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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • 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 encod....

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 sulfox.......

Representative 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.......

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

References

  1. Priori, S. G., Chen, S. R. Inherited dysfunction of sarcoplasmic reticulum Ca2+ handling and arrhythmogenesis. Circulation Research. 108 (7), 871-883 (2011).
  2. Goddard, C. A., et al.

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