Name: Optical Mapping of Action Potentials and Calcium Transients in the Mouse Heart (Video) | JoVE Journal
Uploaded: 2011-09-13T13:00:00.000+00:00
Duration: 8 min 13 s
Description: Learn optical mapping of action potentials and calcium transients in isolated Langendorff perfused mouse hearts. This technique enables high spatial resolution imaging of cardiac electrophysiology for arrhythmia research.

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5 minutes

NIH grants R01 HL085369.

1. Advanced preparation of stock solutions
<ol>
	<li>Prepare two stock solutions of Tyrode's solution (16x) in advance in deionized water and store them at 4°C:
	<ol class="lalpha">
		<li>Stock I (119.872 g/L NaCl, 3.056 g/L CaCl2 (2H2O), 5.6 g/L KCl, 2.6274 g/L NaH2PO4, 3.408 g/L MgCl2 (6H2O), (Fisher Scientific, Fair Lawn, NJ));</li>
		<li>Stock II (26.88 g/L NaHCO3, (Fisher Scientific, Fair Lawn, NJ)).</li>
	</ol>
	</li>
	<li>Prepare stock solutions of fluorescent dyes. To avoid repeated freezing and thawing, we store 30 μl aliquots of both dyes at -20°C, which is sufficient for one experiment:
	<ol class="lalpha">
		<li>Voltage-sensitive dye RH237 (Invitrogen, Carlsbad, CA) stock solution, 1.25 mg/mL solution in dimethyl sulfoxide (DMSO, Sigma, St. Louis, MO);</li>
		<li>Calcium indicator Rhod-2AM (Invitrogen, Carlsbad, CA) stock solution, 1 mg/mL solution in DMSO.</li>
	</ol>
	</li>
	<li>Prepare excitation-contraction uncoupler blebbistatin stock solution (Tocris Bioscience, St. Louis, MO, 2 mg/mL solution in DMSO) in advance and store the dissolved blebbistatin at 4°C.</li>
</ol>
2. Prepare perfusion solutions and experimental setup7
<ol start="4">
	<li>Freshly prepare 2L Tyrode's solution (128.2mM NaCl, 1.3mM CaCl2 (2H2O), 4.7mM KCl, 1.05mM MgCl2 (6H2O), 1.19mM NaH2PO4, 20mM NaHCO3, 11.1mM D-Glucose in deionized water, pH=7.35±0.05). If stock solution is being used to make 2L of Tyrode's solution (sufficient for one experiment) take 1750 mL of deionized water and mix in 125 mL of Stock I, 125 mL of Stock II, and 4g of glucose.</li>
	<li>Turn on the two pumps of the perfusion systems. Set the peristaltic pump (Peri-Star, WPI, Sarasota, USA) that is used for retrograde perfusion to 40 mL/min. Set the other peristaltic pump (Cole-Parmer Masterflex Peristalic Pump L/S, Cole-Parmer Instrument Company, Vernon Hills, Illinois) that is used for superfusion and to return the perfusate back to the holding reservoir to 80 mL/min.</li>
	<li>Wash the perfusion system with 70% ethanol for 30 min and then with 2L deionized water.</li>
	<li>Once all the deionized water is evacuated from the chamber, circulate the Tyrode's solution and pass it through a 5-μm filter (Millipore, Billerica, MA, USA). Warm the perfusate to +37°C with a water jacket and circulator (ThermoNESLAB EX7, Newtown, USA) and oxygenate the perfusate by bubbling O2/CO2 (95% / 5%) gas into the solution. Monitor the pH of the solution with a pH meter (Oakton Instruments, Vernon Hills, IL) and adjust the rate of O2/CO2 bubbling to keep the pH at 7.35 ± .05. Continue monitoring pH and temperature during the experiment.</li>
	<li>The dual optical mapping apparatus consists of two MiCAM Ultima-L CMOS cameras (SciMedia, Costa Mesa, CA) that have high spatial (100x100 pixels, 230±20 μm per pixel) and temporal (1,000-3,000 frames/sec) resolution. Fix a band-pass filter (590±15 nm, Thorlabs, Newton, NJ) in front of the designated calcium imaging camera; while, a long-pass filter (&gt;700 nm, Thorlabs, Newton, NJ) needs to be positioned in front of the designated voltage imaging camera. The cameras are arranged perpendicular to one another by a holder, which contains a dichroic mirror (635 nm cutoff, Omega Optical, Brattleboro, VT). Immediately below the dual camera holder there is a lens (Nikon NIKKOR 55 mm 1:1.4 235052), which focuses the emission light coming from the heart onto the dichroic mirror. The working distance is approximately 3cm. 
	The excitation light is generated by a halogen lamp (Newport Oriel Instruments, Stratford, CT; SciMedia, Costa Mesa, CA) and is passed through a heat filter, shutter, and band-pass filter (520±45 nm). A flexible light guide directs the band-pass filtered light onto the preparation, and a shutter is used to ensure that the preparation is exposed to light only during image acquisition to avoid photobleaching of the dyes.</li>
	<li>Prepare Ag/AgCl2 electrodes for pacing and sensing in advance and install them in the chamber prior to placing the heart. Make sure that amplifiers and filters are adjusted to appropriate levels.</li>
</ol>
3. Harvest the mouse heart, cannulate, and set up Langendorff perfusion
<ol start="10">
	<li>Anesthetize the mouse with ketamine/xylazine (ketamin, 80mg/kg bodyweight; xylazine, 10 mg/kg bodyweight) and heparin (100 units) by intraperitoneal injection. Assure appropriate level of anesthesia by the lack of pain reflex.</li>
	<li>After a mid-sternal incision, quickly remove the heart and wash it in oxygenated (95% O2, 5% CO2), constant-temperature (37±1°C) Tyrode's solution.</li>
	<li>Using a dissecting microscope, rapidly identify the aorta and make a clean cut across the ascending aorta below the right subclavian artery. A short section of aorta is then attached to a custom made 21-gauge cannula with a flared tip. 4-0 black-braided silk suture (Surgical Specialties Corporation, Reading, PA) is used to fix the heart onto the cannula. After cannulation, the heart is retrogradely perfused and superfused with Tyrode's solution. The retrograde perfusion rate is adjusted in the range of 2-5 mL/min to keep the aortic pressure between 60 and 80 mmHg (Pressure transducer, World precision instruments Inc (WPI), Sarasota, USA; Bridge Amplifier TBM4M, WPI, Sarasota, USA).</li>
	<li>After the heart is cannulated, lung, thymus, and fat tissue are then dissected and removed.</li>
	<li>The isolated heart is pinned (Fine Science Tools) at the apex to the bottom of the perfusion chamber (Sylgard coated) to prevent stream-induced movement. The right and left atrial appendages are also stretched and pinned (Fine Science Tools, Inc, Foster City, CA) to the bottom of the chamber, which provides maximal surface area for optical measurements of the atria. 
	Very important! A small silicon tube is inserted into the left ventricle through the pulmonary veins and fixed by silk suture to nearby connective tissue. This prevents solution congestion and acidification of the perfusate trapped in the left ventricle, which is especially important after the suppression of ventricular contractions with an excitation-contraction uncoupler (see Part 4 step 19).</li>
	<li>A custom made electrode is placed on the surface of the heart to conduct the pacing stimulations, which is generated by Master-8 (A.M.P Instruments Ltd, Jerusalem, Israel) or PowerLab 26T (AD Instruments, Sydney, Australia).</li>
	<li>A small cover glass is fixed on the surface of the solution over the heart to reduce motion artifact from the vibrating solution.</li>
	<li>Focus the excitation light on the heart. In addition, adjust the distance between the dual camera apparatus and the heart so that maximum resolution will be obtained.</li>
	<li>Turn off all lights in the room and commence electrical recordings using PowerLab 26T.</li>
</ol>
4. Load voltage and calcium sensitive dyes and excitation-contraction uncoupler
<ol start="19">
	<li>Warm-up 0.6 mL of blebbistatin. Mix 0.5 mL of the blebbistatin with the perfusate in the holding reservoir. Dilute the remaining 0.1 mL of blebbistatin in 1 mL of Tyrode's solution and slowly inject it (over a 20 minute period) through a drug port located near the cannula. Observe as blebbistatin gradually reduces the motion artifact.</li>
	<li>Dilute 30 μL of voltage sensitive dye RH237 stock solution in 1 mL Tyrode's solution and slowly inject over 5-7 minutes into the same injection port as blebbistatin.</li>
	<li>30 μL calcium indicator Rhod-2 AM is 1:1 mixed with Pluronic F127 (Invitrogen, Carlsbad, CA, 20% solution in DMSO) and then diluted in 1 mL Tyrode's solution and slowly applied over 5-10 min through the same injection port.</li>
	<li>Wait for 5-10 minutes for blebbistatin and dyes to reach the cell membrane and cytosol. Continue with the protocol when motion is completely suppressed.</li>
	<li>Continuously monitor ECG recordings over the entire procedure to assure normal electrical function of the heart.</li>
	<li>Commence fluorescent signal recordings using the SciMedia custom software (SciMedia, Costa Mesa, CA).</li>
</ol>
5. Representative results:
<img alt="Optical microscope setup for fluorescence imaging with filters; analyzes Ca²⁺ levels in cells." src="/files/ftp_upload/3275/3275fig1.jpg" data-altupdatedat="1788235205000" data-alt="Figure 1"> 
Figure 1. Experimental setup for the perfusion, electrical recordings and optical mapping. 
EM=emission; Lp=longpass
<img alt="ECG study, heart stimulation setup; includes atrium, ventricle signals, electrode, ECG diagram." src="/files/ftp_upload/3275/3275fig2.jpg" data-altupdatedat="1788235205000" data-alt="Figure 2"> 
Figure 2. Experimental preparation and signal examples recorded during ventricular pacing. Left: ECG signals are collected from Ag/AgCl2 disk electrodes (Top) and an example of S1S1 stimulation protocol is shown (Bottom). Center: Langendorff mouse heart preparation. Right: Representative optical action potentials and calcium transient signals from atria (Top) and ventricles (Bottom) are shown. The yellow arrow (Top) points to the fluorescent signal scattering coming from the ventricles, which is seen in the atrial recordings. 
LV=left ventricle; RV=Right ventricle; LA=left atrium; RA=right atrium
<img alt="Heart signal recording using electrodes; Vm and CaT graph analysis; electrode setup, result chart." src="/files/ftp_upload/3275/3275fig3.jpg" data-altupdatedat="1788235205000" data-alt="Figure 3"> 
Figure 3. Representative optical recordings of Vm and CaT from the ventricles of a wild type mouse heart. A. The experimental preparation with an array of evenly spaced locations marked by black dots whose optical recordings can be seen in (C). B. Example tracing of Vm and CaT from a central location on the array (see box in (C)). C. Vm (blue) and CaT (red) from the array of evenly spaced locations. Signals were binned 3x3.
<img alt="Heart activation time and voltage dynamics diagram; includes pacing, Vm, dV/dt graphs." src="/files/ftp_upload/3275/3275fig4.jpg" data-altupdatedat="1788235205000" data-alt="Figure 4"> 
Figure 4. Activation map and conduction. A. An example activation map from a wild type mouse heart with transverse (T) and longitudinal (L) directions indicated by white arrows. B. Vm signals (Top) and dV/dt (Bottom) corresponding to the three points seen in A (T1, T2, T3; L1, L2, L3).
<img alt="Heart map comparison of APD80 and CaD80 under control and ISO; action potential, calcium transient." src="/files/ftp_upload/3275/3275fig5.jpg" data-altupdatedat="1788235205000" data-alt="Figure 5"> 
Figure 5. Action potential and calcium transient duration analysis. A. Action potential duration at 80% repolarization (APD80) and calcium transient duration at 80% relaxation (CaD80) maps are shown from a heart under control conditions (Left) and after 30 nM isoproterenol application (Right). The yellow/green color in the ventricles (Right) indicates isoproterenol shortened APD80 and CaD80. B. Example tracings of APD80 and CaD80 from the wild type mouse ventricles (Top) and atria (Bottom).

<ol><li>Efimov, I. R., Rendt, J. M., Salama, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optical+maps+of+intracellular+%5BCa2%2B%5Di+transients+and+action-potentials+from+the+surface+of+perfused+guinea-pig+hearts%5BTitle%5D)+AND+%22Circulation%22%5BJournal%5D)">Optical maps of intracellular [Ca2+]i transients and action-potentials from the surface of perfused guinea-pig hearts.</a> Circulation. 90, 1-1 (1994).</li>
<li>Efimov, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optical+mapping+of+repolarization+and+refractoriness+from+intact+hearts%5BTitle%5D)+AND+%22Circulation%22%5BJournal%5D)">Optical mapping of repolarization and refractoriness from intact hearts.</a> Circulation. 90, 1469-1480 (1994).</li>
<li>Choi, B. R., Salama, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Simultaneous+maps+of+optical+action+potentials+and+calcium+transients+in+guinea-pig+hearts%3A+mechanisms+underlying+concordant+alternans%5BTitle%5D)+AND+%22J+Physiol%22%5BJournal%5D)">Simultaneous maps of optical action potentials and calcium transients in guinea-pig hearts: mechanisms underlying concordant alternans.</a> J Physiol. 529, 171-188 (2000).</li>
<li>Pruvot, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Role+of+calcium+cycling+versus+restitution+in+the+mechanism+of+repolarization+alternans%5BTitle%5D)+AND+%22Circ+Res%22%5BJournal%5D)">Role of calcium cycling versus restitution in the mechanism of repolarization alternans.</a> Circ Res. 94, 1083-1090 (2004).</li>
<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, 21-33 (2004).</li>
<li>Fast, V. G. Recording action potential using voltage sensitive dyes. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aFast%2C+VG+%22Practical+methods+in+cardiovascular+research%22">Practical methods in cardiovascular research</a>. Dhein, S., Delmoar, M. , Springer. New York. 233-255 (2005).</li>
<li>Glukhov, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Differential+K%28ATP%29+channel+pharmacology+in+intact+mouse+heart%5BTitle%5D)+AND+%22J+Mol+Cell+Cardiol%22%5BJournal%5D)">Differential K(ATP) channel pharmacology in intact mouse heart.</a> J Mol Cell Cardiol. , (2009).</li>
<li>Baker, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Enhanced+dispersion+of+repolarization+and+refractoriness+in+transgenic+mouse+hearts+promotes+reentrant+ventricular+tachycardia%5BTitle%5D)+AND+%22Circ+Res%22%5BJournal%5D)">Enhanced dispersion of repolarization and refractoriness in transgenic mouse hearts promotes reentrant ventricular tachycardia.</a> Circ Res. 86, 396-407 (2000).</li>
<li>Sutherland, F. J., Hearse, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+isolated+blood+and+perfusion+fluid+perfused+heart%5BTitle%5D)+AND+%22Pharmacol+Res%22%5BJournal%5D)">The isolated blood and perfusion fluid perfused heart.</a> Pharmacol Res. 41, 613-627 (2000).</li>
<li>Efimov, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Virtual+electrode+polarization+in+the+far+field%3A+implications+for+external+defibrillation%5BTitle%5D)+AND+%22Am+J+Physiol+Heart+Circ+Physiol%22%5BJournal%5D)">Virtual electrode polarization in the far field: implications for external defibrillation.</a> Am J Physiol Heart Circ Physiol. 279, 1055-1070 (2000).</li>
<li>Hammouda, M., Kinosita, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+coronary+circulation+in+the+isolated+heart%5BTitle%5D)+AND+%22J+Physiol%22%5BJournal%5D)">The coronary circulation in the isolated heart.</a> J Physiol. 61, 615-628 (1926).</li>
<li>Salama, G., Hwang, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Simultaneous+optical+mapping+of+intracellular+free+calcium+and+action+potentials+from+Langendorff+perfused+hearts%5BTitle%5D)+AND+%22Curr+Protoc+Cytom%22%5BJournal%5D)">Simultaneous optical mapping of intracellular free calcium and action potentials from Langendorff perfused hearts.</a> Curr Protoc Cytom. 12, 17-17 (2009).</li>
<li>Lou, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantitative+panoramic+imaging+of+epicardial+electrical+activity%5BTitle%5D)+AND+%22Ann+Biomed+Eng%22%5BJournal%5D)">Quantitative panoramic imaging of epicardial electrical activity.</a> Ann Biomed Eng. 36, 1649-1658 (2008).</li>
<li>Salama, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Properties+of+new%2C+long-wavelength%2C+voltage-sensitive+dyes+in+the+heart%5BTitle%5D)+AND+%22J+Membr+Biol%22%5BJournal%5D)">Properties of new, long-wavelength, voltage-sensitive dyes in the heart.</a> J Membr Biol. 208, 125-140 (2005).</li>
</ol>

No conflicts of interest declared.

In this experiment we modified the Langendorff perfusion method by adding a small silicon tube, which is especially crucial after the suppression of ventricular contractions with an excitation-contraction uncoupler. The silicon tube is used to prevent solution congestion, acidification of the perfusion solution, and development of ischemia in the left ventricle. The mouse heart is very sensitive to hypothermia; thus, temperature variations across the heart will cause artificial differences in action potential durations. Consequently, a heating system was implemented in the perfusion chamber in order to maintain a constant temperature of 37&deg;C during the entirety of the experiment8. Since a Langendorff model does not retain innervation of the heart, one needs to consider adding neurotransmitters to the perfusate in order to investigate physiological sympathetic and parasympathetic tone9. Besides retrograde perfusion, addition of superfusion of the heart helps to maintain suitable environmental parameters such as pH and temperature. In this method, the Langendorff perfused heart was horizontally placed. A vertical Langendorff perfusion setup can also be used10, but may result in slightly different cardiac mechanics11. In addition to CMOS cameras, alternative detectors are also available and can be applied to map Vm and CaT simultaneously12.
Application of CMOS cameras of high spatio-temporal resolution assures the accuracy of the recordings; however, optical mapping signals are not from a single cell. Rather, each fluorescent signal comes from hundreds or thousands of cells, depending on optical magnification. The much larger ventricular fluorescence can distort atrial signals by optical scattering; therefore, careful interpretation of the optically recorded signals is required. Another limitation of the mouse preparation is the signal distortion and noise induced by the curvature of the surface due to the small size of the heart13. Conduction velocity measurements can be altered not only from the curvature of the mouse heart but also from electrode polarity and virtual electrodes. To achieve accuracy for conduction velocity, activation anisotropy, and repolarization maps, correct focusing of the camera at the surface of the heart is essential. 
In this method, real time ECG recordings can supplement the optical investigation of cardiac electrophysiology. Voltage-sensitive dye (RH237) and calcium indicator (Rhod-2AM) are used in the protocol because of their fast response, similar excitation, and distinct emission spectra3,7. There are alternative combinations of dyes that can be used to measure Vm and CaT other than RH237 and Rhod-2AM3. A novel voltage-sensitive dye, PGHI, with a large Stoke's shift (&gt;200nm) was found to allow better Vm and CaT signals because of the greater separation of the emission wavelengths between PGHI and Rhod-2AM14. Future improvements may focus on exploring novel fluorescent probes, development of new imaging detectors, and improved image processing software. Higher resolution and novel optical imaging modalities for 3D optical mapping are also important future directions of optical mapping5.

<table><tbody><tr><td>NaCl</td><td>Fisher Scientific</td><td>S271-1</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt; (2H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Fisher Scientific</td><td>C79-500</td><td></td></tr><tr><td>KCl</td><td>Fisher Scientific</td><td>S217-500</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt; (6H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Fisher Scientific</td><td>M33-500</td><td></td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt; (H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Fisher Scientific</td><td>S369-500</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Fisher Scientific</td><td>S233-3</td><td></td></tr><tr><td>D-Glucose</td><td>Fisher Scientific</td><td>D16-1</td><td></td></tr><tr><td>Blebbistatin</td><td>Tocris Bioscience</td><td>1760</td><td></td></tr><tr><td>RH237</td><td>Invitrogen</td><td>S1109</td><td></td></tr><tr><td>Rhod-2AM</td><td>Invitrogen</td><td>R1244</td><td></td></tr><tr><td>Pluronic F127</td><td>Invitrogen</td><td>P3000MP</td><td></td></tr><tr><td>Dimethyl sulphoxide (DMSO)</td><td>Sigma-Aldrich</td><td>D2650</td><td></td></tr><tr><td>&lt;a href="http://www.adinstruments.com/products/hardware/corporate/product/ML856/" target="_blank"&gt;PowerLab 26T &lt;/a&gt;</td><td><a href="http://www.adinstruments.com/" target="_blank"><img src="/img/new_window.png"> ADInstruments</a></td><td></td><td></td></tr></tbody></table>

optical mapping of action potentials and calcium transients in the mouse heart

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

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. Cardiovascular physiological phenotyping of the mouse heart can be easily done using fluorescence imaging employing various probes for transmembrane potential (Vm), calcium transients (CaT), and other parameters. Excitation-contraction coupling is characterized by action potential and intracellular calcium dynamics; therefore, it is critically important to map both Vm and CaT simultaneously from the same location on the heart1-4. Simultaneous optical mapping from Langendorff perfused mouse hearts has the potential to elucidate mechanisms underlying heart failure, arrhythmias, metabolic disease, and other heart diseases. Visualization of activation, conduction velocity, action potential duration, and other parameters at a myriad of sites cannot be achieved from cellular level investigation but is well solved by optical mapping1,5,6. In this paper we present the instrumentation setup and experimental conditions for simultaneous optical mapping of Vm and CaT in mouse hearts with high spatio-temporal resolution using state-of-the-art CMOS imaging technology. Consistent optical recordings obtained with this method illustrate that simultaneous optical mapping of Langendorff perfused mouse hearts is both feasible and reliable.

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.

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. ...

The overall goal of this procedure is to simultaneously optically map transmembrane potential and intracellular calcium transient in intact isolated LOR perfused mouse hearts. This is accomplished by first excising the intact mouse heart, then cannulating the aorta and beginning LOR perfusion. The next step is to immobilize the heart by loading an excitation contraction on coupler and then pinning the heart to the perfusion chamber.The final steps are to load the optical dyes and make preparations to reduce motion artifacts during the experiment. Optical images are obtained by collecting the light emitted from the heart with my cam Ultima LC Moss cameras. Hey, my name's di.I'm a graduate student in Washington University. The main advantage of this technique over existing method, like micro electrode, is that optical mapping allows for greater spatial resolution over the entire surface of the heart. Hi, I'm Matt Sulkin.I'm a graduate student at Washington University. This method, if done correctly, can help answer many questions in cardiac electrophysiology such as the basis of arrhythmogenesis. Begin by configuring one peristaltic pump to circulate the solutions at 40 milliliters per minute.Then set a second peristaltic pump to 80 milliliters per minute. This pump returns the Perfuse eight back to the holding and oxygenating reservoir. Turn on the pumps and wash the system with one liter, 70%ethanol for 30 minutes with recirculation.Follow this wash with two liters of deionized water without recirculation. Once the water is evacuated, circulate two liters of Tyro solution and pass it through a five micron filter. Warm the perfuse eight to 37 degrees Celsius with a water jacket and circulator bubble oxygenated gas into the solution.Adjust the rate of gas bubbling to maintain the pH at 7.35 plus or minus 0.05. Monitor both the pH and temperature during experiment. The dual optical mapping apparatus consists of two high speed C moss cameras that can capture a 100 by 100 pixel image at 3000 frames per second.Attach a band pass filter to the calcium imaging camera and attach a long pass Filter to the voltage imaging camera on a dual camera holder. Attach a diic mirror and lens so that emission light coming from the heart is focused onto the mirror by the lens. Also, attach two cameras to the holder and arrange them so they are perpendicular to one another.Next, set up the excitation light. The light is generated by a halogen lamp and directed through a flexible light guide. Pass the light through a heat filter, a shutter to limit exposure, and an excitation band pass filter.Finally, prepare silver and silver chloride electrodes by installing them in the chamber. After double checking and adjusting the levels on the amplifiers and filters, the setup is ready for a heart. Use a cocktail of ketamine and xylazine with 100 units of heparin to anesthetize a mouse before proceeding.Assure that the animal has no pain reflex using a toe pinch. After making a mid sternal incision, quickly remove the heart and the surrounding tissues. Then using dissecting microscope rapidly, identify the aorta and make a clean cut across the ascending aorta below the right subclavian artery.Now attach a short section of aorta to a custom made 21 gauge cannula using four oh black braided silk sutures After cannulation retro greatly perfu and super fuse the heart. With Tyro solution, adjust the flow rate to keep the aortic pressure between 60 and 80 millimeters. Mercury as measured by a pressure transducer.Now dissect and remove the surrounding lung thymus and fat tissue. Now insert a silicone tube into the left ventricle through the pulmonary veins. Suture the tube to nearby connective tissue.This very important step prevents congestion and acidification of Perfuse eight trapped in the left ventricle, especially after the suppression of ventricular contractions. To prevent Perfuse eight induced heart movements, pin the heart to the bottom of the sill guard coated perfusion chamber at its apex. Furthermore, stretch and pin down the right and left atrial appendages to increase the surface area of the atria in the upright configuration.This will maximize recording surface area. To conduct the pacing stimulations, attach a custom made electrode to the surface of the heart. To reduce motion artifacts, fix a small cover glass over the heart on the solution surface.Do this by wedging the glass between several pins. As final preparations, focus the excitation light on the heart and position the cameras to get an optical image then commence with loading the dye while loading dies. Continuously monitor ECG recordings to assure normal electrical function of the heart.Begin with warming up BLEs statin to room temperature. Then mix one half milliliter of BLEs statin into the profuse eight in the holding reservoir, and dilute the remaining one 10th milliliter of BLEs statin into one milliliter of Tyros solution. Over the next 20 minutes, slowly inject the BLEs statin in tyros through a drug port near the cannula.The BLEs statin will gradually reduce the motion artifact. Next, dilute 30 microliters of voltage sensitive dye into one milliliter tyro solution, and slowly inject this mixture over five to seven minutes into the same drug port. Then make the same mixture again.This time, adding an additional 30 microliters of calcium indicator. Slowly apply this mixture over five to 10 minutes through the same port. After the calcium indicator is injected, wait an additional five to 10 minutes for the BLEs statin to fully suppress motion, and for the dyes to enter the cells before proceeding with the experiment.This heart preparation was made. According to the protocol, ECG signals were recorded during ventricular pacing from S one S one stimulation. Action potentials and calcium transient signals were recorded from the atria on the left and ventricles on the right.The arrow shows where signal scattering from the ventricles occurred by binning the recordings from the camera in units of three pixels by three pixels, an array of calcium transients and action potentials can be displayed from the data from the array data. A figure showing action potential conduction can be constructed. Data from three consecutive points on the transverse and longitudinal axis show the amplitude change with the propagation of the action potential across a ventricular surface.Likewise, a figure showing action potential and calcium transient durations can be constructed. This shows a control condition on the left on the right. An injection of isoproterenol was given to the heart, which clearly shortens the duration of both calcium transients and action potentials.Once mastered, this technique can be completed in two to three hours Following this procedure. Other methods like pacing protocol can be performed in order to answer additional questions such as those regarding refractory period of AV node.

optical-mapping-action-potentials-calcium-transients-mouse

Research

JoVE Journal

Bioengineering

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 Langendorff-perfused Rat Hearts

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 article describes the technique used to perform dual channel optical mapping in cultured HL-1 atrial cell monolayers. This unique protocol allows the simultaneous visualization of both calcium (Ca) and voltage (Vm) activity in the same area for the detailed detection and analysis of electrophysiological properties of culture monolayers.

Voltage and Calcium Dual Channel Optical Mapping of Cultured HL-1 Atrial Myocyte Monolayer

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

We present the methodology for the isolation of murine myocytes and how to obtain voltage or calcium traces simultaneously with sarcomere shortening traces using fluorescence photometry with simultaneous digital cell geometry measurements.

Optical Imaging of Isolated Murine Ventricular Myocytes

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 describes the procedure for sectioning and culturing human cardiac slices for preclinical drug testing and details the use of optical mapping for recording transmembrane voltage and intracellular calcium signals simultaneously from these slices.

Preclinical Cardiac Electrophysiology Assessment by Dual Voltage and Calcium Optical Mapping of Human Organotypic Cardiac Slices

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 and calcium transients in isolated Langendorff perfused mouse hearts. This technique enables high spatial resolution imaging of cardiac electrophysiology for arrhythmia research.

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

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