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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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.

Protocol

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 previously15,16.
      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.

Results

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 illuminat...

Discussion

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...

Disclosures

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

Acknowledgements

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.

Materials

NameCompanyCatalog NumberComments
0.2 μm syringe filterMedical equipment factory of Shanghai Medical Instruments Co., Ltd., Shanghai, ChinaN/ATo filter solution
15 mL centrifuge tubeGuangzhou Jet Bio-Filtration Co., Ltd. ChinaCFT011150
1 mL Pasteur pipetteBeijing Labgic Technology Co., Ltd. China00900026
1 mL SyringeB. Braun Medical Inc.YZB/GER-5474-2014
200 μL PCR tubeSangon Biotech Co., Ltd. Shanghai. ChinaF611541-0010Aliquote the stock solutions  to avoid repeated freezing and thawing
50 mL centrifuge tubeGuangzhou Jet Bio-Filtration Co., Ltd. ChinaCFT011500Store Tyrode's solution at 4 °C for follow-up heart isolation
585/40 nm filterChroma TechnologyN/AFilter for calcium signal
630 nm long-pass filterChroma TechnologyG15604AJFilter for voltage signal
Avertin (2,2,2-tribromoethanol)Sigma-Aldrich Poole, Dorset, United KingdomT48402-100GTo minimize suffering and pain reflex
BlebbistatinTocris Bioscience, Minneapolis, MN, United StatesSLBV5564Excitation-contraction uncoupler to  eliminate motion artifact during mapping
CaCl2Sigma-Aldrich, St. Louis, MO, United StatesSLBK1794VFor Tyrode's solution
Custom-made thermostatic bathMappingLab, United KingdomTBC-2.1To keep temperature of perfusion solution
Dimethyl sulfoxide (DMSO)Sigma-Aldrich(RNBT7442)Solvent for dyes
Dumont forcepsMedical equipment factory of Shanghai Medical Instruments Co.,Ltd.,Shanghai, ChinaYAF030
ElectroMap softwareUniversity of BirminghamN/AQuantification of electrical parameters
EMCCD cameraEvolve 512 Delta, Photometrics, Tucson, AZ, United StatesA18G150001Acquire images for optical signals
ET525/36 sputter coated filterChroma Technology319106Excitation filter
GlucoseSigma-Aldrich, St. Louis, MO, United StatesSLBT4811VFor Tyrode's solution
Heparin SodiumChengdu Haitong Pharmaceutical Co., Ltd., Chengdu, China(H51021209)To prevent blood clots in the coronary artery
 Iris forcepsMedical equipment factory of Shanghai Medical Instruments Co.,Ltd.,Shanghai, ChinaYAA010
IsoproterenolMedChemExpress, Carlsbad, CA, United StatesHY-B0468/CS-2582
KClSigma-Aldrich, St. Louis, MO, United StatesSLBS5003For Tyrode's solution
MacroLEDCairn Research, Faversham, United Kingdom7355/7356The excitation light of fluorescence probes
MacroLED light sourceCairn Research, Faversham, United Kingdom7352Control the LEDs
Mayo scissorsMedical equipment factory of Shanghai Medical Instruments Co.,Ltd.,Shanghai, ChinaYBC010
MetaMorphMolecular DevicesN/AOptical signals sampling
MgCl2Sigma-Aldrich, St. Louis, MO, United StatesBCBS6841VFor Tyrode's solution
MICRO3-1401Cambridge Electronic Design limited, United KingdomM5337Connect the electrical stimulator and Spike2 software
MyoPacer EP field stimulatorIon Optix Co, Milton, MA, United StatesS006152Electric stimulator
NaClSigma-Aldrich, St. Louis, MO, United StatesSLBS2340VFor Tyrode's solution
NaH2PO4 Sigma-Aldrich, St. Louis, MO, United StatesBCBW9042For Tyrode's solution
NaHCO3Sigma-Aldrich, St. Louis, MO, United StatesSLBX3605For Tyrode's solution
NeuroLog SystemDigitimerNL905-229For ECG amplifier
OmapScope5MappingLab, United KingdomN/ACalcium alternans and arrhythmia analysis
Ophthalmic scissorsHuaian Teshen Medical Instruments Co., Ltd., Jiang Su, ChinaT4-3904
OptoSplitCairn Research, Faversham, United Kingdom6970Split the emission light for detecting Ca2+ and Vm  simultaneously
Peristalic pumpLonger Precision Pump Co., Ltd., Baoding, China,BT100-2JTo pump the solution
Petri dishBIOFILTCD010060
Pluronic F127Invitrogen, Carlsbad, CA, United States1899021To enhance the loading with Rhod2AM
RH237Thermo Fisher Scientifific, Waltham, MA, United States1971387Voltage-sensitive dye
Rhod-2 AMInvitrogen, Carlsbad, CA, United States1890519Calcium indicator
Silica gel tubeLonger Precision Pump Co., Ltd., Baoding, China,96402-16Connect with the peristaltic pump
Silk sutureYuankang Medical Instrument Co., Ltd.,Yangzhou, China20172650032To fix the aorta
Spike2Cambridge Electronic Design limited, United KingdomN/ATo record and analyze ECG data
Stimulation electrodeMappingLab, United KingdomSE1600-35-2020
T510lpxrChroma Technology312461For light source
T565lpxrChroma Technology321343For light source

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