Name: high-resolution optical mapping of the mouse sino-atrial node
Uploaded: 2016-12-02T11:30:00
Description: Watch this Scientific Journal Video about High-resolution Optical Mapping of the Mouse Sino-atrial Node at JoVE.com

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.
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	<li>Efimov, I. R., Nikolski, V. P., Salama, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+imaging+of+the+heart.">Optical imaging of the heart.</a> Circ Res. 95 (1), 21-33 (2004).</li><li>Herron, T. J., Lee, P., Jalife, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+imaging+of+voltage+and+calcium+in+cardiac+cells+&amp;+tissues.">Optical imaging of voltage and calcium in cardiac cells &amp; tissues.</a> Circ Res. 110 (4), 609-623 (2012).</li><li>Fedorov, V. V., Glukhov, A. V., Chang, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conduction+barriers+and+pathways+of+the+sino-atrial+pacemaker+complex:+their+role+in+normal+rhythm+and+atrial+arrhythmias.">Conduction barriers and pathways of the sino-atrial pacemaker complex: their role in normal rhythm and atrial arrhythmias.</a> Am J Physiol Heart Circ Physiol. , (2012).</li><li>Boyett, M. R., Honjo, H., Kodama, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+sinoatrial+node,+a+heterogeneous+pacemaker+structure.">The sinoatrial node, a heterogeneous pacemaker structure.</a> Cardiovasc Res. 47 (4), 658-687 (2000).</li><li>Glukhov, A. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sinoatrial+Node+Reentry+in+a+Canine+Chronic+Left+Ventricular+Infarct+Model:+The+Role+of+Intranodal+Fibrosis+and+Heterogeneity+of+Refractoriness.">Sinoatrial Node Reentry in a Canine Chronic Left Ventricular Infarct Model: The Role of Intranodal Fibrosis and Heterogeneity of Refractoriness.</a> Circ Arrhythm Electrophysiol. , (2013).</li><li>Glukhov, A. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calsequestrin+2+deletion+causes+sinoatrial+node+dysfunction+and+atrial+arrhythmias+associated+with+altered+sarcoplasmic+reticulum+calcium+cycling+and+degenerative+fibrosis+within+the+mouse+atrial+pacemaker+complex.">Calsequestrin 2 deletion causes sinoatrial node dysfunction and atrial arrhythmias associated with altered sarcoplasmic reticulum calcium cycling and degenerative fibrosis within the mouse atrial pacemaker complex.</a> Eur Heart J. 36 (11), 686-697 (2015).</li><li>John, R. M., Kumar, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sinus+Node+and+Atrial+Arrhythmias.">Sinus Node and Atrial Arrhythmias.</a> Circulation. 133 (19), 1892-1900 (2016).</li><li>Swaminathan, P. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidized+CaMKII+causes+cardiac+sinus+node+dysfunction+in+mice.">Oxidized CaMKII causes cardiac sinus node dysfunction in mice.</a> J Clin Invest. 121 (8), 3277-3288 (2011).</li><li>Glukhov, A. V., Fedorov, V. V., Anderson, M. E., Mohler, P. J., Efimov, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+anatomy+of+the+murine+sinus+node:+high-resolution+optical+mapping+of+ankyrin-B+heterozygous+mice.">Functional anatomy of the murine sinus node: high-resolution optical mapping of ankyrin-B heterozygous mice.</a> Am J Physiol Heart Circ Physiol. 299 (2), H482-H491 (2010).</li><li>Egom, E. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+sinoatrial+node+function+and+increased+susceptibility+to+atrial+fibrillation+in+mice+lacking+natriuretic+peptide+receptor+C.">Impaired sinoatrial node function and increased susceptibility to atrial fibrillation in mice lacking natriuretic peptide receptor C.</a> J Physiol. 593 (5), 1127-1146 (2015).</li><li>Torrente, A. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Burst+pacemaker+activity+of+the+sinoatrial+node+in+sodium-calcium+exchanger+knockout+mice.">Burst pacemaker activity of the sinoatrial node in sodium-calcium exchanger knockout mice.</a> Proc Natl Acad Sci U S A. 112 (31), 9769-9774 (2015).</li><li>Lang, D., Sulkin, M., Lou, Q., Efimov, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+mapping+of+action+potentials+and+calcium+transients+in+the+mouse+heart.">Optical mapping of action potentials and calcium transients in the mouse heart.</a> J Vis Exp. (55), e3275 (2011).</li><li>Fedorov, V. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+blebbistatin+as+an+excitation-contraction+uncoupler+for+electrophysiologic+study+of+rat+and+rabbit+hearts.">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><li>Swift, L. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+of+blebbistatin+for+cardiac+optical+mapping+and+other+imaging+applications.">Properties of blebbistatin for cardiac optical mapping and other imaging applications.</a> Pflugers Arch. 464 (5), 503-512 (2012).</li><li>Laughner, J. I., Ng, F. S., Sulkin, M. S., Arthur, R. M., Efimov, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Processing+and+analysis+of+cardiac+optical+mapping+data+obtained+with+potentiometric+dyes.">Processing and analysis of cardiac optical mapping data obtained with potentiometric dyes.</a> Am J Physiol Heart Circ Physiol. 303 (7), H753-H765 (2012).</li><li>Liu, J., Dobrzynski, H., Yanni, J., Boyett, M. R., Lei, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organisation+of+the+mouse+sinoatrial+node:+structure+and+expression+of+HCN+channels.">Organisation of the mouse sinoatrial node: structure and expression of HCN channels.</a> Cardiovasc Res. 73 (4), 729-738 (2007).</li><li>Verheijck, E. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+features+of+the+mouse+sinoatrial+node+in+relation+to+connexin+distribution.">Electrophysiological features of the mouse sinoatrial node in relation to connexin distribution.</a> Cardiovasc Res. 52 (1), 40-50 (2001).</li><li>Krishnaswamy, P. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+parasympathetic+nervous+system+regulation+of+the+sinoatrial+node+in+Akita+diabetic+mice.">Altered parasympathetic nervous system regulation of the sinoatrial node in Akita diabetic mice.</a> J Mol Cell Cardiol. 82, 125-135 (2015).</li><li>Nygren, A., Lomax, A. E., Giles, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+of+action+potential+durations+in+isolated+mouse+left+and+right+atria+recorded+using+voltage-sensitive+dye+mapping.">Heterogeneity of action potential durations in isolated mouse left and right atria recorded using voltage-sensitive dye mapping.</a> Am J Physiol Heart Circ Physiol. 287 (6), H2634-H2643 (2004).</li><li>Efimov, I. R., Fedorov, V. V., Joung, B., Lin, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+cardiac+pacemaker+circuits:+methodological+puzzles+of+the+sinoatrial+node+optical+mapping.">Mapping cardiac pacemaker circuits: methodological puzzles of the sinoatrial node optical mapping.</a> Circ Res. 106 (2), 255-271 (2010).</li><li>Aschar-Sobbi, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+atrial+arrhythmia+susceptibility+induced+by+intense+endurance+exercise+in+mice+requires+TNFalpha.">Increased atrial arrhythmia susceptibility induced by intense endurance exercise in mice requires TNFalpha.</a> Nat Commun. 6, 6018 (2015).</li><li>Hansen, B. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atrial+fibrillation+driven+by+micro-anatomic+intramural+re-entry+revealed+by+simultaneous+sub-epicardial+and+sub-endocardial+optical+mapping+in+explanted+human+hearts.">Atrial fibrillation driven by micro-anatomic intramural re-entry revealed by simultaneous sub-epicardial and sub-endocardial optical mapping in explanted human hearts.</a> Eur Heart J. 36 (35), 2390-2401 (2015).</li><li>Lang, D., Petrov, V., Lou, Q., Osipov, G., Efimov, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatiotemporal+control+of+heart+rate+in+a+rabbit+heart.">Spatiotemporal control of heart rate in a rabbit heart.</a> J Electrocardiol. 44 (6), 626-634 (2011).</li><li>Kanlop, N., Sakai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+mapping+study+of+blebbistatin-induced+chaotic+electrical+activities+in+isolated+rat+atrium+preparations.">Optical mapping study of blebbistatin-induced chaotic electrical activities in isolated rat atrium preparations.</a> J Physiol Sci. 60 (2), 109-117 (2010).</li><li>Glukhov, A. V., Flagg, T. P., Fedorov, V. V., Efimov, I. R., Nichols, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+K(ATP)+channel+pharmacology+in+intact+mouse+heart.">Differential K(ATP) channel pharmacology in intact mouse heart.</a> J Mol Cell Cardiol. 48 (1), 152-160 (2010).</li><li>Wu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+sinoatrial+node+function+and+intra-atrial+conduction+in+murine+gain-of-function+Scn5a+/DeltaKPQ+hearts+suggest+an+overlap+syndrome.">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>

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.

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			<td height="21" style="height:21px;width:395px;">water jacket</td>
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			<td colspan="2" style="width:409px;">1660 Series Tissue Bath for Large Organ or Single Cell Isolation Procedures</td>
		</tr>
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			<td height="21" style="height:21px;">water bath / circulator</td>
			<td>Fisher Scientific</td>
			<td>1016S</td>
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			<td height="21" style="height:21px;">pressure amplifier</td>
			<td>AD Instruments</td>
			<td>MLT0670</td>
			<td></td>
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			<td height="21" style="height:21px;">EMD Millipore Nylon Net Filters</td>
			<td>Fisher Scientific</td>
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			<td height="21" style="height:21px;">Pressure transducer</td>
			<td>AD Instruments</td>
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			<td>World Precision Instruments</td>
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			<td height="21" style="height:21px;">Vannas Tubingen scissors&#160;</td>
			<td>World Precision Instruments</td>
			<td>503379</td>
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			<td height="21" style="height:21px;">Dumont forceps</td>
			<td>World Precision Instruments</td>
			<td>501201, 500085</td>
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			<td>World Precision Instruments</td>
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			<td>World Precision Instruments</td>
			<td>501241</td>
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			<td>World Precision Instruments</td>
			<td>15917</td>
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			<td height="21" style="height:21px;">Iris scissors</td>
			<td>World Precision Instruments</td>
			<td>501263</td>
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			<td height="21" style="height:21px;">ECG 12 mm needle (29-gauge) electrodes (monopolar)&#160;</td>
			<td>AD Instruments</td>
			<td>MLA1203</td>
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			<td height="21" style="height:21px;">in-line Luer injection port</td>
			<td>Ibidi</td>
			<td>10820</td>
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			<td height="21" style="height:21px;">Ultima-L CMOS camera&#160;</td>
			<td>SciMedia</td>
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			<td>Moritex USA Inc</td>
			<td>MHAB-150W</td>
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			<td height="20" style="height:20px;width:395px;">NaCl</td>
			<td style="width:200px;">Fisher Scientific</td>
			<td style="width:243px;">S271-1</td>
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			<td height="20" style="height:20px;width:395px;">CaCl2 (2H2O)</td>
			<td style="width:200px;">Fisher Scientific</td>
			<td style="width:243px;">C79-500</td>
			<td style="width:167px;"></td>
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			<td height="20" style="height:20px;width:395px;">KCl</td>
			<td style="width:200px;">Fisher Scientific</td>
			<td style="width:243px;">S217-500</td>
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			<td height="20" style="height:20px;width:395px;">MgCl2 (6H2O)</td>
			<td style="width:200px;">Fisher Scientific</td>
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			<td height="20" style="height:20px;width:395px;">NaH2PO4 (H2O)</td>
			<td style="width:200px;">Fisher Scientific</td>
			<td style="width:243px;">S369-500</td>
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			<td height="20" style="height:20px;width:395px;">NaHCO3</td>
			<td style="width:200px;">Fisher Scientific</td>
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			<td height="20" style="height:20px;width:395px;">D-Glucose</td>
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			<td height="20" style="height:20px;width:395px;">Blebbistatin</td>
			<td style="width:200px;">Tocris Bioscience</td>
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			<td height="20" style="height:20px;width:395px;">RH237</td>
			<td>ThermoFisher Scientific</td>
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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 ...

Watch this Scientific Journal Video about High-resolution Optical Mapping of the Mouse Sino-atrial Node at JoVE.com

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

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.

Research

JoVE Journal

Biology

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