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>

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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 al...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.2 &#956;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 &#956;L PCR tube</td>
			<td>Sangon Biotech Co., Ltd. Shanghai. China</td>
			<td>F611541-0010</td>
			<td>Aliquote the stock solutions&#160; 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&#39;s solution at 4 &#176;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&#160; eliminate motion artifact during mapping</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma-Aldrich, St. Louis, MO, United States</td>
			<td>SLBK1794V</td>
			<td>For Tyrode&#39;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&#39;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>&#160;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&#39;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>MgCl2</td>
			<td>Sigma-Aldrich, St. Louis, MO, United States</td>
			<td>BCBS6841V</td>
			<td>For Tyrode&#39;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&#39;s solution</td>
		</tr>
		<tr>
			<td>NaH2PO4&#160;</td>
			<td>Sigma-Aldrich, St. Louis, MO, United States</td>
			<td>BCBW9042</td>
			<td>For Tyrode&#39;s solution</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma-Aldrich, St. Louis, MO, United States</td>
			<td>SLBX3605</td>
			<td>For Tyrode&#39;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 Ca2+ and Vm&#160; 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 illuminat...

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

Research

JoVE Journal

Biology

1.0K Views.  Southwest Medical University. 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.

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

Proper Care and Cleaning of the Microscope

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a microscope causes reduction in contrast and resolution. DIC is especially sensitive to contamination and scratches on the lens surfaces. This protocol details the procedure for keeping the microscope clean.

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a microscope causes reduction in contrast and resolution. DIC is especially sensitive to contamination and scratches on the lens surfaces. This protocol details the procedure for keeping the microscope clean.

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a ...

Major Components of the Light Microscope

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for different applications, and how it should be maintained as required to obtain maximum image-forming capacity and resolution. The components of the microscope are described in detail here.

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for different applications, and how it should be maintained as required to obtain maximum image-forming capacity and resolution. The components of the microscope are described in detail here.

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for ...

Phase Contrast and Differential Interference Contrast (DIC) Microscopy

Phase-contrast microscopy is often used to produce contrast for transparent, non light-absorbing, biological specimens. The technique was discovered by Zernike, in 1942, who received the Nobel prize for his achievement. DIC microscopy, introduced in the late 1960s, has been popular in biomedical research because it highlights edges of specimen structural detail, provides high-resolution optical sections of thick specimens including tissue cells, eggs, and embryos and does not suffer from the  phase halos  typical of phase-contrast images. This protocol highlights the principles and practical applications of these microscopy techniques.

Phase Contrast and Differential Interference Contrast DIC Microscopy

This protocol highlights the principles and practical applications of Phase and Differential Interference Contrast (DIC) Microscopy

Phase-contrast microscopy is often used to produce contrast for transparent, non light-absorbing, biological specimens. The technique was discovered by ...

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 ...

Semi-automated Optical Heartbeat Analysis of Small Hearts

We have developed a method for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts (Fink, et. al., 2009). Our Semi-automatic Optical Heartbeat Analysis (SOHA) uses a novel movement detection algorithm that is able to detect cardiac movements associated with individual contractile and relaxation events. The program provides a host of physiologically relevant readouts including systolic and diastolic intervals, heart rate, as well as qualitative and quantitative measures of heartbeat arrhythmicity. The program also calculates heart diameter measurements during both diastole and systole from which fractional shortening and fractional area changes are calculated. Output is provided as a digital file compatible with most spreadsheet programs. Measurements are made for every heartbeat in a record increasing the statistical power of the output. We demonstrate each of the steps where user input is required and show the application of our methodology to the analysis of heart function in all three genetically tractable heart models.

We have developed a Semi-automated Optical Heartbeat Analysis method (SOHA) for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts. We demonstrate the application of our methodology to the analysis of heart function in fruit fly and embryonic mouse hearts.

We have developed a method for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts (Fink, et. al., 2009). Our ...

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

Optical mapping has proven to be a valuable technique to detect cardiac electrical activity on both intact ex vivo hearts and in cultured myocyte monolayers. HL-1 cells have been widely used as a 2-Dimensional cellular model for studying diverse aspects of cardiac physiology. However, it has been a great challenge to optically map calcium (Ca) transients and action potentials simultaneously from the same field of view in a cultured HL-1 atrial cell monolayer. This is because special handling and care is required to prepare healthy cells that can be electrically captured and optically mapped. Therefore, we have developed an optimal working protocol for dual channel optical mapping. In this manuscript, we have described in detail how to perform the dual channel optical mapping experiment. This protocol is a useful tool to enhance the understanding of action potential propagation and Ca kinetics in arrhythmia development.

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

Optical mapping has proven to be a valuable technique to detect cardiac electrical activity on both intact ex vivo hearts and in cultured myocyte ...

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.

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

Subtype-specific Optical Action Potential Recordings in Human Induced Pluripotent Stem Cell-derived Ventricular Cardiomyocytes

Cardiomyocytes generated from human induced pluripotent stem cells (iPSC-CMs) are an emerging tool in cardiovascular research. Rather than being a homogenous population of cells, the iPSC-CMs generated by current differentiation protocols represent a mixture of cells with ventricular-, atrial-, and nodal-like phenotypes, which complicates phenotypic analyses. Here, a method to optically record action potentials specifically from ventricular-like iPSC-CMs is presented. This is achieved by lentiviral transduction with a construct in which a genetically-encoded voltage indicator is under the control of a ventricular-specific promoter element. When iPSC-CMs are transduced with this construct, the voltage sensor is expressed exclusively in ventricular-like cells, enabling subtype-specific optical membrane potential recordings using time-lapse fluorescence microscopy.

Here we present a method to optically image action potentials, specifically in ventricular-like induced pluripotent stem cell-derived cardiomyocytes. The method is based on the promoter-driven expression of a voltage-sensitive fluorescent protein.

Cardiomyocytes generated from human induced pluripotent stem cells (iPSC-CMs) are an emerging tool in cardiovascular research. Rather than being a ...

Induction of Right Ventricular Failure by Pulmonary Artery Constriction and Evaluation of Right Ventricular Function in Mice

The mechanism of right ventricular failure (RVF) requires clarification due to the uniqueness, high morbidity, high mortality, and refractory nature of RVF. Previous rat models imitating RVF progression have been described. Compared with rats, mice are more accessible, economical, and widely used in animal experiments. We developed a pulmonary artery constriction (PAC) approach which is comprised of banding the pulmonary trunk in mice to induce right ventricular (RV) hypertrophy. A special surgical latch needle was designed that allows for easier separation of the aorta and the pulmonary trunk. In our experiments, the use of this fabricated latch needle reduced the risk of arteriorrhexis and improved the surgical success rate to 90%. We used different padding needle diameters to precisely create quantitative constriction, which can induce different degrees of RV hypertrophy. We quantified the degree of constriction by evaluating the blood flow velocity of the PA, which was measured by noninvasive transthoracic echocardiography. RV function was precisely evaluated by right heart catheterization at 8 weeks after surgery. The surgical instruments made inhouse were composed of common materials using a simple process that is easy to master. Therefore, the PAC approach described here is easy to imitate using instruments made in the lab and can be widely used in other labs. This study presents a modified PAC approach that has a higher success rate than other models and an 8-week postsurgery survival rate of 97.8%. This PAC approach provides a useful technique for studying the mechanism of RVF and will enable an increased understanding of RVF.

Here, we provide a useful approach for studying the mechanism of right ventricular failure. A more convenient and efficient approach to pulmonary artery constriction is established using surgical instruments made inhouse. In addition, methods to evaluate the quality of this approach by echocardiography and catheterization are provided.

The mechanism of right ventricular failure (RVF) requires clarification due to the uniqueness, high morbidity, high mortality, and refractory nature of ...