted. Tissue is immediately placed in ice-cold Krebs-Henseleit solution contained in a 50 mL beaker on ice.</li>
<li>The ascending aorta is quickly identified and dissected from the surrounding tissue. An appropriately-sized cannula (Harvard Apparatus) is inserted into the aorta, taking care to avoid interrupting obligatory perfusion of the coronary arteries by inserting the cannula too far into the aortic root. The cannula is secured to the ascending aorta with 5-0 silk (Ethicon).</li>
<li>The rat heart is then placed on the Langendorff apparatus without introducing air bubbles into the cannula. Retrograde coronary vascular perfusion is now established with the warm, oxygenated Krebs-Henseleit solution from the pressure-head described in step 1.6.</li>
<li>Extra tissue, including the lungs, is now removed and the heart is perfused for 20 minutes to permit recovery of function and stabilize the rhythm. During this time, a very thin thermocouple temperature probe (Cole-Parmer) is introduced into the left ventricular cavity and sutured in place with 5-0 Prolene suture (Ethicon). The probe is connected to a thermo-controller (Digi-Sense) to ensure the temperature of the heart is maintained at 37° C by adjusting the settings of the water circulatory pumps. Motion from perfusate dripping from the cardiac apex is minimized by placing a piece of gauze in the effluent receptacle.</li>
</ol>
Part 3: Load heart with potentiometric dye and acquire electrographic and optical signals
<ol>
<li>Di-8-ANEPPS (Invitrogen) is loaded into the heart by switching to the perfusion line containing the Krebs-Henseleit mixed with the fluorescent dye using a stopcock. In addition, an 18G cannula is placed into the left and/or right atrium and additional 50 mL of dye solution is slowly administered into each of these chambers because they are not sufficiently perfused with dye introduced through the coronary arteries.</li>
<li>During the loading procedure, 3 ECG leads (Harvard Apparatus) are gently placed on the surface of the heart that is not facing the optics used for mapping. Electrode No 1 is positioned on the posterior apical portion of the left ventricle, No 2 on the left atrium, and No 3 as a reference electrode on the aortic root (Figure 1). The atrial and ventricular electrographic signals are subsequently amplified, digitized, and displayed alongside optical signals using the software (RedShirt Imaging) (Figure 2). An oscilloscope (Tektronix model TDS 1002) is also used to visualize the surface ECG s in real-time and to assure adequate pacing.</li>
<li>ECG amplifier (Hugo Sachs Elektronik) settings:
High Pass Filter: 0.1 Hz Low Pass Filter: 150 Hz</li>
<li>The CMOS camera (RedShirt Imaging) and macroscope is positioned using the X-Y-Z adjustments so that the surface of the heart is in focus and centered in the acquisition frame. The camera and optics are mounted on a vibration isolation table (Minus K Technology) to minimize resonant frequencies. At the same time, a coaxial pacing electrode (Harvard Apparatus) controlled with an isolated, S48 electrical-stimulation unit (Grass), is placed on the right atrium and the heart is paced at 300 beats per minute (Figure 1).</li>
<li>Electrical stimulation (Grass) settings: Rate: 5 pulses per second Delay: 0.2 ms Duration: 2 ms Volts: 6-12 V Mode: Repeat Pulse: Single</li>
<li>For optical recordings which lack motion artifacts from contraction, the heart needs to be electromechanically uncoupled. We do this by again switching perfusion lines to Krebs-Henseleit solution that contains 11 mMol/L BDM. Between acquisitions, the heart is perfused with unadulterated Krebs-Henseleit to help preserve viability of the preparation.</li>
<li>The recording parameters are set using the software (RedShirt Imaging) with the following acquisition settings: Configuration: 2,000 Hz; 128x128 pixel array Frame Interval: 0.5 ms Camera Amplifier Gain: 5x On-Chip Gain: 8x-12Me-well Shutter: 500 ms delay Number of Frames: 4000 Duration: 2000 ms</li>
<li>All room and equipment lights are turned off or shielded to eliminate background noise during recording. The LED light illuminates the heart only during the optical recording to reduce photo-bleaching and dye toxicity. The light source shutter is controlled with a 5 V pulse delivered through the control panel by way of a D-to-A board in the computer (RedShirt Imaging).</li>
</ol>
Part 4: Analyze acquisition information using RedShirt Imaging Software
<ol>
<li>Following acquisition, data is processed using different filter settings. We generally use the default settings except when adjusting the Band Stop/Pass filter, which is set with the left boundary at 44.0 and the right boundary at 98.0. Afterward the recorded information is processed and a movie is generated (RedShirt Imaging).</li>
<li>Data from one acquisition corresponds to the local electrical activation at 16,384 sites on the heart surface over a period of 2 seconds. The software allows these local signals to be directly compared with one another and with the atrial and ventricular electrographic recordings. Data is then visualized by mapping local electrical activation to color and rendering this information as an animation showing the spatiotemporal electrical activation on the cardiac surface. To create such an animation, we use the software to: 
<ul>
<li>temporally and/or spatially filter the data</li>
<li>select a start and end time for the animation</li>
<li>map optical signals to color based on resting light intensity of each pixel</li>
<li>overlay the resulting color data with a picture of the heart </li>
<li>generate the animation</li>
</ul>
</li>
</ol>
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).
<img src="/files/ftp_upload/1138/Figure3.jpg" alt="Langendorff heart setup diagram for cardiac pacing study, highlighting numbered components." data-altupdatedat="1754904545000" data-alt="Figure 1"> 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.
<img src="/files/ftp_upload/1138/Figure4thumb.jpg" alt="Biological process diagram with signals; data analysis of fluorescence microscopy results." border="0" data-altupdatedat="1754904545000" data-alt="Figure 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 <a href="https://www.jove.com/files/ftp_upload/1138/Figure4.jpg">click here</a> to see a larger version of this figure.

<ol><li>Efimov, I. R., Nikolski, V. P., Salama, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optical+imaging+of+the+heart%5BTitle%5D)+AND+%22Circ+Res%22%5BJournal%5D)">Optical imaging of the heart.</a> Circ Res. 95 (1), 21-33 (2004).</li>
<li>Hucker, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Images+in+cardiovascular+medicine.+Optical+mapping+of+the+human+atrioventricular+junction%5BTitle%5D)+AND+%22Circulation%22%5BJournal%5D)">Images in cardiovascular medicine. Optical mapping of the human atrioventricular junction.</a> Circulation. 117 (11), 1474-147 (2008).</li>
<li>Entcheva, E., Bien, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Macroscopic+optical+mapping+of+excitation+in+cardiac+cell+networks+with+ultra-high+spatiotemporal+resolution%5BTitle%5D)+AND+%22Prog+Biophys+Mol+Biol%22%5BJournal%5D)">Macroscopic optical mapping of excitation in cardiac cell networks with ultra-high spatiotemporal resolution.</a> Prog Biophys Mol Biol. 92 (2), 232-257 (2006).</li>
<li>Entcheva, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fluorescence+imaging+of+electrical+activity+in+cardiac+cells+using+an+all-solid-state+system%5BTitle%5D)+AND+%22IEEE+Trans+Biomed+Eng%22%5BJournal%5D)">Fluorescence imaging of electrical activity in cardiac cells using an all-solid-state system.</a> IEEE Trans Biomed Eng. 51 (2), 333-341 (2004).</li>
<li>Stamm, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rapid+endotoxin-induced+alterations+in+myocardial+calcium+handling%3A+obligatory+role+of+cardiac+TNF-alpha%5BTitle%5D)+AND+%22Anesthesiology%22%5BJournal%5D)">Rapid endotoxin-induced alterations in myocardial calcium handling: obligatory role of cardiac TNF-alpha.</a> Anesthesiology. 95 (6), 1396-1405 (2001).</li>
<li>Choi, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cardiac+conduction+through+engineered+tissue%5BTitle%5D)+AND+%22Am+J+Pathol%22%5BJournal%5D)">Cardiac conduction through engineered tissue.</a> Am J Pathol. 169 (1), 72-85 (2006).</li>
<li>Skrzypiec-Spring, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolated+heart+perfusion+according+to+Langendorff---still+viable+in+the+new+millennium%5BTitle%5D)+AND+%22J+Pharmacol+Toxicol+Methods%22%5BJournal%5D)">Isolated heart perfusion according to Langendorff---still viable in the new millennium.</a> J Pharmacol Toxicol Methods. 55 (2), 113-126 (2007).</li>
<li>Fedorov, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Application+of+blebbistatin+as+an+excitation-contraction+uncoupler+for+electrophysiologic+study+of+rat+and+rabbit+hearts%5BTitle%5D)+AND+%22Heart+Rhythm%22%5BJournal%5D)">Application of blebbistatin as an excitation-contraction uncoupler for electrophysiologic study of rat and rabbit hearts.</a> Heart Rhythm. 4 (5), 619-626 (2007).</li>
</ol>

Experiments on animals were performed in accordance with the guidelines and regulations set forth by the Institutional Animal Care and Use Committee at Children's Hospital Boston.

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 ...

<table><tbody><tr><td>CardioCMOS-SM128f</td><td>Equipment</td><td>RedShirtImaging, LLC</td><td></td><td></td></tr><tr><td>CardioPlex Software</td><td>Equipment</td><td>RedShirtImaging, LLC</td><td></td><td></td></tr><tr><td>LUXEON LED Light Source 460-490 nm </td><td>Equipment</td><td>Lumileds Lighting, US, LLC, San Jose, CA 95131 USA</td><td>LXHL-PB02</td><td></td></tr><tr><td>ECG Amplifier Type 689 Hugo Sachs Elektronik</td><td>Equipment</td><td>Harvard Apparatus</td><td>730149</td><td></td></tr><tr><td>Dichroic Mirror 505 nm</td><td>Equipment</td><td>Semrock</td><td>FF505-SDi01-25x36</td><td></td></tr><tr><td>Emission Filter 605 nm Long Pass</td><td>Equipment</td><td>SciMedia</td><td></td><td></td></tr><tr><td>THT Sideways</td><td>Equipment</td><td>SciMedia</td><td>25 BM-8</td><td></td></tr><tr><td>Mini Ball Joint Holder</td><td>Equipment</td><td>Harvard Apparatus</td><td>BS4 73-0177</td><td></td></tr><tr><td>Small Stimulation Electrode Set</td><td>Equipment</td><td>Harvard Apparatus</td><td>BS4 73-0160</td><td></td></tr><tr><td>BM-6 Benchtop Vibration Isolation Platform</td><td>Equipment</td><td>Technology Inc., Inglewood, CA 90301</td><td>25 BM-6</td><td></td></tr><tr><td>Monopolar ECG Electrode</td><td>Equipment</td><td>Harvard Apparatus</td><td>BS4 73-0200</td><td></td></tr><tr><td>Roller Pump SCI 400</td><td>Equipment</td><td>Watson-Marlow Pumps Group</td><td>401U/D1</td><td></td></tr><tr><td>Roller Pump MasterFlex Easy Load II</td><td>Equipment</td><td>Cole-Parmer</td><td>Model 77201-60</td><td></td></tr><tr><td>Tubing Marprene #14</td><td>Equipment</td><td>Watson-Marlow Pumps Group</td><td>902.0016.016</td><td></td></tr><tr><td>MasterFlex Tubing</td><td>Equipment</td><td>PharMed, Westlake, OH 44145 USA</td><td>06485-25</td><td></td></tr><tr><td>S48 Square Pulse Stimulator</td><td>Equipment</td><td>Grass Technologies</td><td>Model S48</td><td></td></tr><tr><td>SIU5 RF TRANSFORMER ISOLATION UNIT</td><td>Equipment</td><td>Grass Technologies</td><td>Model SIU5</td><td></td></tr><tr><td>5 Liter Water Jacketed Reservoir</td><td>Equipment</td><td>Radnoti Glass Technology Inc.</td><td>120142-5</td><td></td></tr><tr><td>2 Liter Water Jacketed Reservoir</td><td>Equipment</td><td>Radnoti Glass Technology Inc.</td><td>120142-2</td><td></td></tr><tr><td>0.5 Liter Water Jacketed Reservoir</td><td>Equipment</td><td>Radnoti Glass Technology Inc.</td><td>120142-0</td><td></td></tr><tr><td>0.25 Liter Water Jacketed Reservoir</td><td>Equipment</td><td>Radnoti Glass Technology Inc.</td><td>120142-025</td><td></td></tr><tr><td>10 ml Heating Coil</td><td>Equipment</td><td>Radnoti Glass Technology Inc.</td><td>158822</td><td></td></tr><tr><td>Compliance Bubble Trap</td><td>Equipment</td><td>Radnoti Glass Technology Inc.</td><td>130149</td><td></td></tr><tr><td>Luer Disconnect Cannula</td><td>Equipment</td><td>Harvard Apparatus</td><td>72-1444</td><td></td></tr><tr><td>3-Way stopcock, FLL to MLT, No Port Covers</td><td>Equipment</td><td>Harvard Apparatus</td><td>BS4 72-2630</td><td></td></tr><tr><td>Thermocouple Thermometer</td><td>Equipment</td><td>Cole-Parmer</td><td>WU-91100-40</td><td></td></tr><tr><td>Ultra Fine IT-Series Flexible Microprobe </td><td>Equipment</td><td>PhysiTemp Instruments Inc., Clifton, NJ 07013 USA</td><td>IT-24P</td><td></td></tr><tr><td>Oscilloscope Tektronix TDS 1002</td><td>Equipment</td><td>Tektronix, Inc.</td><td>TDS 1002B</td><td></td></tr><tr><td>2,3-Butanedione monoxime </td><td>Reagent</td><td>Sigma-Aldrich</td><td>B0753</td><td></td></tr><tr><td>Ketamine HCl</td><td>Reagent</td><td>Hospira Inc.</td><td>RL-0065</td><td></td></tr><tr><td>Xylazine</td><td>Reagent</td><td>Lloyd, Inc.</td><td>LB15705A</td><td></td></tr><tr><td>E-TOXA-CLEAN&amp;#174;</td><td>Reagent</td><td>Sigma-Aldrich</td><td>E9029</td><td></td></tr><tr><td>Di-8-ANEPPS</td><td>Reagent</td><td>Invitrogen</td><td>D-3167</td><td></td></tr></tbody></table>

optical mapping of langendorff-perfused rat hearts

Optical Mapping of Langendorff-perfused Rat Hearts

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&deg;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 &mu;Mol/L) and briefly illuminated the preparation with 475&plusmn;15 nm excitation light. During a typical 2 second period of illumination, &gt;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.

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).

Optical mapping of the cardiac surface with voltage-sensitive fluorescent dyes has become an important tool to investigate electrical excitation in ...

Hi, I am Yon Ziv from the laboratory of Dr.Cohen in the Department of Anesthesiology at Children's Hospital and Howard Medical School. Today we'll show a procedure of optical mapping in Lal perfused red hearts. We use this procedure in our laboratory to study the spread of electrical activity on the cardiac surface.So let's get started. On the morning of the experiment, mix up four liters of Krebs Hensel light solution. After mixing up the solution, remove undissolved particles with a 200 micron filter using vacuum filtration.First, use one liter of the prepared Krebs Henzel lite solution to dissolve 11 millimoles of BDM. Second, take another 100 milliliters of Krebs Henzel lite and mix in 10 millimolar D eight NFS in DMSO to a final concentration of five Micromolar D eight annex in solution. The water jacketed, glass reservoirs, the bubbler and the glassware used here are all from red.Naughty.Transfer the two solutions to the reservoirs pre-war to 41 degrees Celsius. Oxygenate the solutions with 0.2 micron filtered gas using a submerged bubbler pump the solutions from the three stock reservoirs through the wall-mounted Ling Endorf system with peristaltic pumps and low absorption silicone tubing to harvest the rat heart. First intraperitoneal inject a 200 to 250 gram Lewis rat with ketamine and xylazine to induce deep general anesthesia and with heparin to prevent blood coagulation and myocardial ischemia during explanation.Once the rat is completely anesthetized and does not respond to a toe pinch, proceed with removing the heart. Begin the surgery by first removing the anterior chest wall to easily access the heart and great vessels. Then carefully dissect the surrounding tissue and open the pericardial sac.After identifying the inferior vena cava, ligate this vessel with five O Proline sutures and explan the entire heart lung block. Immediately place the tissue in ice cold Krebs Henzel light solution in a 50 milliliter beaker on ice, quickly identify the ascending aorta and dissect it from the surrounding tissue. Insert an appropriately sized cannula into the aorta.Taking care to avoid inserting the cannula too far into the aortic root and interrupting obligatory perfusion of the coronary arteries. Secure the cannula to the ascending aorta. Then place the rat heart on the Ling Endorf apparatus without introducing air bubbles into the cannula.Retrograde coronary vascular perfusion is now established with the warm oxygenated Krebs Hensel lite solution. From the pressure head of the Ling Endorf apparatus, now remove extra tissue including the lungs after cleaning, perfuse the heart for 20 minutes to permit recovery of function and stabilize the rhythm as the heart is perfusing. Introduce a very thin thermocouple temperature probe into the left ventricular cavity.Suture the probe in place with five oh prolene sutures and adjust the settings of the water circulatory pumps to maintain the temperature of the heart. At 37 degrees Celsius, minimize motion from the Perfuse eight dripping from the cardiac apex. By placing a piece of gauze in the effluent receptacle, the heart is now ready to be loaded with potentia metric dye to acquire electrographic and optical signals to load the potential metric dye dye eight NEPs into the heart.Switch over to the profusion line that contains Krebs, hence light mixed with fluorescent dye. Because the atria are not sufficiently perfused with dye introduced through the coronary arteries, an 18 gauge cannula is placed in the left and or right atrium. And an additional 50 milliliters of dye solution is slowly administered into each of these chambers.During the loading procedure, gently place three ECG leads on the surface of the heart. That is not facing the optics used for mapping. The first electrode is positioned on the posterior apical portion of the left ventricle.The second is placed on the left atrium and the third acts as a reference electrode on the aortic root. The atrial and ventricular electrographic signals are subsequently amplified, digitized, and displayed optical signals using the software. An oscilloscope is also used to visualize the surface ECGs in real time and to assure adequate pacing.Set the ECG amplifier at 0.1 hertz for the high pass filter and 150 hertz for the low pass filter position the CMOs camera using the X, Y, Z adjustments. A piece of glass that is positioned in the focal plane of the camera is placed on the surface of the heart so that the area of interest is in focus and centered in the acquisition frame. The camera and optics are mounted on a vibration isolation table to minimize resonant frequencies.At the same time. On the right atrium place a coaxial pacing electrode controlled with an isolated S 48 electrical stimulation unit. This will pace the heart at 300 beats per minute, set the electrical stimulation as follows.The rate should be five pulses per second, a delay of 0.2 milliseconds, a two millisecond stimulation duration with a strength of six to 12 volts and mode set to repeat and single pulse. For optical recordings that lack motion artifacts from contraction, the heart needs to be electro mechanically uncoupled. Do this by again, switching perfusion lines to Krebs Hensel solution containing 11 millimolar BDM.Between acquisitions. Perfuse the heart with unadulterated Krebs HENSEL light to help preserve the viability of the preparation. Configure the software recording parameters to 2000 hertz with a 1 28 by 1 28 pixel array.The frame interval should be set to 0.5 milliseconds and gains should be set to five x for the camera amplifier and eight x for the on-chip gain. The on-chip gain should also be set to 12. Set the software's camera control for a shutter delay of 500 milliseconds and record 4, 000 frames at a duration of 2000 milliseconds.Turn off or shield all room and equipment lights to eliminate background noise during the recording, the LED light illuminates the heart only during the optical recording. To reduce photobleaching and dye toxicity, control the light source shutter with a five volt pulse delivered through the control panel by way of a D two A board in the computer following acquisition. Process the data using different filter settings.The default settings are generally used except when adjusting the band stop pass filter, which is set with the left boundary at 44.0 and the right boundary at 98.0. Afterwards, the recorded information is processed and used to generate a movie. Data from one acquisition corresponds to the local electrical activation at 16, 384 sites on the heart surface over a period of two seconds.The software allows these local signals to be directly compared with one another and with the atrial and ventricular electrographic recordings data by mapping local electrical activation to color and render this information as an animation showing the spatiotemporal electrical activation on the cardiac surface. To create such an animation, first, use the software to temporally and or spatially filter the data. Next, select a start and end time for the animation.Third, map optical signals to color based on resting light intensity of each pixel. Fourth, overlay the resulting color data with a picture of the heart, and finally generate the animation 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 D eight anep. These real time imaging movies demonstrate an excitation wavefront propagating across the epicardial surface of the heart, as well as the simultaneously acquired electrographic recordings.We've just demonstrated a high resolution technique to optically image action potential movement on the surface of Alanor Perfuse Red Heart. When doing this procedure, it is important to quickly remove the heart from the anesthetized red. To avoid myocardial ischemia, the myocardial cells need to be adequately perfused and the heart must be completely loaded with water sensitive dye.So that's it. Thank you for watching and good luck with your experiments.

optical-mapping-of-langendorff-perfused-rat-hearts

Research

JoVE Journal

Biology

This report provides a detailed description of the methodology and results of simultaneous endocardial and epicardial optical mapping of electrical excitation in the intact left atrium of a Langendorff-perfused sheep heart during stretch-induced atrial fibrillation.

High-Resolution Endocardial and Epicardial Optical Mapping in a Sheep Model of Stretch-Induced Atrial Fibrillation

This article describes the basic procedures for conducting optical mapping experiments in the Langendorff-perfused rabbit heart using the panoramic imaging system, and the dual (voltage and calcium) imaging modality.

Multiparametric Optical Mapping of the Langendorff-perfused Rabbit Heart

This paper details the dissection procedure, instrumental setup, and experimental conditions during optical mapping of transmembrane potential (Vm) and intracellular calcium transient (CaT) in intact isolated Langendorff perfused mouse hearts.

Optical Mapping of Action Potentials and Calcium Transients in the Mouse Heart

In this video, we perform Langendorff retrograde perfusion of an isolated mouse heart. During the procedure, the cardiac activity of the heart is maintained by perfusing it retrogradely with a perfusion solution via the coronary arteries using a cannula inserted into the ascending aorta. The model is used to study the cardiac pathophysiology of the heart.

Langendorff Retrograde Heart Perfusion: An Ex Vivo Technique to Perfuse Isolated Mouse Heart

The isolated rat heart is an enduring model for ischemia reperfusion injury. Here, we describe the process of harvesting the beating heart from a rat via in situ aortic cannulation, Langendorff perfusion of the heart, simulated ischemia-reperfusion injury, and infarct staining to confirm the extent of ischemic insult.

Induction and Assessment of Ischemia-reperfusion Injury in Langendorff-perfused Rat Hearts

This article describes the detailed protocol and equipment necessary for dual optical mapping of transmembrane potential (Vm) and free intra-sarcoplasmic reticulum (SR) Ca2+ in the Langendorff-perfused rabbit heart. This method allows for direct observation and quantification of Vm and SR Ca2+ dynamics in the intact heart.

Optical Mapping of Intra-Sarcoplasmic Reticulum Ca2+ and Transmembrane Potential in the Langendorff-perfused Rabbit Heart

Here, we present a protocol for optical mapping of electrical activity from the mouse right atrium and especially the sino-atrial node, at a high spatial and temporal resolution.

High-resolution Optical Mapping of the Mouse Sino-atrial Node

This protocol describes the electrophysiological evaluation of murine atria utilizing an optical mapping system with a high temporal and spatial resolution, including dual recordings of the membrane voltage and Ca2+ transient under programmed stimulation through a specialized electrode catheter.

Electrophysiological Assessment of Murine Atria with High-Resolution Optical Mapping

The objective of this study was to establish a method for investigating cardiac dynamics using a translational animal model. The described experimental approach incorporates dual-emission optocardiography in conjunction with an electrophysiological study to assess electrical activity in an isolated, intact porcine heart model.

Optocardiography and Electrophysiology Studies of Ex Vivo Langendorff-perfused Hearts

This protocol introduces dual-dye optical mapping of mouse hearts obtained from wild-type and knock-in animals affected by catecholaminergic polymorphic ventricular tachycardia, including electrophysiological measurements of transmembrane voltage and intracellular Ca2+ transients with high temporal and spatial resolution.

Dual-Dye Optical Mapping of Hearts from RyR2R2474S Knock-In Mice of Catecholaminergic Polymorphic Ventricular Tachycardia

Learn optical mapping of action potentials on Langendorff-perfused rat hearts using di-8-ANEPPS dye. This high-resolution technique enables detailed study of cardiac electrical activity and arrhythmia mechanisms.

Optical Mapping of Langendorff-perfused Rat Hearts

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