Name: dual-dye optical mapping of hearts from ryr2r2474s knock-in mice of catecholaminergic polymorphic ventricular tachycardia
Uploaded: 2023-12-22T12:20:00.000+00:00
Duration: 9 min 36 s
Description: Watch this Scientific Journal Video about Dual-Dye Optical Mapping of Hearts from RyR2R2474S Knock-In Mice of Catecholaminergic Polymorphic Ventricular Tachycardia at JoVE.com

This study is supported by the National Natural Science Foundation of China (81700308 to XO and 31871181 to ML, and 82270334 to XT), Sichuan Province Science and Technology Support Program (CN) (2021YJ0206 to XO, 23ZYZYTS0433, and 2022YFS0607 to XT, and 2022NSFSC1602 to TC) and State Key Laboratory for Chemistry and Molecular Engineering of Medicinal Resources (Guangxi Normal University) (CMEMR2017-B08 to XO), MRC (G10031871181 to ML02647, G1002082, ML), BHF (PG/14/80/31106, PG/16/67/32340, PG/12/21/29473, PG/11/59/29004 ML), BHF CRE at Oxford (ML) grants.

Male 10&#160;to 14-week-old wild-type mice or RyR2-R2474S/+ mice (C57BL/6 background) weighing 20-25 g are used for the experiments. All procedures have been approved by the animal care and use committee of Southwest Medical University, Sichuan, China (approval NO:20160930) in conformity with the national guidelines under which the institution operates.
1. Preparation
<ol>
	<li>Stock solutions
	<ol>
		<li>Blebbistatin stock solution: Add 1 mL of 100% dimethyl sulfoxide (DMSO) in the original flask containing 2.924 mg of (-) blebbistatin powder to reach a concentration of 10 mM.</li>
		<li>Voltage indicator RH237 stock solution: Add 1 mL of 100% DMSO in the original flask with 1 mg of RH237 powder to achieve&#160;a concentration of 2.01 mM.</li>
		<li>Calcium indicator Rhod-2 AM stock solution: Add 1 mL of 100% DMSO into 1 mg of Rhod-2 AM powder to reach a concentration of 0.89 mM.</li>
		<li>Pluronic F127 stock solution: Add 1 mL of 100% DMSO into 200&#160;mg of Pluronic F127&#160;to reach a concentration of 20% w/v (0.66 mM).</li>
		<li>Aliquot the stock solutions into 200-&#181;L PCR tubes in 21-51 &#181;L (21 &#181;L of RH237, 31 &#181;L of Rhod-2 AM, and 51 &#181;L of blebbistatin) for single or double use to avoid repeated freezing and thawing. Then, wrap the solutions with aluminum foil and store&#160;at -20 &#176;C, except for the Pluronic F127 stock solution placed in a dark room at ambient temperature.</li>
	</ol>
	</li>
	<li>Perfusion solution
	<ol>
		<li>Krebs solution (in mM): Prepare 1 L of Krebs solution (NaCl 119, NaHCO3 25, NaH2PO4 1.0, KCl 4.7, MgCl2 1.05, CaCl2 1.35, and glucose 10).</li>
		<li>Filter the solution with a 0.22 &#181;m aseptic needle filter and oxygenate with 95% O2/5% CO2.</li>
		<li>Take 40 mL of Krebs solution into a 50 mL centrifugal tube and store it at 4 &#176;C for follow-up heart isolation.</li>
	</ol>
	</li>
	<li>The Langendorff perfusion system and optical mapping device
	<ol>
		<li>Set up the Langendorff perfusion system.
		<ol>
			<li>Turn on the water bath and set the temperature to 37 &#176;C.</li>
			<li>Wash the Langendorff perfusion system with 1 L of deionized water.</li>
			<li>Perfuse the solution from the intake tract and adjust the outflow rate to 3.5-4 mL/min. Then, oxygenate the perfusate with O2/CO2 (95%/5%) gas at 37 &#176;C. 
			NOTE: A bubble is never allowed in the perfusion system.</li>
		</ol>
		</li>
		<li>Prepare the optical mapping system.
		<ol>
			<li>Install the electron multiplying charge-coupled device (EMCCD) camera (512 &#215; 512 pixels), lens (40x magnification), wavelength splitter light-emitting diodes (LEDs), electrocardiogram (ECG) monitor, and stimulation electrode (Figure 1).</li>
			<li>Adjust the proper working distance from the lens to the heart position.</li>
			<li>Set two LEDs at the diagonal position of the thermostatic bath for even illumination, providing a wavelength of 530 nm for generating excitation light. Use an ET525/50 sputter-coated filter to remove any out-of-band light for the LEDs.</li>
			<li>Adjust the handle switch to achieve an equal square of the target surface, making the voltage and calcium images appear adequately on the acquiring interface.</li>
			<li>Turn the aperture of the lens to the maximum diameter to avoid any leaking of voltage or calcium signals.</li>
			<li>Adjust the camera lens at a proper height, as it serves a fine working distance to the thermostatic bath, 10 cm is mostly used.</li>
			<li>Turn on the camera for stable sampling temperature at -50 &#176;C.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Procedures
<ol>
	<li>Mouse heart harvest, cannulation, and perfusion
	<ol>
		<li>Intraperitoneally inject the animals with avertin solution (1.2%, 0.5-0.8 mL) and heparin (200 units) to minimize suffering and pain reflex and prevent blood clot formation. After 15 mins, sacrifice the animals by cervical dislocation.</li>
		<li>Open the chest with scissors, harvest the heart carefully, and place it into the cold Krebs solution (4 &#176;C, 95% O2, 5% CO2) to slow down the metabolism and protect the heart.</li>
		<li>Remove the surrounding tissue of the aorta, cannulate the aorta using a custom-made cannulating needle (outer diameter: 0.8 mm, inner diameter: 0.6 mm, length: 27 mm) and fix it with a 4-0 silk suture.</li>
		<li>Perfuse the heart with the Langendorff system at a constant speed of 3.5-4.0 mL/min and keep the temperature at 37 &#177; 1 &#176;C. 
		NOTE: All the subsequent procedures are performed in this condition.</li>
		<li>Insert a small plastic tube (0.7 mm diameter, 20 mm length) into the left ventricle to release the congestion of solution in the chamber to avoid overpreload.</li>
	</ol>
	</li>
	<li>Uncoupler of excitation-contraction and dual-dye loading
	<ol>
		<li>Put two leads into the perfusate in the bath, turn on the powers of the ECG amplifier box and the electric stimulation controller, and then start the referenced ECG software and monitor ECG continuously.</li>
		<li>Perform the subsequent steps in the dark when the heart reaches a stable state condition (the heart is beating rhythmically at ~400 bpm).</li>
		<li>Mix 50 &#181;L of 10 mM blebbistatin stock solution with 50 mL of Krebs solution to reach a concentration of 10 &#181;M. Constantly perfuse the blebbistatin-Krebs solution mixture into the heart for 10 mins to uncouple contraction from excitation and avoid contraction artifacts during filming.</li>
		<li>Use a red flashlight to check whether the heart contraction stops totally because contraction will influence the dye loading quality.</li>
		<li>After uncoupling&#160;excitation-contraction, mix 15 &#181;L of Rhod-2 AM stock solution with 15 &#181;L of Pluronic F127 stock solution in 50 mL of Krebs solution to achieve the final concentrations of 0.267 &#181;M Rhod-2 AM and 0.198 &#181;M Pluronic F127. Then, perfuse the heart continuously with Rhod-2 AM working solution for 15 mins in the Langendorff perfusion system.</li>
		<li>Keep the oxygen supply during intracellular calcium dye loading. Since bubbles are easily formed in Pluronic F127, insert a bubble trap into the perfusion system to avoid gas embolization of the coronaries.</li>
		<li>Dilute 10 &#181;L of RH237 stock solution into 50 mL of the perfusate to reach the final concentration at 0.402 &#181;M and perform loading for 10 mins.</li>
		<li>At the end of dual-dye loading, take a sequence of photos to ensure both voltage and calcium signals are adequate for analysis (no interaction between two signals).</li>
	</ol>
	</li>
	<li>Optical mapping and arrhythmia induction 
	NOTE: Optical mapping starts after contraction cessation and an appropriate dye-loading, and the heart is consecutively perfused as in the steps described above at 2.1 (4).
	<ol>
		<li>Turn on the two LEDs for excitation lights and adjust their intensity at a proper range (strong enough for illumination and relatively straightforward filming but not too robust for overexposure).</li>
		<li>Put the heart beneath the detection device, ensure it is under adequate illumination of two LEDs, and adjust the light spot diameter to 2 cm.</li>
		<li>Set the working distance from the lens to the heart to 10 cm, giving a sampling rate of nearly 500 Hz and a spatial resolution of 120 x 120 &#181;m per pixel.</li>
		<li>Open the signal sampling software to control the camera digitally to capture voltage and calcium signals simultaneously.</li>
		<li>Start the myopacer field stimulator, and set the pacing pattern at Transistor Transistor Logic (TTL), 2 ms pacing duration for each pulse, and 0.3 V as an initial intensity.</li>
		<li>Use 30 consecutive 10 Hz S1 stimuli to test the diastolic voltage threshold of the heart driven by the ECG recording software. Gradually increase the voltage amplitude until 1:1 capture is realized (check QRS wave from the ECG monitor, action potential (AP), and calcium transients (CaT) signals).</li>
		<li>After determining the voltage threshold, pace the heart at an intensity of 2x the diastolic voltage threshold with a pair of platinum electrodes attached to the epicardial of the left ventricle (LV) apex (ELVA).</li>
		<li>Implement the S1S1 protocol to measure calcium or action potential alternans and restitution properties. Pace the heart consecutively at a basic cycle length of 100 ms, decreasing 10 ms of the cycle length every following sequence until 50 ms is reached. Each episode includes 30 consecutive stimuli with a 2 ms pulse width. At the same time, start optical mapping before stimulation (sampling time includes ~10 sinus rhythms and pacing duration).</li>
		<li>To measure the ventricular effective refractory period (ERP) by using the S1S2 stimulus protocol, begin with an S1S1 pacing cycle length of 100 ms with an S2 coupled at 60 ms with a 2 ms step decrement until S2 fails to capture ectopic QRS complex.</li>
		<li>For arrhythmia induction, conduct perpetual 50 Hz burst pacing (50 continuous electrical stimulations with a 2 ms pulse width), and perform the same pacing episode after a 2 s interval of resting.</li>
		<li>Observe ECG recordings carefully during the continuous high-frequency pacing period so that the simultaneous optical mapping recordings can start promptly when an interesting arrhythmic ECG wave generates (since most cardiac arrhythmias are induced by electrical pacing, the optical signals are sampled 2-3 s before burst pacing in case of losing important cardiac events).</li>
		<li>Image using EMCCD camera (sampling rate: 500 Hz, pixel size: 64 x 64).</li>
	</ol>
	</li>
	<li>Data analysis
	<ol>
		<li>Image loading and signal processing
		<ol>
			<li>Press Select Folder and Load Images to load the images into the image acquisition software for semi-automatically massive video data analysis according to the setup and protocol described previously15,16.</li>
			<li>Enter the correct sampling parameters (such as Pixel Size and Framerate).</li>
			<li>Set the image threshold by manual input and select the region of interest (ROI).</li>
			<li>Implement a 3 x 3 pixel Gaussian spatial filter, a Savitzky-Goaly filter, and a top-hat baseline correction.</li>
			<li>Press Process Images to remove the baseline and calculate the electrophysiological parameters, such as APD80 and CaTD50.</li>
		</ol>
		</li>
		<li>Electrophysiological parameters analysis
		<ol>
			<li>Set the initiation time of APD at the peak and the terminal point at 80% repolarization (APD80) for calculation of APD80. Similarly, CaTD start time is defined as the peak, and the terminal point is defined as the 80% relaxation.</li>
			<li>Measurement of conduction velocity (CV) depends on the pixel size and the action potential conduction time between two pixels or more. Calculate the average velocity from all the chosen pixels-this is the mean conduction velocity of the selected region. Generate corresponding isochronal maps simultaneously for a clear view of the conduction direction. 
			NOTE: O&#39;Shea et al.15 reported the CV measurement in detail.</li>
			<li>For alternans and arrhythmia analysis, calcium alternans are defined as a continuous large and small peak amplitude appearing alternatively. Use the peak amplitude ratio to assess the severity of frequency-dependent alternans (1-A2/A1). Apply phase maps to analyze complex arrhythmias like ventricular tachycardia (VT). Look for the rotors appearing distinctly at a specific region as the rotors shift.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Priori, S. G., Chen, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inherited+dysfunction+of+sarcoplasmic+reticulum+Ca2++handling+and+arrhythmogenesis.">Inherited dysfunction of sarcoplasmic reticulum Ca2+ handling and arrhythmogenesis.</a> Circulation Research. 108 (7), 871-883 (2011).</li><li>Goddard, C. A., 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=Physiological+consequences+of+the+P2328S+mutation+in+the+ryanodine+receptor+(RyR2)+gene+in+genetically+modified+murine+hearts.">Physiological consequences of the P2328S mutation in the ryanodine receptor (RyR2) gene in genetically modified murine hearts.</a> Acta Physiologica. 194 (2), 123-140 (2008).</li><li>Sabir, I. N., 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=Alternans+in+genetically+modified+langendorff-perfused+murine+hearts+modeling+catecholaminergic+polymorphic+ventricular+tachycardia.">Alternans in genetically modified langendorff-perfused murine hearts modeling catecholaminergic polymorphic ventricular tachycardia.</a> Frontiers in Physiology. 1, 126 (2010).</li><li>Zhang, Y., Matthews, G. D., Lei, M., Huang, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abnormal+Ca2++homeostasis,+atrial+arrhythmogenesis,+and+sinus+node+dysfunction+in+murine+hearts+modeling+RyR2+modification.">Abnormal Ca2+ homeostasis, atrial arrhythmogenesis, and sinus node dysfunction in murine hearts modeling RyR2 modification.</a> Frontiers in Physiology. 4, 150 (2013).</li><li>Leenhardt, A., 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=Catecholaminergic+polymorphic+ventricular+tachycardia+in+children.+A+7-year+follow-up+of+21+patients.">Catecholaminergic polymorphic ventricular tachycardia in children. A 7-year follow-up of 21 patients.</a> Circulation. 91 (5), 1512-1519 (1995).</li><li>Priori, S. 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=Mutations+in+the+cardiac+ryanodine+receptor+gene+(hRyR2)+underlie+catecholaminergic+polymorphic+ventricular+tachycardia.">Mutations in the cardiac ryanodine receptor gene (hRyR2) underlie catecholaminergic polymorphic ventricular tachycardia.</a> Circulation. 103 (2), 196-200 (2001).</li><li>Wehrens, X. H., 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=FKBP12.6+deficiency+and+defective+calcium+release+channel+(ryanodine+receptor)+function+linked+to+exercise-induced+sudden+cardiac+death.">FKBP12.6 deficiency and defective calcium release channel (ryanodine receptor) function linked to exercise-induced sudden cardiac death.</a> Cell. 113 (7), 829-840 (2003).</li><li>Novak, A., 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=Functional+abnormalities+in+iPSC-derived+cardiomyocytes+generated+from+CPVT1+and+CPVT2+patients+carrying+ryanodine+or+calsequestrin+mutations.">Functional abnormalities in iPSC-derived cardiomyocytes generated from CPVT1 and CPVT2 patients carrying ryanodine or calsequestrin mutations.</a> Journal of Cellular and Molecular Medicine. 19 (8), 2006-2018 (2015).</li><li>Napolitano, C., Mazzanti, A., Bloise, R., Priori, S. G., Adam, M. P., 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=CACNA1C-related+disorders.">CACNA1C-related disorders.</a> GeneReviews. , (1993).</li><li>Makita, N., 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=Novel+calmodulin+mutations+associated+with+congenital+arrhythmia+susceptibility.">Novel calmodulin mutations associated with congenital arrhythmia susceptibility.</a> Circulation. Cardiovascular Genetics. 7 (4), 466-474 (2014).</li><li>Gomez-Hurtado, N., 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=Novel+CPVT-associated+calmodulin+mutation+in+CALM3+(CALM3-A103V)+activates+arrhythmogenic+Ca+waves+and+sparks.">Novel CPVT-associated calmodulin mutation in CALM3 (CALM3-A103V) activates arrhythmogenic Ca waves and sparks.</a> Circulation, Arrhythmia and Electrophysiology. 9 (8), (2016).</li><li>Wleklinski, M. J., Kannankeril, P. J., Knollmann, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+and+tissue+mechanisms+of+catecholaminergic+polymorphic+ventricular+tachycardia.">Molecular and tissue mechanisms of catecholaminergic polymorphic ventricular tachycardia.</a> Journal of Physiology. 598 (14), 2817-2834 (2020).</li><li>Neco, P., 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=Paradoxical+effect+of+increased+diastolic+Ca2++release+and+decreased+sinoatrial+node+activity+in+a+mouse+model+of+catecholaminergic+polymorphic+ventricular+tachycardia.">Paradoxical effect of increased diastolic Ca2+ release and decreased sinoatrial node activity in a mouse model of catecholaminergic polymorphic ventricular tachycardia.</a> Circulation. 126 (4), 392-401 (2012).</li><li>Bogdanov, K. Y., Vinogradova, T. M., Lakatta, E. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sinoatrial+nodal+cell+ryanodine+receptor+and+Na(+)-Ca(2+)+exchanger:+molecular+partners+in+pacemaker+regulation.">Sinoatrial nodal cell ryanodine receptor and Na(+)-Ca(2+) exchanger: molecular partners in pacemaker regulation.</a> Circulation Research. 88 (12), 1254-1258 (2001).</li><li>O'Shea, C., 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=ElectroMap:+High-throughput+open-source+software+for+analysis+and+mapping+of+cardiac+electrophysiology.">ElectroMap: High-throughput open-source software for analysis and mapping of cardiac electrophysiology.</a> Scientific Reports. 9 (1), 1389 (2019).</li><li>O'Shea, C., 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=High-throughput+analysis+of+optical+mapping+data+using+ElectroMap.">High-throughput analysis of optical mapping data using ElectroMap.</a> Journal of Visualized Experiments: JoVE. (148), e59663 (2019).</li><li>Choi, B. R., 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=Simultaneous+maps+of+optical+action+potentials+and+calcium+transients+in+guinea-pig+hearts:+mechanisms+underlying+concordant+alternans.">Simultaneous maps of optical action potentials and calcium transients in guinea-pig hearts: mechanisms underlying concordant alternans.</a> Journal of Physiology. 529, 171-188 (2000).</li><li>Rybashlykov, D., Brennan, J., Lin, Z., Efimov, I. R., Syunyaev, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Open-source+low-cost+cardiac+optical+mapping+system.">Open-source low-cost cardiac optical mapping system.</a> PLoS One. 17 (3), 0259174 (2022).</li><li>Lucas-Lopez, C., 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=Absolute+stereochemical+assignment+and+fluorescence+tuning+of+the+small+molecule+tool,+(-)-blebbistatin.">Absolute stereochemical assignment and fluorescence tuning of the small molecule tool, (-)-blebbistatin.</a> European Journal of Organic Chemistry. 2005 (9), 1736-1740 (2005).</li><li>Ponsaerts, 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=The+myosin+II+ATPase+inhibitor+blebbistatin+prevents+thrombin-induced+inhibition+of+intercellular+calcium+wave+propagation+in+corneal+endothelial+cells.">The myosin II ATPase inhibitor blebbistatin prevents thrombin-induced inhibition of intercellular calcium wave propagation in corneal endothelial cells.</a> Investigative Ophthalmology &amp; Visual Science. 49 (11), 4816-4827 (2008).</li><li>Jou, C., Spitzer, K., Tristani-Firouzi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blebbistatin+effectively+uncouples+the+excitation-contraction+process+in+zebrafish+embryonic+heart.">Blebbistatin effectively uncouples the excitation-contraction process in zebrafish embryonic heart.</a> Cellular Physiology &amp; Biochemistry. 25 (4-5), 419-424 (2010).</li><li>Brack, K. E., Narang, R., Winter, J., Ng, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanical+uncoupler+blebbistatin+is+associated+with+significant+electrophysiological+effects+in+the+isolated+rabbit+heart.">The mechanical uncoupler blebbistatin is associated with significant electrophysiological effects in the isolated rabbit heart.</a> Experimental Physiology. 98 (5), 1009-1027 (2013).</li><li>O'Shea, C., 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=High-throughput+analysis+of+optical+mapping+data+using+ElectroMap.">High-throughput analysis of optical mapping data using ElectroMap.</a> Journal of Visualized Experiments: JoVE. (148), e59663 (2019).</li><li>He, 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=A+dataset+of+dual+calcium+and+voltage+optical+mapping+in+healthy+and+hypertrophied+murine+hearts.">A dataset of dual calcium and voltage optical mapping in healthy and hypertrophied murine hearts.</a> Scientific Data. 8 (1), 314 (2021).</li><li>Lei, M., Huang, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+arrhythmogenesis:+a+tale+of+two+clocks.">Cardiac arrhythmogenesis: a tale of two clocks.</a> Cardiovascular Research. 116 (14), e205-e209 (2020).</li><li>Mal Baudot, ., 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=Concomitant+genetic+ablation+of+L-type+Cav1.3+&#945;1D+and+T-type+Cav3.1+&#945;1G+Ca2++channels+disrupts+heart+automaticity.">Concomitant genetic ablation of L-type Cav1.3 &#945;1D and T-type Cav3.1 &#945;1G Ca2+ channels disrupts heart automaticity.</a> Scientific Reports. 10 (1), 18906 (2020).</li><li>Dai, W., 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=ZO-1+regulates+intercalated+disc+composition+and+atrioventricular+node+conduction.">ZO-1 regulates intercalated disc composition and atrioventricular node conduction.</a> Circulation Research. 127 (2), e28-e43 (2020).</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+complex1.">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 complex1.</a> European Heart Journal. 36 (11), 686-697 (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> Proceedings of the National Academy of Sciences of the United States of America. 112 (31), 9769-9774 (2015).</li><li>Yang, B., 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=Ventricular+SK2+upregulation+following+angiotensin+II+challenge:+Modulation+by+p21-activated+kinase-1.">Ventricular SK2 upregulation following angiotensin II challenge: Modulation by p21-activated kinase-1.</a> Journal of Molecular and Cellular Cardiology. 164, 110-125 (2022).</li><li>Dong, 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=A+protocol+for+dual+calcium-voltage+optical+mapping+in+murine+sinoatrial+preparation+with+optogenetic+pacing.">A protocol for dual calcium-voltage optical mapping in murine sinoatrial preparation with optogenetic pacing.</a> Frontiers in Physiology. 10, 954 (2019).</li><li>He, 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=A+protocol+for+transverse+cardiac+slicing+and+optical+mapping+in+murine+heart.">A protocol for transverse cardiac slicing and optical mapping in murine heart.</a> Frontiers in Physiology. 10, 755 (2019).</li><li>Hoeker, G. S., Katra, R. P., Wilson, L. D., Plummer, B. N., Laurita, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+calcium+release+in+tissue+from+the+failing+canine+heart.">Spontaneous calcium release in tissue from the failing canine heart.</a> American Journal of Physiology. Heart and Circulatory Physiology. 297 (4), H1235-H1242 (2009).</li><li>Laurita, K. R., Singal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+action+potentials+and+calcium+transients+simultaneously+from+the+intact+heart.">Mapping action potentials and calcium transients simultaneously from the intact heart.</a> American Journal of Physiology. Heart and Circulatory Physiology. 280 (5), H2053-H2060 (2001).</li><li>Johnson, P. L., Smith, W., Baynham, T. C., Knisley, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Errors+caused+by+combination+of+Di-4+ANEPPS+and+Fluo3/4+for+simultaneous+measurements+of+transmembrane+potentials+and+intracellular+calcium.">Errors caused by combination of Di-4 ANEPPS and Fluo3/4 for simultaneous measurements of transmembrane potentials and intracellular calcium.</a> Annals of Biomedical Engineering. 27 (4), 563-571 (1999).</li></ol>

None of the authors has any conflicts of interest to declare.

Based on our experience, the keys to a successful dual-dye optical mapping of a mouse heart include a well-prepared solution and heart, dye loading, achieving the best signal-to-noise ratio, and reducing the motion artifact.
Preparation of solution 
Krebs solution is essential for a successful heart experiment. MgCl2 and CaCl2 stock solutions (1 mol/L) are prepared in advance considering their water absorption and added to the Krebs solution after all other components are dissolved in pure water because Mg2+ and Ca2+ can easily precipitate with CO32+. The Krebs solution is bubbled with 95% O2/5% CO2 for at least 30 mins to ensure oxygenation. Because the mouse heart is particularly sensitive to pH, the solution pH should be around 7.4 after oxygenation. Even if tiny particles are in the solution, the experimental results may be affected because these particles may block the capillaries and affect the perfusion effect. Hence, the solution is filtered using a 0.22 &#181;m aseptic needle filter before use.
Heart preparation 
Before hearts are harvested, the mice are first heparinized to avoid clot formation in the coronary artery system, preventing poor dye perfusion caused by cardiac congestion from affecting the subsequent imaging. The shorter the heart&#39;s ischemic time, the better the heart&#39;s condition. Therefore, the ischemic time is controlled within 2-3 mins from hearts being harvested to cannulation via the aorta on the Langendorff system. In addition, well-maintained perfusion pressure is also essential. Hence, a thin silicone (plastic) tube is inserted into the left ventricular cavity to avoid the left ventricular pressure being too high during ventricular contraction after the left ventricular outlet is ligated, which could result in poor myocardial perfusion and anoxic tissue acidification.
Dye loading 
These experiments perform dye loading by perfusing the heart in the Langendorff system. It is crucial to monitor the heart rhythm because poor dye loading will occur when the abnormal rhythm is caused by surgical operations or ischemia-reperfusion damage. The heart must be healthy enough to perform the subsequent steps. Rhod-2 AM, a Ca2+-sensitive dye, is an acetyl methyl ester derivative of Rhod 2, which is easily loaded into cells in its AM form. A 100-fold increase in the molecule&#39;s fluorescence intensity results from Ca2+ chelation17. Pluronic F127 is incorporated into the Rhod-2 AM loading solution to prevent Rhod-2 AM from polymerizing in the buffer and help it enter cells. Pluronic F127 can reduce the stability of Rhod-2 AM, so it is only recommended to add it when preparing the working solution but not in the storage solution for long-term storage. The voltage-sensitive dye RH237 is used in this study due to its favorable spectral properties for use with Ca2+ indicator Rhod-2 AM.
Achieving the best signal-to-noise ratio 
Obtaining images with high signal-to-noise ratios is the target of imaging, but noise is like a shadowy ghost that always causes trouble. Due to weak signals, lower noise is particularly important in some high-speed microscopic imaging applications, such as optical mapping. The signal-to-noise ratio (SNR) is calculated as the root mean square amplitude ratio to the root mean square noise, where the noise amplitude is evaluated at resting potential18. Some factors, such as light source, optical filters, focusing optics, and photodetectors, are essential to achieve the best SNR. In the study, the background region of&#160;the sample is examined for noise, which often fluctuates at a tiny level. The optical signal detected by each pixel is the average of emitted light from its surface area. AP and calcium activities oscillate during arrhythmia, and both signals&#39; amplitude is relatively low. Even minor interference may lead to distortion of optical signals and result in mistakes in data analysis. Therefore, the interpretation of optical signals should be careful when the local heterogeneity is caused by electrical function during arrhythmias like VT.
Reduce the motion artifact 
Compared with electrode recording, optical signals are often influenced by contraction activity of the Langendorff perfused hearts because of motion artifact. To capture accurate optical signals, pharmacological inhibitors of excitation-contraction are mostly used. To minimize the motion artifact during imaging, blebbistatin is adopted to stop the heart from beating. It is a selective inhibitor of the ATPase activity of non-muscle myosin II and effectively uncouples the excitation-contraction process of the heart19,20,21. Although some studies imply some side effects using the compound22, we utilize the lowest working concentration at 10 &#181;M to minimize the possible damage to the heart.
ElectroMap software for analysis of cardiac optical mapping datasets 
ElectroMap is a high-throughput open-source software for the analysis of cardiac optical mapping datasets. It provides an analysis of main cardiac electrophysiology parameters, including AP and CaT morphology, CV, diastolic interval, dominant frequency, time-to-peak, and relaxation constant (&#964;) 15,23. The software allows multiple filtering options, including the Gaussian filter, Savitzky-Goaly filter, and Top-hat baseline correction. Gaussian filter is a two-dimensional smoothing by calculating the weighted average smoothing of each channel and adjacent channels. It is commonly used for spike glitch noise. Savitzky-Goaly filter fits a lower polynomial and continuous subset of adjacent datasets through the least square method, which meets the need for various smooth filtering and is also effective for processing non-periodic and non-linear datasets derived from noise. Top-hat baseline correction can adjust the optical signals to the same height according to the peaks of the traces, calculating parameters such as action potential duration (APD) and calcium transient duration (CaTD) much more accurately. Baseline drift occasionally occurs when sampling voltage and calcium fluorescence signals. It is also useful when calculating calcium alternans and amplitude. Both ventricles were selected for electrophysiological investigation.
Advantages and disadvantages of dual-dye mapping and methods to limit interference 
In recent years, it has been realized that it is vital to clarify cell depolarization or repolarization and intercellular conduction heterogeneity in the whole heart, as well as coupling of the membrane clock and calcium clock, which is critical for understanding the mechanism of diseases such as arrhythmia24,25. Optical mapping has a high spatiotemporal resolution to determine the ventricular activation and repolarization properties of the heart of transgenic mice26,27,28,29. It can also detect multi-parameter imaging, for example, measurement of membrane potential and intracellular calcium of the same heart24,30 or tissue31,32 loaded with voltage and calcium-sensitive dye. Dual-dye imaging is beneficial for studying the relationship between action potential and calcium, such as the relationship between the membrane (M) clock and Ca2+ (C)-clock or spontaneous calcium release and delayed after depolarization (DAD). Normal cardiac excitation then requires the cyclic events in the two clocks to be aligned. Disruption in this alignment leads to arrhythmia25. The relationship between spontaneous calcium release and DAD is the mechanism of triggering activities in heart failure 33. However, the combination of dyes should be carefully selected. The combination of RH-237/Rhod-2 or di-4-ANEPPS/Indo-1 allows simultaneous recording, while Fluo-3/4/di-4-ANEPPS will lead to errors due to overlapping emission spectra of two dyes30,34,35. This experiment selected RH237 and Rhod-2 AM to load the heart and acquired good imaging quality.
In addition, the camera used in this protocol has two target surfaces, which enables it to capture the split signals on one sampling interface and allows a single camera to detect two different emission wavelengths. Such simultaneous mapping of optical AP and CaT combining various photoelectron spectroscopy (PES) protocols will allow us to determine the interrelationship between&#160;abnormal [Ca2+]i and electrical instability under stress conditions and the effect of post-activation potentiation on these anomalies. The spatially heterogeneous nature of SR Ca2+ cycling and how this affects the emergence, severity, and concordance of electrical alternans and arrhythmogenic behavior, such as spatially discordant alternans and consequent VTs, will be studied in the intact heart in different groups. SR Ca2+ alternans, RyR2 refractoriness and their role in SR Ca2+ and APD alternans will be explored.

Inherited cardiac disorder catecholaminergic polymorphic ventricular tachycardia (CPVT) manifests as polymorphic ventricular tachycardia (PVT) episodes following physical activity, stress, or catecholamine challenge, which can deteriorate into potentially fatal ventricular fibrillation1,2,3,4. Recent evidence following its first report as a clinical syndrome in 1995 implicated mutations in seven genes, all involved in sarcoplasmic reticular (SR) store Ca2+ release in this condition: the most frequently reported RYR2 encoding ryanodine receptor 2 (RyR2) of Ca2+ release channels5,6, FKBP12.67, CASQ2 encoding cardiac calsequestrin8, TRDN encoding the junctional SR protein triadin9, and CALM19, CALM210, and CALM3 identically encoding calmodulin11,12. These genotypic patterns attribute the arrhythmic events to the unregulated pathological release of SR store Ca2+12.
Spontaneous Ca2+ release from SR can be detected as Ca2+ sparks or Ca2+ waves, which activates the Na+/Ca2+ exchanger (NCX). The exchanger of one Ca2+ for three Na+ generates an inward current, which speeds up the diastolic depolarization and drives the membrane voltage to the threshold of action potential (AP). In RyR2 knock-in mice, the increased activity of RyR2R4496C in the sinoatrial node (SAN) leads to an unanticipated decrease in SAN automaticity by Ca2+-dependent decrease of ICa,L and SR Ca2+ depletion during diastole, identifying subcellular pathophysiologic alterations contributing to the SAN dysfunction in CPVT patients13,14. Occurrence of the related cardiomyocyte cytosolic Ca2+ waves is more likely following increases in background cytosolic [Ca2+] following RyR sensitization by catecholamine, including isoproterenol (ISO), challenge.
Detailed kinetic changes in Ca2+ signaling following RyR2-mediated Ca2+ release in response to action potential (AP) activation that may be the cause of the observed ventricular arrhythmias in intact cardiac CPVT models remain to be determined for the full range of reported RyR2 genotypes12. This paper presents the instrumentation setup and experimental procedure for high-throughput mapping of Ca2+ signals and transmembrane potentials (Vm) in murine wild-type (WT) and heterozygous RyR2-R2474S/+ hearts, combined with programmed electrical pacing before and after isoproterenol challenge. This protocol provides a method for the mechanistic study of CPVT disease in isolated mouse hearts.

<table><tbody><tr><td>0.2 &amp;mu;m syringe filter</td><td>Medical equipment factory of Shanghai Medical Instruments Co., Ltd., Shanghai, China</td><td>N/A</td><td>To filter solution</td></tr><tr><td>15 mL centrifuge tube</td><td>Guangzhou Jet Bio-Filtration Co., Ltd. China</td><td>CFT011150</td><td></td></tr><tr><td>1 mL Pasteur pipette</td><td>Beijing Labgic Technology Co., Ltd. China</td><td>00900026</td><td></td></tr><tr><td>1 mL Syringe</td><td>B. Braun Medical Inc.</td><td>YZB/GER-5474-2014</td><td></td></tr><tr><td>200 &amp;mu;L PCR tube</td><td>Sangon Biotech Co., Ltd. Shanghai. China</td><td>F611541-0010</td><td>Aliquote the stock solutions&amp;nbsp; to avoid repeated freezing and thawing</td></tr><tr><td>50 mL centrifuge tube</td><td>Guangzhou Jet Bio-Filtration Co., Ltd. China</td><td>CFT011500</td><td>Store Tyrode&#039;s solution at 4 &amp;deg;C for follow-up heart isolation</td></tr><tr><td>585/40 nm filter</td><td>Chroma Technology</td><td>N/A</td><td>Filter for calcium signal</td></tr><tr><td>630 nm long-pass filter</td><td>Chroma Technology</td><td>G15604AJ</td><td>Filter for voltage signal</td></tr><tr><td>Avertin (2,2,2-tribromoethanol)</td><td>Sigma-Aldrich Poole, Dorset, United Kingdom</td><td>T48402-100G</td><td>To minimize suffering and pain reflex</td></tr><tr><td>Blebbistatin</td><td>Tocris Bioscience, Minneapolis, MN, United States</td><td>SLBV5564</td><td>Excitation-contraction uncoupler to&amp;nbsp; eliminate motion artifact during mapping</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich, St. Louis, MO, United States</td><td>SLBK1794V</td><td>For Tyrode&#039;s solution</td></tr><tr><td>Custom-made thermostatic bath</td><td>MappingLab, United Kingdom</td><td>TBC-2.1</td><td>To keep temperature of perfusion solution</td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Sigma-Aldrich</td><td>(RNBT7442)</td><td>Solvent for dyes</td></tr><tr><td>Dumont forceps</td><td>Medical equipment factory of Shanghai Medical Instruments Co.,Ltd.,Shanghai, China</td><td>YAF030</td><td></td></tr><tr><td>ElectroMap software</td><td>University of Birmingham</td><td>N/A</td><td>Quantification of electrical parameters</td></tr><tr><td>EMCCD camera</td><td>Evolve 512 Delta, Photometrics, Tucson, AZ, United States</td><td>A18G150001</td><td>Acquire images for optical signals</td></tr><tr><td>ET525/36 sputter coated filter</td><td>Chroma Technology</td><td>319106</td><td>Excitation filter</td></tr><tr><td>Glucose</td><td>Sigma-Aldrich, St. Louis, MO, United States</td><td>SLBT4811V</td><td>For Tyrode&#039;s solution</td></tr><tr><td>Heparin Sodium</td><td>Chengdu Haitong Pharmaceutical Co., Ltd., Chengdu, China</td><td>(H51021209)</td><td>To prevent blood clots in the coronary artery</td></tr><tr><td>&amp;nbsp;Iris forceps</td><td>Medical equipment factory of Shanghai Medical Instruments Co.,Ltd.,Shanghai, China</td><td>YAA010</td><td></td></tr><tr><td>Isoproterenol</td><td>MedChemExpress, Carlsbad, CA, United States</td><td>HY-B0468/CS-2582</td><td></td></tr><tr><td>KCl</td><td>Sigma-Aldrich, St. Louis, MO, United States</td><td>SLBS5003</td><td>For Tyrode&#039;s solution</td></tr><tr><td>MacroLED</td><td>Cairn Research, Faversham, United Kingdom</td><td>7355/7356</td><td>The excitation light of fluorescence probes</td></tr><tr><td>MacroLED light source</td><td>Cairn Research, Faversham, United Kingdom</td><td>7352</td><td>Control the LEDs</td></tr><tr><td>Mayo scissors</td><td>Medical equipment factory of Shanghai Medical Instruments Co.,Ltd.,Shanghai, China</td><td>YBC010</td><td></td></tr><tr><td>MetaMorph</td><td>Molecular Devices</td><td>N/A</td><td>Optical signals sampling</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich, St. Louis, MO, United States</td><td>BCBS6841V</td><td>For Tyrode&#039;s solution</td></tr><tr><td>MICRO3-1401</td><td>Cambridge Electronic Design limited, United Kingdom</td><td>M5337</td><td>Connect the electrical stimulator and Spike2 software</td></tr><tr><td>MyoPacer EP field stimulator</td><td>Ion Optix Co, Milton, MA, United States</td><td>S006152</td><td>Electric stimulator</td></tr><tr><td>NaCl</td><td>Sigma-Aldrich, St. Louis, MO, United States</td><td>SLBS2340V</td><td>For Tyrode&#039;s solution</td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&amp;nbsp;&lt;/sub&gt;</td><td>Sigma-Aldrich, St. Louis, MO, United States</td><td>BCBW9042</td><td>For Tyrode&#039;s solution</td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma-Aldrich, St. Louis, MO, United States</td><td>SLBX3605</td><td>For Tyrode&#039;s solution</td></tr><tr><td>NeuroLog System</td><td>Digitimer</td><td>NL905-229</td><td>For ECG amplifier</td></tr><tr><td>OmapScope5</td><td>MappingLab, United Kingdom</td><td>N/A</td><td>Calcium alternans and arrhythmia analysis</td></tr><tr><td>Ophthalmic scissors</td><td>Huaian Teshen Medical Instruments Co., Ltd., Jiang Su, China</td><td>T4-3904</td><td></td></tr><tr><td>OptoSplit</td><td>Cairn Research, Faversham, United Kingdom</td><td>6970</td><td>Split the emission light for detecting Ca&lt;sup&gt;2+&lt;/sup&gt; and V&lt;sub&gt;m&lt;/sub&gt;&amp;nbsp; simultaneously</td></tr><tr><td>Peristalic pump</td><td>Longer Precision Pump Co., Ltd., Baoding, China,</td><td>BT100-2J</td><td>To pump the solution</td></tr><tr><td>Petri dish</td><td>BIOFIL</td><td>TCD010060</td><td></td></tr><tr><td>Pluronic F127</td><td>Invitrogen, Carlsbad, CA, United States</td><td>1899021</td><td>To enhance the loading with Rhod2AM</td></tr><tr><td>RH237</td><td>Thermo Fisher Scientifific, Waltham, MA, United States</td><td>1971387</td><td>Voltage-sensitive dye</td></tr><tr><td>Rhod-2 AM</td><td>Invitrogen, Carlsbad, CA, United States</td><td>1890519</td><td>Calcium indicator</td></tr><tr><td>Silica gel tube</td><td>Longer Precision Pump Co., Ltd., Baoding, China,</td><td>96402-16</td><td>Connect with the peristaltic pump</td></tr><tr><td>Silk suture</td><td>Yuankang Medical Instrument Co., Ltd.,Yangzhou, China</td><td>20172650032</td><td>To fix the aorta</td></tr><tr><td>Spike2</td><td>Cambridge Electronic Design limited, United Kingdom</td><td>N/A</td><td>To record and analyze ECG data</td></tr><tr><td>Stimulation electrode</td><td>MappingLab, United Kingdom</td><td>SE1600-35-2020</td><td></td></tr><tr><td>T510lpxr</td><td>Chroma Technology</td><td>312461</td><td>For light source</td></tr><tr><td>T565lpxr</td><td>Chroma Technology</td><td>321343</td><td>For light source</td></tr></tbody></table>

dual-dye optical mapping of hearts from ryr2r2474s knock-in mice of catecholaminergic polymorphic ventricular tachycardia

The pro-arrhythmic cardiac disorder catecholaminergic polymorphic ventricular tachycardia (CPVT) manifests as polymorphic ventricular tachycardia episodes following physical activity, stress, or catecholamine challenge, which can deteriorate into potentially fatal ventricular fibrillation. The mouse heart is a widespread species for modeling inherited cardiac arrhythmic diseases, including CPVT. Simultaneous optical mapping of transmembrane potential (Vm) and calcium transients (CaT) from Langendorff-perfused mouse hearts has the potential to elucidate mechanisms underlying arrhythmogenesis. Compared with the cellular level investigation, the optical mapping technique can test some electrophysiological parameters, such as the determination of activation, conduction velocity, action potential duration, and CaT duration. This paper presents the instrumentation setup and experimental procedure for high-throughput optical mapping of CaT and Vm in murine wild-type and heterozygous RyR2-R2474S/+ hearts, combined with programmed electrical pacing before and during the isoproterenol challenge. This approach has demonstrated a feasible and reliable method for mechanistically studying CPVT disease in an ex vivo mouse heart preparation.

Optical mapping has been a popular approach in studying complex cardiac arrhythmias in the past decade. The optical mapping setup consists of an EMCCD camera, giving a sampling rate of up to 1,000 Hz and a spatial resolution of 74 x 74 &#181;m for each pixel. It enables a rather high signal-noise ratio during signal sampling (Figure 1). Once the Langendorff-perfused heart reaches a stable state and the dye loading finishes, the heart is placed in the homoeothermic chamber under the illumination of two 530 nm LEDs, which are used for excitation of the voltage indicator RH237 and Ca2+ indicator Rhod-2 AM. The emission light is split into two wavelengths of 600 nm (for Ca2+) and 670 nm (for Vm), which are detected simultaneously using the EMCCD camera.&#160;After perfusion of avertin and heparin for 15 mins, use the surgical instruments (Figure 2A) to open the chest and quickly extract the heart, then transfer it to the cold Krebs solution (4 &#176;C, 95% O2, 5% CO2) (Figure 2B). Clean out the surrounding tissues carefully, fix the aorta with a 4-0 suture, and a 0.7 mm plastic tube is inserted into the left ventricle (Figure 2C) to release the congestion of the perfusate in the left ventricular chamber. Put the ECG leads into the perfusate (Figure 2D) and ensure that the heart beats rhythmically according to ECG monitoring driven by the ECG recording software. Then, perform dual-dye loading in the dark (Figure 2E).
After contraction artifacts have been minimized by blebbistatin (10 &#181;M) and an adequate dye loading has been completed, the filming started for about 10 sinus beats before the S1S1 pacing protocol to evaluate frequency-depend electrophysiological parameter restitution properties and calcium alternans after isoproterenol (1 &#181;M ISO) challenge (Figure 3A). Figure 3B exhibits a representative ECG wavefront of VT and corresponding action potential (AP) and CaT traces induced by a 50 Hz burst pacing sequence in a CPVT mouse. Optical signal imaging software is used to complete a semi-automatical analysis of massive video data.
Figure 4A,B show typical traces and heat maps of APD80 and CaTD80, respectively. ISO shortens APD80 in WT and CPVT mice, but no difference was found between WT and CPVT mice before and after the ISO challenge (Figure 4C, **P &#60; 0.01. n = 5/6). Figure 4D indicates that CaTD80 in CPVT mice are longer than in WT after the ISO challenge, while there was no significance before ISO treatment (**P &#60; 0.01. n =6.).
For conduction measurement, Figure 5A presents a single vector algorithm for the quantification of CV. According to the voltage signals, the WT and CPVT hearts possess the same conduction ability across the epicardium at baseline and after ISO intervention (Figure 5B). Figure 5C,D show the representative activation maps of voltage and calcium in WT and CPVT hearts before and after the ISO challenge.
Calcium alternans is a critical parameter for arrhythmia. Calcium amplitude alternans is calculated according to the formulation as shown in Figure 6A. Calcium signals in WT hearts stay stable at baseline during consecutive S1S1 pacing at 14.29 and 16.67 Hz (Figure 6B), while CPVT hearts show frequency-dependent alternans (Figure 6C). After the ISO challenge, CPVT hearts exhibit frequency-dependent alternans in calcium signal during S1S1 pacing, while WT hearts are not influenced (Figure 6D,E). After continuous S1S1 pacing, a burst pacing protocol is performed to induce lethal arrhythmias. WT and CPVT hearts exhibit normal conduction during 50 Hz burst pacing at baseline (Figure 7A). After perfusion with ISO, CPVT hearts show high-frequency rotors after 50 Hz burst pacing, while WT hearts maintain normal conduction (Figure 7B).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65082/65082fig01v2.jpg" /> 
Figure 1: Optical mapping apparatus. The system includes a custom-designed EMCCD camera with a high spatial-temporal resolution (sampling rate up to 1,000 Hz, minimal sampling pixel 74 x 74 &#181;m). An electric stimulation controller is used for sampling and output electric stimulation protocol. Two green LEDs are used for the excitation light of fluorescence probes. A long-pass dichroic mirror (610 nm) and corresponding emitters split the voltage and calcium fluorescence emission lights. RH237, the voltage-sensitive dye, has an emission light at a peak wavelength of 670 nm, while Rhod-2 AM, the calcium-sensitive dye, possesses an emission light at a peak wavelength of 600 nm. Minor changes in both fluorescence signals could be captured by the camera simultaneously because of the camera sensor&#39;s high sampling rate and sensitivity. Abbreviations: EMCCD = electron-multiplying charge-coupled device; LED = light-emittting diode; ECG = electrocardiogram. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65082/65082fig01largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65082/65082fig02.jpg" /> 
Figure 2: Preparation and dual-dye loading. (A)&#160;The surgical instruments. (B) Harvest of the mouse heart.(C) Cut off the unnecessary tissue carefully for a clear view of the aorta and insert a 0.7 mm plastic tube from the aorta into the left ventricle. (D)&#160;The heart is removed quickly to the Langendorff perfusion system. (E) Dual-dye loading and excitation-contraction cessation in the dark. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65082/65082fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65082/65082fig03.jpg" /> 
Figure 3: S1S1 protocol and arrhythmia induction protocol. (A)&#160;Representative ECG wavefront and corresponding AP and calcium signal traces using S1S1 pacing protocol after ISO challenge. (B)&#160;VT induction by a 50 Hz burst pacing sequence after perfusion of ISO in a CPVT mouse. Abbreviations: ECG = electrocardiogram; AP = action potential; ISO = isoproterenol; VT = ventricular tachycardia; CPVT = catecholaminergic polymorphic ventricular tachycardia. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65082/65082fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65082/65082fig04.jpg" /> 
Figure 4: APD80 and CaTD80 analysis at 10 Hz before and after ISO challenge. (A) Representative AP traces and APD80 heat maps of WT and CPVT hearts before and after ISO treatment. (B) Typical CaT traces and CaTD80 heat maps of WT and CPVT hearts before and after the ISO challenge. (C) ISO shortens APD80 in WT and CPVT mice, but no difference is found between WT and CPVT mice before and after the ISO challenge. (D) CaTD80 in CPVT mice are longer than in WT after ISO challenge, while there was no significance before ISO treatment. (* P &#60; 0.05, **P &#60; 0.01. n =5/6.) Abbreviations: AP = action potential; APD80 = peak at 80% repolarization; ISO = isoproterenol; CPVT = catecholaminergic polymorphic ventricular tachycardia; WT = wild type. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65082/65082fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/65082/65082fig05.jpg" /> 
Figure 5: Conduction velocity analysis at 10 Hz. (A) The single vector algorithm of conduction velocity. (B)&#160;No difference in the CV of AP in WT and CPVT mice. (C) Representative heat maps demonstrate that CPVT mice have the same conduction ability as WT mice before and after the ISO challenge according to voltage signals. (D)&#160;No significant difference is found in the two groups for action potential-induced CaT80 isochrones before and after the ISO challenge. Abbreviations: AP = action potential; ISO = isoproterenol; CPVT = catecholaminergic polymorphic ventricular tachycardia; WT = wild type; AT = activation time; CV = conduction velocity. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65082/65082fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/65082/65082fig06.jpg" /> 
Figure 6:&#160;Calcium amplitude alternans analysis. (A) The algorithm of calculating calcium amplitude alternans. (B) Calcium signals in WT hearts stay stable at baseline during consecutive S1S1 pacing at 14.29 and 16.67 Hz, while (C) CPVT hearts show frequency-dependent alternans. (D) WT hearts are not influenced by the ISO challenge, while (E) after the ISO challenge, CPVT hearts exhibit frequency-dependent alternans in calcium signal during S1S1 pacing. Abbreviations: ISO = isoproterenol; CPVT = catecholaminergic polymorphic ventricular tachycardia; WT = wild type. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65082/65082fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/65082/65082fig07.jpg" /> 
Figure 7: Tachyarrhythmia analysis using phase maps. (A) WT and CPVT hearts exhibit normal conduction during 50 Hz burst pacing at baseline. (B) After perfusion with ISO, CPVT hearts show high-frequency rotors after 50 Hz burst pacing, while WT hearts maintain normal conduction. Abbreviations: ISO = isoproterenol; CPVT = catecholaminergic polymorphic ventricular tachycardia; WT = wild type. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65082/65082fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Dual-Dye Optical Mapping of Hearts from RyR2R2474S Knock-In Mice of Catecholaminergic Polymorphic Ventricular Tachycardia at JoVE.com

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

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

The pro-arrhythmic cardiac disorder catecholaminergic polymorphic ventricular tachycardia (CPVT) manifests as polymorphic ventricular tachycardia episodes ...

dual-dye-optical-mapping-hearts-from-ryr2r2474s-knock-mice

Nanyang Technological University

Research

JoVE Journal

Biology

1.1K Views.  Southwest Medical University. Deze methode zal ons helpen om de elektrofysiologische eigenschappen en mechanismen van ventriculaire uitstrijkjes te onthullen die verband houden met ziekten zoals catecholaminerge polymorfe ventriculaire tachycardie. Met behulp van deze techniek kunnen we de membraanpotentiaal en intercellulaire calciumsignalen tegelijkertijd verkrijgen onder verschillende geprogrammeerde elektrische pacingprotocollen, wat vooral geschikt is voor het onderzoeken van de onderliggende mechanismen en dynamiek ...

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

Multiparametric Optical Mapping of the Langendorff-perfused Rabbit Heart

Optical imaging and fluorescent probes have significantly advanced research methodology in the field of cardiac electrophysiology in ways that could not have been accomplished by other approaches1. With the use of the calcium- and voltage-sensitive dyes, optical mapping allows measurement of transmembrane action potentials and calcium transients with high spatial resolution without the physical contact with the tissue. This makes measurements of the cardiac electrical activity possible under many conditions where the use of electrodes is inconvenient or impossible1. For example, optical recordings provide accurate morphological changes of membrane potential during and immediately after stimulation and defibrillation, while conventional electrode techniques suffer from stimulus-induced artifacts during and after stimuli due to electrode polarization1. 
The Langendorff-perfused rabbit heart is one of the most studied models of human heart physiology and pathophysiology. Many types of arrhythmias observed clinically could be recapitulated in the rabbit heart model. It was shown that wave patterns in the rabbit heart during ventricular arrhythmias, determined by effective size of the heart and the wavelength of reentry, are very similar to that in the human heart2. It was also shown that critical aspects of excitation-contraction (EC) coupling in rabbit myocardium, such as the relative contribution of sarcoplasmic reticulum (SR), is very similar to human EC coupling3. Here we present the basic procedures of optical mapping experiments in Langendorff-perfused rabbit hearts, including the Langendorff perfusion system setup, the optical mapping systems setup, the isolation and cannulation of the heart, perfusion and dye-staining of the heart, excitation-contraction uncoupling, and collection of optical signals. These methods could be also applied to the heart from species other than rabbit with adjustments to flow rates, optics, solutions, etc.
Two optical mapping systems are described. The panoramic mapping system is used to map the entire epicardium of the rabbit heart4-7. This system provides a global view of the evolution of reentrant circuits during arrhythmogenesis and defibrillation, and has been used to study the mechanisms of arrhythmias and antiarrhythmia therapy8,9. The dual mapping system is used to map the action potential (AP) and calcium transient (CaT) simultaneously from the same field of view10-13. This approach has enhanced our understanding of the important role of calcium in the electrical alternans and the induction of arrhythmia14-16.

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.

Optical imaging and fluorescent probes have significantly advanced research methodology in the field of cardiac electrophysiology in ways that could not ...

Bioengineering

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. ...

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

Atrial fibrillation (AF) is a complex cardiac arrhythmia with high morbidity and mortality.1,2 It is the most common sustained cardiac rhythm disturbance seen in clinical practice and its prevalence is expected to increase in the coming years.3 Increased intra-atrial pressure and dilatation have been long recognized to lead to AF,1,4 which highlights the relevance of using animal models and stretch to study AF dynamics. Understanding the mechanisms underlying AF requires visualization of the cardiac electrical waves with high spatial and temporal resolution. While high-temporal resolution can be achieved by conventional electrical mapping traditionally used in human electrophysiological studies, the small number of intra-atrial electrodes that can be used simultaneously limits the spatial resolution and precludes any detailed tracking of the electrical waves during the arrhythmia. The introduction of optical mapping in the early 90's enabled wide-field characterization of fibrillatory activity together with sub-millimeter spatial resolution in animal models5,6 and led to the identification of rapidly spinning electrical wave patterns (rotors) as the sources of the fibrillatory activity that may occur in the ventricles or the atria.7-9 Using combined time- and frequency-domain analyses of optical mapping it is possible to demonstrate discrete sites of high frequency periodic activity during AF, along with frequency gradients between left and right atrium. The region with fastest rotors activates at the highest frequency and drives the overall arrhythmia.10,11 The waves emanating from such rotor interact with either functional or anatomic obstacles in their path, resulting in the phenomenon of fibrillatory conduction.12 Mapping the endocardial surface of the posterior left atrium (PLA) allows the tracking of AF wave dynamics in the region with the highest rotor frequency. Importantly, the PLA is the region where intracavitary catheter-based ablative procedures are most successful terminating AF in patients,13 which underscores the relevance of studying AF dynamics from the interior of the left atrium. Here we describe a sheep model of acute stretch-induced AF, which resembles some of the characteristics of human paroxysmal AF. Epicardial mapping on the left atrium is complemented with endocardial mapping of the PLA using a dual-channel rigid borescope c-mounted to a CCD camera, which represents the most direct approach to visualize the patterns of activation in the most relevant region for AF maintenance.

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.

 Atrial fibrillation (AF) is a complex cardiac arrhythmia with high morbidity and mortality.1,2 It is the most common sustained cardiac rhythm disturbance ...

Medicine

Optical Mapping of Langendorff-perfused Rat Hearts

Optical mapping of the cardiac surface with voltage-sensitive fluorescent dyes has become an important tool to investigate electrical excitation in experimental models that range in scale from cell cultures to whole-organs[1, 2]. Using state-of-the-art optical imaging systems, generation and propagation of action potentials during normal cardiac rhythm or throughout initiation and maintenance of arrhythmias can be visualized almost instantly[1]. The latest commercially-available systems can provide information at exceedingly high spatiotemporal resolutions and were based on custom-built equipment initially developed to overcome the obstacles imposed by more conventional electrophysiological methods[1]. Advancements in high-resolution and high-speed complementary metal-oxide-semiconductor (CMOS) cameras and intensely-bright, light-emitting diodes (LEDs) as well as voltage-sensitive dyes, optics, and filters have begun to make electrical signal acquisition practical for cardiovascular cell biologists who are more accustomed to working with microscopes. Although the newest generation of CMOS cameras can acquire 10,000 frames per second on a 16,384 pixel array, depending on the type of sample preparation, long-established fluorescence acquisition technologies such as photodiode arrays, laser scanning systems, and cooled charged-coupled device (CCD) cameras still have some distinct advantages with respect to dynamic range, signal-to-noise ratio, and quantum efficiency[1, 3]. In the present study, Lewis rat hearts were perfused ex vivo with a crystalloid perfusate (Krebs-Henseleit solution) at 37&deg;C on a modified Langendorff apparatus. After a 20 minute stabilization period, the hearts were intermittently perfused with 11 mMol/L 2,3-butanedione monoxime to eliminate contraction-associated motion during image acquisition. For optical mapping, we loaded hearts with the fast-response potentiometric probe di-8-ANEPPS[4] (5 &mu;Mol/L) and briefly illuminated the preparation with 475&plusmn;15 nm excitation light. During a typical 2 second period of illumination, &gt;605 nm light emitted from the cardiac preparation was imaged with a high-speed CMOS camera connected to a horizontal macroscope. For this demonstration, hearts were paced at 300 beats per minute with a coaxial electrode connected to an isolated electrical stimulation unit. Simultaneous bipolar electrographic recordings were acquired and analyzed along with the voltage signals using readily-available software. In this manner, action potentials on the surface of Langendorff-perfused rat hearts can be visualized and registered with electrographic signals.

This article describes a high temporal and spatial resolution technique to optically image action potential movement on the surface of Langendorff-perfused rat hearts using a potentiometric dye (di-8-ANEPPS).

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

Optical Imaging of Isolated Murine Ventricular Myocytes

The ability to isolate adult cardiac myocytes has permitted researchers to study a variety of cardiac pathologies at the single cell level. While advances in calcium sensitive dyes have permitted the robust optical recording of single cell calcium dynamics, recording of robust transmembrane optical voltage signals has remained difficult. Arguably, this is because of the low single to noise ratio, phototoxicity, and photobleaching of traditional potentiometric dyes. Therefore, single cell voltage measurements have long been confined to the patch clamp technique which while the gold standard, is technically demanding and low throughput. However, with the development of novel potentiometric dyes, large, fast optical responses to changes in voltage can be obtained with little to no phototoxicity and photobleaching. This protocol describes in detail how to isolate adult murine myocytes which can be used for cellular shortening, calcium, and optical voltage measurements. Specifically, the protocol describes how to use a ratiometric calcium dye, a single-excitation calcium dye, and a single excitation voltage dye. This approach can be used to assess the cardiotoxicity and arrhythmogenicity of various chemical agents. While phototoxicity is still an issue at the single cell level, methodology is discussed on how to reduce it.

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.

The ability to isolate adult cardiac myocytes has permitted researchers to study a variety of cardiac pathologies at the single cell level. While advances ...

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

Human cardiac slice preparations have recently been developed as a platform for human physiology studies and therapy testing to bridge the gap between animal and clinical trials. Numerous animal and cell models have been used to examine the effects of drugs, yet these responses often differ in humans. Human cardiac slices offer an advantage for drug testing in that they are directly derived from viable human hearts. In addition to having preserved multicellular structures, cell-cell coupling, and extracellular matrix environments, human cardiac tissue slices can be used to directly test the effect of innumerable drugs on adult human cardiac physiology. What distinguishes this model from other heart preparations, such as whole hearts or wedges, is that slices can be subjected to longer-term culture. As such, cardiac slices allow for studying the acute as well as chronic effects of drugs. Furthermore, the ability to collect several hundred to a thousand slices from a single heart makes this a high-throughput model to test several drugs at varying concentrations and combinations with other drugs at the same time. Slices can be prepared from any given region of the heart. In this protocol, we describe the preparation of left ventricular slices by isolating tissue cubes from the left ventricular free wall and sectioning them into slices using a high precision vibrating microtome. These slices can then either be subjected to acute experiments to measure baseline cardiac electrophysiological function or cultured for chronic drug studies. This protocol also describes dual optical mapping of cardiac slices for simultaneous recordings of transmembrane potentials and intracellular calcium dynamics to determine the effects of the drugs being investigated.

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.

Human cardiac slice preparations have recently been developed as a platform for human physiology studies and therapy testing to bridge the gap between ...

Electrophysiological Assessment of Murine Atria with High-Resolution Optical Mapping

Recent genome-wide association studies targeting atrial fibrillation (AF) have indicated a strong association between the genotype and electrophysiological phenotype in the atria. That encourages us to utilize a genetically-engineered mouse model to elucidate the mechanism of AF. However, it is difficult to evaluate the electrophysiological properties in murine atria due to their small size. This protocol describes the electrophysiological evaluation of atria using an optical mapping system with a high temporal and spatial resolution in Langendorff perfused murine hearts. The optical mapping system is assembled with dual high-speed complementary metal oxide semiconductor cameras and high magnification objective lenses, to detect the fluorescence of a voltage-sensitive dye and Ca2+ indicator. To focus on the assessment of murine atria, optical mapping is performed with an area of 2 mm &#215; 2 mm or 10 mm x 10 mm, with a 100 &#215; 100 resolution (20 &#181;m/pixel or 100 &#181;m/pixel) and sampling rate of up to 10 kHz (0.1 ms) at maximum. A 1-French size quadripolar electrode pacing catheter is placed into the right atrium through the superior vena cava avoiding any mechanical damage to the atrium, and pacing stimulation is delivered through the catheter. An electrophysiological study is performed with programmed stimulation including constant pacing, burst pacing, and up to triple extrastimuli pacing. Under a spontaneous or pacing rhythm, the optical mapping recorded the action potential duration, activation map, conduction velocity, and Ca2+ transient individually in the right and left atria. In addition, the programmed stimulation also determines the inducibility of atrial tachyarrhythmias. Precise activation mapping is performed to identify the propagation of the excitation in the atrium during an induced atrial tachyarrhythmia. Optical mapping with a specialized setting enables a thorough electrophysiological evaluation of the atrium in murine pathological models.

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.

Recent genome-wide association studies targeting atrial fibrillation (AF) have indicated a strong association between the genotype and ...

Advanced Cardiac Rhythm Management by Applying Optogenetic Multi-Site Photostimulation in Murine Hearts

Ventricular tachyarrhythmias are a major cause of mortality and morbidity worldwide. Electrical defibrillation using high-energy electric shocks is currently the only treatment for life-threatening ventricular fibrillation. However, defibrillation may have side-effects, including intolerable pain, tissue damage, and worsening of prognosis, indicating a significant medical need for the development of more gentle cardiac rhythm management strategies. Besides energy-reducing electrical approaches, cardiac optogenetics was introduced as a powerful tool to influence cardiac activity using light-sensitive membrane ion channels and light pulses. In the present study, a robust and valid method for successful photostimulation of Langendorff perfused intact murine hearts will be described based on multi-site pacing applying a 3 x 3 array of micro light-emitting diodes (micro-LED). Simultaneous optical mapping of epicardial membrane voltage waves allows the investigation of the effects of region-specific stimulation and evaluates the newly induced cardiac activity directly on-site. The obtained results show that the efficacy of defibrillation is strongly dependent on the parameters chosen for photostimulation during a cardiac arrhythmia. It will be demonstrated that the illuminated area of the heart plays a crucial role for termination success as well as how the targeted control of cardiac activity during illumination for modifying arrhythmia patterns can be achieved. In summary, this technique provides a possibility to optimize the on-site mechanism manipulation on the way to real-time feedback control of cardiac rhythm and, regarding the region specificity, new approaches in reducing the potential harm to the cardiac system compared to the usage of non-specific electrical shock applications.

This work reports a method for controlling the cardiac rhythm of intact murine hearts of transgenic channelrhodopsin-2 (ChR2) mice using local photostimulation with a micro-LED array and simultaneous optical mapping of epicardial membrane potential.

Ventricular tachyarrhythmias are a major cause of mortality and morbidity worldwide. Electrical defibrillation using high-energy electric shocks is ...

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

Sarcoplasmic reticulum (SR) Ca2+ handling plays a key role in normal excitation-contraction coupling and aberrant SR Ca2+ handling is known to play a significant role in certain types of arrhythmia. Because arrhythmias are spatially distinct, emergent phenomena, they must be investigated at the tissue level. However, methods for directly probing SR Ca2+ in the intact heart remain limited. This article describes the protocol for dual optical mapping of transmembrane potential (Vm) and free intra-SR [Ca2+] ([Ca2+]SR) in the Langendorff-perfused rabbit heart. This approach takes advantage of the low-affinity Ca2+ indicator Fluo-5N, which has minimal fluorescence in the cytosol where intracellular [Ca2+] ([Ca2+]i) is relatively low but exhibits significant fluorescence in the SR lumen where [Ca2+]SR is in the millimolar range. In addition to revealing SR Ca2+ characteristics spatially across the epicardial surface of the heart, this approach has the distinct advantage of simultaneous monitoring of Vm, allowing for investigations into the bidirectional relationship between Vm and SR Ca2+ and the role of SR Ca2+ in arrhythmogenic phenomena.

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

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.

Sarcoplasmic reticulum (SR) Ca2+ handling plays a key role in normal excitation-contraction coupling and aberrant SR Ca2+ handling is known to play a ...

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.

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.

Dual-Dye Optical Mapping of Hearts from RyR2^R2474S Knock-In Mice of Catecholaminergic Polymorphic Ventricular Tachycardia

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