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

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 fibrillation^1,2,3,4. 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 channels^5,6, FKBP12.6⁷, CASQ2 encoding cardiac calsequestrin⁸, TRDN encoding the junctional SR protein triadin⁹, and CALM1⁹, CALM2¹⁰, and CALM3 identically encoding calmodulin^11,12. 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 RyR2^R4496C in the sinoatrial node (SAN) leads to an unanticipated decrease in SAN automaticity by Ca²⁺-dependent decrease of I_Ca,L and SR Ca²⁺ depletion during diastole, identifying subcellular pathophysiologic alterations contributing to the SAN dysfunction in CPVT patients^13,14. 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 (V_m) 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.

Male 10 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

1. Stock solutions
   1. 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.
   2. Voltage indicator RH237 stock solution: Add 1 mL of 100% DMSO in the original flask with 1 mg of RH237 powder to achieve a concentration of 2.01 mM.
   3. 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.
   4. Pluronic F127 stock solution: Add 1 mL of 100% DMSO into 200 mg of Pluronic F127 to reach a concentration of 20% w/v (0.66 mM).
   5. Aliquot the stock solutions into 200-µL PCR tubes in 21-51 µL (21 µL of RH237, 31 µL of Rhod-2 AM, and 51 µL of blebbistatin) for single or double use to avoid repeated freezing and thawing. Then, wrap the solutions with aluminum foil and store at -20 °C, except for the Pluronic F127 stock solution placed in a dark room at ambient temperature.
2. Perfusion solution
   1. 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).
   2. Filter the solution with a 0.22 µm aseptic needle filter and oxygenate with 95% O₂/5% CO₂.
   3. Take 40 mL of Krebs solution into a 50 mL centrifugal tube and store it at 4 °C for follow-up heart isolation.
3. The Langendorff perfusion system and optical mapping device
   1. Set up the Langendorff perfusion system.
      1. Turn on the water bath and set the temperature to 37 °C.
      2. Wash the Langendorff perfusion system with 1 L of deionized water.
      3. 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 °C.
         NOTE: A bubble is never allowed in the perfusion system.
   2. Prepare the optical mapping system.
      1. Install the electron multiplying charge-coupled device (EMCCD) camera (512 × 512 pixels), lens (40x magnification), wavelength splitter light-emitting diodes (LEDs), electrocardiogram (ECG) monitor, and stimulation electrode (Figure 1).
      2. Adjust the proper working distance from the lens to the heart position.
      3. 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.
      4. 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.
      5. Turn the aperture of the lens to the maximum diameter to avoid any leaking of voltage or calcium signals.
      6. Adjust the camera lens at a proper height, as it serves a fine working distance to the thermostatic bath, 10 cm is mostly used.
      7. Turn on the camera for stable sampling temperature at -50 °C.

2. Procedures

1. Mouse heart harvest, cannulation, and perfusion
   1. 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.
   2. Open the chest with scissors, harvest the heart carefully, and place it into the cold Krebs solution (4 °C, 95% O₂, 5% CO₂) to slow down the metabolism and protect the heart.
   3. 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.
   4. Perfuse the heart with the Langendorff system at a constant speed of 3.5-4.0 mL/min and keep the temperature at 37 ± 1 °C.
      NOTE: All the subsequent procedures are performed in this condition.
   5. 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.
2. Uncoupler of excitation-contraction and dual-dye loading
   1. 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.
   2. Perform the subsequent steps in the dark when the heart reaches a stable state condition (the heart is beating rhythmically at ~400 bpm).
   3. Mix 50 µL of 10 mM blebbistatin stock solution with 50 mL of Krebs solution to reach a concentration of 10 µM. Constantly perfuse the blebbistatin-Krebs solution mixture into the heart for 10 mins to uncouple contraction from excitation and avoid contraction artifacts during filming.
   4. Use a red flashlight to check whether the heart contraction stops totally because contraction will influence the dye loading quality.
   5. After uncoupling excitation-contraction, mix 15 µL of Rhod-2 AM stock solution with 15 µL of Pluronic F127 stock solution in 50 mL of Krebs solution to achieve the final concentrations of 0.267 µM Rhod-2 AM and 0.198 µM Pluronic F127. Then, perfuse the heart continuously with Rhod-2 AM working solution for 15 mins in the Langendorff perfusion system.
   6. 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.
   7. Dilute 10 µL of RH237 stock solution into 50 mL of the perfusate to reach the final concentration at 0.402 µM and perform loading for 10 mins.
   8. 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).
3. 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).
   1. 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).
   2. 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.
   3. 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 µm per pixel.
   4. Open the signal sampling software to control the camera digitally to capture voltage and calcium signals simultaneously.
   5. 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.
   6. 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).
   7. 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).
   8. 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).
   9. 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.
   10. 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.
   11. 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).
   12. Image using EMCCD camera (sampling rate: 500 Hz, pixel size: 64 x 64).
4. Data analysis
   1. Image loading and signal processing
      1. 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 previously^15,16.
      2. Enter the correct sampling parameters (such as Pixel Size and Framerate).
      3. Set the image threshold by manual input and select the region of interest (ROI).
      4. Implement a 3 x 3 pixel Gaussian spatial filter, a Savitzky-Goaly filter, and a top-hat baseline correction.
      5. Press Process Images to remove the baseline and calculate the electrophysiological parameters, such as APD80 and CaTD50.
   2. Electrophysiological parameters analysis
      1. 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.
      2. 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'Shea et al.¹⁵ reported the CV measurement in detail.
      3. 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.

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 µm 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 V_m), which are detected simultaneously using the EMCCD camera. 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 °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 µM) 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 µM 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 < 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 < 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).

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 µm). 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's high sampling rate and sensitivity. Abbreviations: EMCCD = electron-multiplying charge-coupled device; LED = light-emittting diode; ECG = electrocardiogram. Please click here to view a larger version of this figure.

Figure 2: Preparation and dual-dye loading. (A) 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) The heart is removed quickly to the Langendorff perfusion system. (E) Dual-dye loading and excitation-contraction cessation in the dark. Please click here to view a larger version of this figure.

Figure 3: S1S1 protocol and arrhythmia induction protocol. (A) Representative ECG wavefront and corresponding AP and calcium signal traces using S1S1 pacing protocol after ISO challenge. (B) 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. Please click here to view a larger version of this figure.

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 < 0.05, **P < 0.01. n =5/6.) Abbreviations: AP = action potential; APD80 = peak at 80% repolarization; ISO = isoproterenol; CPVT = catecholaminergic polymorphic ventricular tachycardia; WT = wild type. Please click here to view a larger version of this figure.

Figure 5: Conduction velocity analysis at 10 Hz. (A) The single vector algorithm of conduction velocity. (B) 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) 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. Please click here to view a larger version of this figure.

Figure 6: 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. Please click here to view a larger version of this figure.

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. Please click here to view a larger version of this figure.

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 µm 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's ischemic time, the better the heart'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'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 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' 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 heart^19,20,21. Although some studies imply some side effects using the compound²², we utilize the lowest working concentration at 10 µM 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 (τ) ^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 arrhythmia^24,25. Optical mapping has a high spatiotemporal resolution to determine the ventricular activation and repolarization properties of the heart of transgenic mice^26,27,28,29. It can also detect multi-parameter imaging, for example, measurement of membrane potential and intracellular calcium of the same heart^24,30 or tissue^31,32 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 dyes^30,34,35. 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 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.