Name: High-resolution Optical Mapping of the Mouse Sino-atrial Node (Video) | JoVE Journal
Uploaded: 2016-12-02T11:30:00.000+00:00
Duration: 11 min 7 s
Description: Learn high-resolution optical mapping of the mouse sinoatrial node using Langendorff-perfused hearts or isolated atrial preparations. This method enables precise investigation of pacemaker mechanisms and cardiac arrhythmias.

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5 minutes

We are supported by the University of Wisconsin-Madison Medical School start-up (A.V.G.).

All experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Pub. No. 80-23). All methods and protocols used in these studies have been approved by The University of Wisconsin Animal Care and Use Protocol Committee following the Guidelines for Care and Use of Laboratory Animals published by NIH (publication No. 85-23, revised 1996). All animals used in this study received humane care in compliance with the Guide for the Care and Use of Laboratory Animals.
1. Heart Removal and Langendorff Perfusion
<ol>
	<li>Prior to heart isolation, warm Tyrode solution to 37 &#176;C by using a water bath and a water jacket.</li>
	<li>Anesthetize the mouse with 5% isoflurane/95% O2.</li>
	<li>Ensure appropriate level of anesthesia by checking the loss of pain reflex.</li>
	<li>Perform thoracotomy. Open the chest by using curved 5.5&#34; Mayo scissors and 5.5&#34; Kelly hemostatic forceps to make a 1 cm cut on the front of the thorax.
	<ol>
		<li>Quickly extract the heart from the chest (within 30 sec). Use 4&#34; curved Iris forceps to grab the lung tissue and cut out the lung, thymus and heart together with the pericardium using 4.3&#34; Iris scissors. Do not grab the heart directly.</li>
		<li>Wash it in oxygenated (95% O2 / 5% CO2), constant-temperature (36.8 &#177; 0.4&#176;C) modified Tyrode&#39;s solution. Use the following solution composition (in mM): 128.2 NaCl, 4.7 KCl, 1.19 NaH2PO4, 1.05 MgCl2, 1.3 CaCl2, 20.0 NaHCO3, and 11.1 glucose (pH 7.35 &#177; 0.05).</li>
	</ol>
	</li>
	<li>While bathed in the same solution, identify the lung, thymus, and fat tissue. Carefully dissect them from the heart. Use curved 3&#34; Vannas-Tubingen scissors and 4.3&#34; #5 forceps.</li>
	<li>Then identify the aorta and cannulate it onto a custom-made 21 G cannula. Use two curved 4.3&#34; #5B forceps.</li>
	<li>After cannulation, simultaneously perfuse (through the cannula at a flow rate of ~5 ml/min; this should be adjusted based on the aortic pressure, see below) and superfuse (in the bath at a flow rate of ~50 ml/min) the heart with warmed and filtered Tyrode solution throughout the entire experiment. Pass the perfusion solution through an in-line 11 &#181;m nylon filter to prevent clogging of coronary circulation.</li>
	<li>Using a pressure transducer (or a pressure gauge) connected via a 3-way stopcock Luer lock to the perfusion line, monitor the aortic pressure and maintain it between 60 and 80 mmHg. Adjust perfusion speed if required to keep the aortic pressure within this range.</li>
</ol>
2. Optical Mapping of the SAN from the Langendorff-perfused Whole Heart
<ol>
	<li>Instrumentation
	<ol>
		<li>Place the heart horizontally and pin the ventricular apex to the silicon-coated bottom of the chamber using 0.1 mm diameter pins to prevent stream-induced movement during the experiment.12</li>
		<li>Insert a small (.012&#34; I.D. x .005&#34;) silicon tube into the left ventricle (LV) via the pulmonary vein, the left atria (LA) and the mitral valve (MV). Fix the tube by a silk suture (4-0) to the sounding connective tissue.</li>
		<li>Position the heart with its posterior side facing up (Figure 1A, left panel).</li>
		<li>Pin the edge of the RA appendage (RAA) to the silicon bottom of the chamber using 0.1 mm diameter minutien pins. Adjust its level in order to make the posterior surface of the RA flat and allow it to be located the camera&#39;s focal plane. This will enable optical measurements from the maximal surface area of the atrium.</li>
		<li>Noose superior (SVC) and inferior (IVC) vena cava by a silk suture (4-0), stretch and pin the other end of the suture to the bottom of a silicon-coated chamber (see Figure 3A). Make sure the sutures do not block the optical field of view.</li>
		<li>Place the custom-made pacing electrode on the edge of the RAA. To make electrodes, use silicon coated 0.25 mm diameter silver wires, with 0.5 mm inter-electrode distance and devoid of silicon for a length of 1 mm at the pacing end. Then place two ECG 12 mm needle (29-gauge) monopolar electrodes near the base of the right and left ventricles. Place the ground ECG electrode near the apex of the ventricles .</li>
	</ol>
	</li>
	<li>Staining
	<ol>
		<li>Since both fluorescent dyes and electro-mechanical uncoupler blebbistatin are light-sensitive, perform all the procedures described below in a dark room. First prepare voltage-sensitive dye RH-237 or di-4-ANNEPS as a stock solution, 1.25 mg/ml in dimethyl sulfoxide (2.5 mM), aliquot it (30 &#181;l each) and store the aliquots at -20 &#176;C.</li>
		<li>Dilute 5-10 &#181;l of dye stock solution in 1 ml of warmed (37 &#176;C) Tyrode solution and then inject it into the coronary perfusion line over a period of 5-7 min using an in-line Luer injection port.</li>
		<li>Prepare blebbistatin as a stock solution (2 mg/ml in dimethyl sulfoxide, 6.8 mM) in advance and store it at 4&#176;C.</li>
		<li>After 20 min of stabilization, add 0.5 ml of warmed (37 &#176;C) blebbistatin in the perfusate and dilute 0.1 ml of blebbistatin in 1 ml of warmed (37 &#176;C) Tyrode solution. Then inject into the coronary perfusion line over a period of 5-7 min using an in-line Luer injection port.</li>
	</ol>
	</li>
</ol>
3. Optical mapping of the SAN from the Isolated Atrial Preparation
<ol>
	<li>Instrumentation
	<ol>
		<li>For the isolated atrial preparation, isolate and cannulate the heart as described above in steps 1.1-1.5 for whole-heart preparation.</li>
		<li>Dissect the ventricles away from the anterior side. See Figure 1A, right panel, cut #1 for details.</li>
		<li>Open the RA by cutting through the tricuspid valve (TV) along the TV-SVC axis. See Figure 1A, right panel, cut #2 for details.</li>
		<li>Cut the medial limb of the crista terminalis to open the RAA. See Figure 1A, right panel, cut #3.</li>
		<li>Open the anterior atrial free wall by performing a cut from the midline of the previous cut#3 to the edge of the right bottom corner of the RAA, flatten and pin the atrial free wall to the bottom of a silicon-coated chamber. See the direction shown by the hollow arrow in Figure 1A, cut #4. Preserve a rim of ventricular tissue for pinning the preparation to prevent damage to the atria.</li>
		<li>Similarly, open LA by cutting through the MV along the MV-upper corner of the LA appendage (LAA).</li>
		<li>To open the LAA, cut from the middle of the opened LAA, through the anterior atrial free wall until near a middle rim of the LAA.</li>
		<li>Open the anterior atrial free wall along the same direction, then flatten and pin it to the bottom of a Sylgard-coated chamber.</li>
		<li>Partially remove the interatrial septal tissue. This will reduce scattering of the optical signal from tissue that is not in focus. Therefore, both the LA and RA as well as the SAN and atrio-ventricular junction (AVJ) are accessible in this preparation (Figure 1B).</li>
		<li>Lift up the final preparation by about 0.5 mm from the bottom of the silicon-coated chamber in order to allow superfusion from both epicardial and endocardial surfaces.</li>
		<li>Superfuse the preparation with warmed (37 &#176;C) Tyrode solution at a constant rate of ~12 ml/min for a focused flow located near the SVC and ~30 ml/min for a bath superfusion.</li>
		<li>Place the custom-made pacing Ag/AgCl2 electrode (0.25 mm diameter) on the edge of the dissected RAA. Then place two ECG 12 mm needle (29G) electrodes (monopolar) near the RAA and LAA, respectively. Place the ground ECG electrode near the AVJ.</li>
	</ol>
	</li>
	<li>Staining
	<ol>
		<li>Perform a direct application of the voltage-sensitive dye (RH-237 or di-4-ANNEPS). For this, dilute 1-2 &#181;l of the dye stock solution in 1 mm of warmed (37 &#176;C) Tyrode solution and slowly release the diluted dye on the surface of the preparation by using a 1 mm pipette.</li>
		<li>Alternatively, perform atrial staining with the voltage-sensitive dye through a coronary perfusion of the Langendorff-perfused heart similar to that described above for the whole-heart preparation (steps 2.2.1-2.2.2).
		<ol>
			<li>After the coronary perfusion staining, isolate the atrial preparation as described. Perform additional surface staining when needed to reach a satisfactory fluorescence level. Quick atrial isolation does not bleach the dye and does not affect the quality of staining.</li>
		</ol>
		</li>
		<li>Immobilize the preparation with the electro-mechanical uncoupler, blebbistatin. Dilute 3-5 &#181;l blebbistatin stock solution in the warmed (37 &#176;C) Tyrode solution and slowly release the diluted blebbistatin on the surface of the preparation and surrounding solution. Since blebbistatin has been shown to be light sensitive,13,14 avoid long exposure to light during preparation and staining.</li>
		<li>Perform additional blebbistatin application as much as needed to suppress contraction. Normally up to 3 additional applications (3-5 &#181;l per each application) can be used to suppress the motion artifact sufficiently in order to accurately reconstruct SAN and atrial activation.</li>
		<li>Add additional 0.5 ml of warmed blebbistatin in the perfusate. During this procedure, pause the superfusion for 30 sec to allow the dye/blebbistatin to stain the atrial tissue.</li>
	</ol>
	</li>
</ol>
4. Optical Mapping Set Up
NOTE: A detailed description of the optical mapping system is provided elsewhere.12
<ol>
	<li>Use a camera with a temporal resolution of 2,000 frames/sec or higher, and a series of condenser lenses to reach spatial resolution of 100 &#181;m/pixel or higher. This is required to reconstruct SAN activation and intranodal propagation.</li>
	<li>To reduce motion artifact from the vibrating solution, fix a small cover glass on the surface of the solution over the heart/isolated atrial preparation.</li>
	<li>Use excitation light (520 &#177; 45 nm wavelength) provided by a constant-current, low-noise halogen lamp. Direct the filtered light beam onto the preparation by using a flexible bifurcated light guide.</li>
	<li>Filter the emitted fluorescence by a long-pass filter (&#62;715 nm). Collect, digitize and save the acquired fluorescent signal on a computer using software provided by the camera manufacturer.</li>
</ol>
5. Data Processing
NOTE: Optical mapping data is collected and stored as a series of matrices of fluorescent intensity at different time points. Each pixel represents a measure of fluorescent intensity collected from a specific location on the tissue preparation at a specific time point. Background fluorescence is automatically removed per pixel to allow for better visualization of fluorescence changes produced by membrane voltage changes and inverted to correspond with action potentials (APs) measured by microelectrode systems. Details of the different steps in processing optical imaging data, including image segmentation, spatial filtering, temporal filtering, and baseline drift removal, are provided in the focused review.15
<ol>
	<li>Manually or by using the special algorithm (for example, a thresholding or edge detection)15 to determine tissue boundaries, create a binary mask of 1 (included pixels) and 0 (excluded pixels) and apply the mask to each frame in the sequence. This process creates a binary image that highlights areas of interest for further processing.</li>
	<li>To improve signal-to-noise ratio of the optical data, filter the signals within the selected areas of interest. For this, use spatial (i.e. averaging of neighboring fluorescent pixels as defined by a desired convolution bin or kernel, for instance 3 x 3 or, for noisy data, 5 x 5) and/or temporal filtering (for example, Butterworth, Chebyshev type 1, Chebyshev type 2, Elliptic etc.) (Figure 2). Keep in mind the possibility of filtered-induced artifacts when interpreting the data. For more details, see review.15</li>
	<li>Remove drift of baseline in optical recordings when needed (by using high-pass filtering or polynomial fitting to the original signal) and then normalize each pixel signal from 0 (minimum fluorescence) to 1 (maximum fluorescence).</li>
	<li>Select a time window that encompasses the activation times of all the pixels for a single AP propagation. Assign each pixel its activation time as the time of maximum upstroke derivative (dF/dtmax, where F is fluorescence intensity). Using all pixel activation times, reconstruct the isochronal activation map. Each isochron will thus show the pixels activated at the same time.</li>
	<li>To reconstruct the AP duration (APD) distribution map, for each pixel calculate the duration between the activation time and the time at a specific level of repolarization (for example, at 80% of repolarization, APD80). Using all pixel APD values, reconstruct the APD distribution isochronal map. Each isochron will thus show the pixels with the same APD.</li>
	<li>To calculate conduction velocity for AP propagation, fit a surface of the preparation to the activation time data calculated in 5.4. For this, use polynomial surface fitting or local kernel surface smoothing and then calculate local conduction velocity vectors from the gradient of the fitted surface.</li>
	<li>To create the repolarization map, assign each pixel its repolarization time, defined as the maximum second derivative (d2F/dt2) of the optical signal at the end of the OAP, or the time of 90% repolarization.</li>
</ol>

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<li>Kanlop, N., Sakai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optical+mapping+study+of+blebbistatin-induced+chaotic+electrical+activities+in+isolated+rat+atrium+preparations%5BTitle%5D)+AND+%22J+Physiol+Sci%22%5BJournal%5D)">Optical mapping study of blebbistatin-induced chaotic electrical activities in isolated rat atrium preparations.</a> J Physiol Sci. 60 (2), 109-117 (2010).</li>
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<li>Wu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Altered+sinoatrial+node+function+and+intra-atrial+conduction+in+murine+gain-of-function+Scn5a%2B%2FDeltaKPQ+hearts+suggest+an+overlap+syndrome%5BTitle%5D)+AND+%22Am+J+Physiol+Heart+Circ+Physiol%22%5BJournal%5D)">Altered sinoatrial node function and intra-atrial conduction in murine gain-of-function Scn5a+/DeltaKPQ hearts suggest an overlap syndrome.</a> Am J Physiol Heart Circ Physiol. 302 (7), H1510-H1523 (2012).</li>
</ol>

No conflicts of interest are declared.

Here, we presented two types of mouse SAN preparations: 1) intact SAN in the Langendorff-perfused whole heart, and 2) SAN in the isolated, opened atrial preparation. These two types of preparation serve different experimental purposes. In the Langendorff-perfused whole heart preparation, the intact atrial structure is preserved which makes it possible to study complex atrial arrhythmias such as atrial fibrillation as well as interactions between the SAN and atrium during reentrant tachyarrhythmias.6 In contras...

Novel scientific breakthroughs that lead to leaps in the understanding of human physiology are often preceded by technological advances. Fluorescent optical mapping, for example, enables investigation of multiple physiological parameters in both cells and tissues.1,2 It significantly improved our understanding of how the anatomical structure is associated with electrophysiological functions and dysfunctions. In the heart, the natural pacemaker and conduction systems such as the sinoatrial node (SAN) and the atrioventricular junction consist of nodal myocytes that are insulated by the surrounding atrial myocytes.3-5 Such organization creates complex three-dimensional structures with specialized electrical properties. Both structural and functional remodeling of these pacemaker structures has been recognized to form significant electrophysiological heterogeneities.6,7 Understanding the mechanism underlying how such heterogeneities result in SAN dysfunctions and atrial arrhythmogenesis will dramatically benefit the clinical treatment of these diseases. It requires a technique to visualize the propagation of electrical signals at the tissue level, such as optical mapping. 
Recently, accumulating evidence has proven the advance of optical mapping in studies of atrial electrophysiology and pathology.2 However, novel and rigorous studies are dependent on the accurate interpretation of experimental data, whose validity and stability rely on careful experiment protocols. Genetically modified mice are extensively used for research as animal models of human diseases including sick sinus syndrome, pacemaker abnormalities and atrial arrhythmias.6,8-11 Thus, a combination of fluorescent optical mapping with transgenic mouse models provides a powerful tool to study cardiac electrical abnormalities associated with various pathologies. In this paper, we present a protocol for high-resolution optical mapping of the mouse SAN and atrium. Specifically, we discuss and compare different dye loading approaches, time effects on dye bleaching and heart rate stability during the experiment.

<table><tbody><tr><td>water jacket</td><td>Radnoti</td><td>1660 Series Tissue Bath for Large Organ or Single Cell Isolation Procedures</td><td></td></tr><tr><td>water bath / circulator</td><td>Fisher Scientific</td><td>1016S</td><td></td></tr><tr><td>pressure amplifier</td><td>AD Instruments</td><td>MLT0670</td><td></td></tr><tr><td>EMD Millipore Nylon Net Filters</td><td>Fisher Scientific</td><td>NY1102500</td><td></td></tr><tr><td>Pressure transducer</td><td>AD Instruments</td><td>MLT0670</td><td></td></tr><tr><td>Stainless Steel Minutien Pins - 0.1 mm Diameter</td><td>Fine Science Tools</td><td>26002-10&amp;nbsp;</td><td></td></tr><tr><td>Perfusion pump</td><td>World Precision Instruments</td><td>PERIPRO-4LS</td><td></td></tr><tr><td>Superfusion pump</td><td>World Precision Instruments</td><td>PERIPRO-4HS</td><td></td></tr><tr><td>Vannas Tubingen scissors&amp;nbsp;</td><td>World Precision Instruments</td><td>503379</td><td></td></tr><tr><td>Dumont forceps</td><td>World Precision Instruments</td><td>501201, 500085</td><td></td></tr><tr><td>Mayo scissors</td><td>World Precision Instruments</td><td>501750</td><td></td></tr><tr><td>Kelly hemostatic forceps</td><td>World Precision Instruments</td><td>501241</td><td></td></tr><tr><td>Iris forceps</td><td>World Precision Instruments</td><td>15917</td><td></td></tr><tr><td>Iris scissors</td><td>World Precision Instruments</td><td>501263</td><td></td></tr><tr><td>ECG 12 mm needle (29-gauge) electrodes (monopolar)&amp;nbsp;</td><td>AD Instruments</td><td>MLA1203</td><td></td></tr><tr><td>in-line Luer injection port</td><td>Ibidi</td><td>10820</td><td></td></tr><tr><td>Ultima-L CMOS camera&amp;nbsp;</td><td>SciMedia</td><td>MiCAM-05&amp;nbsp;</td><td></td></tr><tr><td>halogen lamp</td><td>Moritex USA Inc</td><td>MHAB-150W</td><td></td></tr><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>ThermoFisher Scientific</td><td>S1109</td><td></td></tr><tr><td>Dimethyl sulphoxide (DMSO)</td><td>Sigma-Aldrich</td><td>D2650</td><td></td></tr></tbody></table>

high-resolution optical mapping of the mouse sino-atrial node

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

Sino-atrial node (SAN) dysfunctions and associated complications constitute important causes of morbidity in patients with cardiac diseases. The development of novel pharmacological therapies to cure these patients relies on the thorough understanding of both normal physiology and pathophysiology of the SAN. Among the studies of cardiac pacemaking, the mouse SAN is widely used due to its feasibility for modifications in the expression of different genes that encode SAN ion channels or calcium handling proteins. Emerging evidence from electrophysiological and histological studies has also proved the representativeness and similarity of the mouse SAN structure and functions to larger mammals, including the presence of specialized conduction pathways from the SAN to the atrium and a complex pacemakers' hierarchy within the SAN. Recently, the technique of optical mapping has greatly facilitated the exploration and investigation of the origin of excitation and conduction within and from the mouse SAN, which in turn has extended the understanding of the SAN and benefited clinical treatments of SAN dysfunction associated diseases. In this manuscript, we have described in detail how to perform the optical mapping of the mouse SAN from the intact, Langendorff-perfused heart and from the isolated atrial preparation. This protocol is a useful tool to enhance the understanding of mouse SAN physiology and pathophysiology.

Optical Mapping of the Intact SAN from the Langendorff-perfused Heart 
A typical example of an RA activation contour map reconstructed for spontaneous sinus rhythm is shown in Figure 3 for a Langendorff-perfused mouse heart. The early activation point is located within the intercaval region near the SVC where the SAN is anatomically defined.3,16 Two RA activation contour maps acquired at 1.0 and 0.5 msec sampling rate are shown in Fi...

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.

Sino-atrial node (SAN) dysfunctions and associated complications constitute important causes of morbidity in patients with cardiac diseases. The ...

The overall goal of this experimental protocol is to perform optical mapping of the mouse sinoatrial node from an intact Langendorff-perfused heart or an isolated atrial preparation. This method can help answer key questions in the sinoatrial node electro-and pathophysiology field about the mechanisms of pacemaker abnormalities, sick sinus syndrome, and various diseases associated atrial arrhythmias. The main advantage of this technique is that it enables the investigation of multiple parameters in the intact tissues with very high spatial and atemporal resolution.After confirming the appropriate level of anesthesia by toe pinch, use curved 5.5 inch Mayo scissors and 5.5 inch Kelly Hemostatic Forceps to make a one centimeter incision on the front of the thorax. Using four inch curved Iris Forceps, quickly grasp the lung tissue and use four thirds inch Iris scissors to cut out the lung, thymus, and heart together, along with the pericardium. Wash the tissues in oxygenated 37 degree Celsius modified Tyrode solution.Then use curved three inch Vannas Tubingen scissors and four thirds inch number five forceps to remove the lung, thymus, and fat tissue from the heart. Next, use a custom made 21 gauge cannula and two curved 4.3 inch number five B forceps to cannulate the aorta and simultaneously perfuse and superfuse the heart with 37 degree Celsius Tyrode solution directing the solution through an inline 11 micron nylon filter prior to the perfusion to prevent clogging of the coronary circulation. Then connect a pressure transducer to a Luer lock on the perfusion line via a three-way stopcock to monitor the aortic pressure adjusting the perfusion speed to maintain the aortic pressure between 60 to 80 millimeters of mercury as necessary.For optical mapping of the sinoatrial node from a Langendorff-perfused heart, position the organ in a horizontal orientation with the posterior side facing up in a glass tissue organ bath chamber and use 0.1 millimeter diameter pins to fix the ventricular apex to the silicone-coated bottom of the chamber to prevent stream-induced movement during the experiment. Insert a small silicone tube into the left ventricle via the pulmonary vein, the left atria, and the mitral valve and use a 40 silk suture to fix the tube to the surrounding connective tissue. Next, use another 40 silk suture to noose the superior and inferior vena cavae and pin the edge of the right atrial appendage to the bottom of the chamber.Stretch and pin the other end of the suture to the bottom of the chamber and place a custom-made pacing electrode on the edge of the right atrial appendage. Then place two electrocardiogram 12 millimeter needle monopolar electrodes near the base of the right and left ventricles and place the ground electrocardiogram electrode near the ventricle apex. To stain the tissue, inject the diluted dye into an inline perfusion port of the coronary perfusion line for delivery over a five to seven minute period.After 20 minutes of stabilization, add 0.5 milliliters of 37 degree Celsius blebbistatin into the perfusate followed by the injection of 0.1 milliliters of blebbistatin diluted in 1 milliliter of 37 degree Celsius Tyrode solution into the coronary perfusion line over another five to seven minutes using an inline Luer lock injection port. Blebbistatin should be used with caution. To prevent precipitation of the inhibitor, first dissolve the blebbistatin in medium that has been warmed to a clear 37 degrees and vigorously stir the mixture.To optically map the sample, first fix a small covered glass onto the surface of the solution over the tissue to reduce any motion artifacts from the vibrating solution. Then using a flexible bifurcated light guide with an excitation light provided by a constant current, low noise, halogen lamp, direct the filtered light beam onto the tissue and image the resulting fluorescent signal. After cannulating the heart as just demonstrated, dissect the ventricles away from the anterior side and open the right atrium through the tricuspid valve along the tricuspid valve superior vena cava axis followed by an incision through the medial limb of the crista terminalis.To open the anterior atrial free wall, make an incision from the midline of the medial limb incision to the edge of the right bottom corner of the right atrial appendage and pin the flattened atrial free wall to the bottom of a silicone-coated chamber. To open the left atrial appendage, cut through the mitral valve along the mitral valve upper corner of the left atrial appendage. Then pin the opened left atrium as it was previously done for the right atrium.Then partially remove the ventricular tissue. Preserve a rim of ventricular tissue for pinning the preparation to prevent damage to the atria and pin the tissue to the bottom of a Sylgard-coated chamber. Now lift the final preparation by about 0.5 millimeters from the bottom of the silicone-coated chamber to allow superfusion from both the epicardial and endocardial surfaces and superfuse the sample with 37 degree Celsius Tyrode solution.Then place a custom-made pacing silver chloride electrode on the edge of the dissected right atrial appendage. Place one electrocardiogram 12 millimeter needle mono polar electrode near each of the right and left atrial appendages, and place the ground electrocardiogram electrode near the atrial ventricular junction. To stain the heart tissue, use a 1 millimeter pipette to slowly release voltage-sensitive dye diluted in 37 degree Celsius Tyrode solution directly onto the tissue.Then immobilize the sample with the direct application of diluted blebbistatin onto the tissue followed by the administration of another 0.5 milliliters of blebbistatin via the perfusate and optically map the sample as just demonstrated. The tissue staining and blebbistatin inhibition steps are critical requiring accurate implementation and a precise staining procedure so that the atrial physiological parameters including the heart rate, allocation of the leading pacemaker and atrial conduction are not affected. Here a typical right atrial activation contour map reconstructed for spontaneous sinus rhythm for a Langendorff-perfused mouse heart with two corresponding right atrial contour maps acquired at 1 and 0.5 millisecond sampling rates are shown.The early activation point is located within the intercaval region near the superior vena cava where the sinoatrial node is anatomically defined. In this experiment, after right atrial appendage pacing for at least one minute at 10 to 12 Hertz, the electrical stimulation was stopped and the sinoatrial node recovery time was calculated as the time interval between the last captured action potential and the first spontaneous action potential. Activation of the isolated atrial preparation during spontaneous sinus rhythm allows the precise area of the leading pacemaker location to be identified.If the surgery and dye loading procedures are followed appropriately, no significant changes in the physiological characteristics of the atrium should be observed. Although arterial staining may require a larger amount of dye, the fluorescent signal in the sinoatrial node area tissue appears to be more stable by this loading method with a signal intensity decay time after coronary staining almost twice as long as that after surface staining. Once mastered, this technique can be completed in 30, 40 minutes if it's performed properly.While attempting this procedure, take care to locate atrial anatomy before making a cut to avoid damaging the atrial tissue and the sinus node. Following this procedure, other measures like conventional glass mitral electrode recordings can be performed to answer additional questions about commutative transmembrane potential characteristics. After its development, this technique paved the way for researchers in the field of intellect physiology to explore atrial arrhythmias including sinoatrial node dysfunction in the heart.After watching this video, you should have a good understanding of how to perform high resolution optical mapping of a mouse sinus node preparation.

high-resolution-optical-mapping-of-the-mouse-sino-atrial-node

Research

JoVE Journal

Biology

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 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

This video demonstrates the procedure to isolate sinoatrial node or SAN from mouse heart. The SAN houses the sinoatrial cells that initiates heartbeat by generating spontaneous action potentials.

Sinoatrial Node Isolation from Mouse Model: A Procedure to Harvest SA Node from Murine Heart

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 provide a step-by-step protocol for whole-mount immunofluorescence staining of the sinoatrial node (SAN) and atrioventricular node (AVN) in murine hearts.

Whole-Mount Immunofluorescence Staining, Confocal Imaging and 3D Reconstruction of the Sinoatrial and Atrioventricular Node in the Mouse

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.

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

We report a method for mesoscopic reconstruction of the whole mouse heart by combining new advancements in tissue transformation and staining with the development of an axially scanned light-sheet microscope.

Mesoscopic Optical Imaging of Whole Mouse Heart

The protocol utilizes advanced light-sheet microscopy along with adapted tissue clearing methods to investigate intricate cardiac structures in rodent hearts, holding great potential for the understanding of cardiac morphogenesis and remodeling.

Light-Sheet Imaging to Reveal Cardiac Structure in Rodent Hearts

Learn high-resolution optical mapping of the mouse sinoatrial node using Langendorff-perfused hearts or isolated atrial preparations. This method enables precise investigation of pacemaker mechanisms and cardiac arrhythmias.

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

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