Dual-Dye optisk kortlægning af hjerter fra RyR2R2474S Knock-In mus af katekolaminerg polymorf ventrikulær takykardi
Summary
Denne protokol introducerer dobbeltfarvet optisk kortlægning af musehjerter opnået fra vildtype- og knock-in-dyr, der er ramt af katekolaminer polymorf ventrikulær takykardi, herunder elektrofysiologiske målinger af transmembranspænding og intracellulære Ca2+ transienter med høj tidsmæssig og rumlig opløsning.
Abstract
Den pro-arytmiske hjertesygdom catecholaminerge polymorfe ventrikulære takykardi (CPVT) manifesterer sig som polymorfe ventrikulære takykardi episoder efter fysisk aktivitet, stress eller catecholamin udfordring, som kan forværres til potentielt dødelig ventrikelflimmer. Musens hjerte er en udbredt art til modellering af arvelige hjertearytmiske sygdomme, herunder CPVT. Samtidig optisk kortlægning af transmembranpotentiale (Vm) og calciumtransienter (CaT) fra Langendorff-perfunderede musehjerter har potentiale til at belyse mekanismerne bag arytmogenese. Sammenlignet med undersøgelsen på celleniveau kan den optiske kortlægningsteknik teste nogle elektrofysiologiske parametre, såsom bestemmelse af aktivering, ledningshastighed, aktionspotentiel varighed og CaT-varighed. Dette papir præsenterer instrumenteringsopsætningen og den eksperimentelle procedure til optisk kortlægning med høj kapacitet af CaT og Vm i murine vildtype og heterozygote RyR2-R2474S / + hjerter kombineret med programmeret elektrisk pacing før og under isoproterenoludfordringen. Denne fremgangsmåde har vist en gennemførlig og pålidelig metode til mekanisk undersøgelse af CPVT-sygdom i et ex vivo-musehjertepræparat.
Introduction
Arvelig hjertesygdom katekolaminminerg polymorf ventrikulær takykardi (CPVT) manifesterer sig som polymorf ventrikulær takykardi (PVT) episoder efter fysisk aktivitet, stress eller catecholamin udfordring, som kan forværres til potentielt dødelig ventrikelflimmer 1,2,3,4 . Nylige beviser efter sin første rapport som et klinisk syndrom i 1995 implicerede mutationer i syv gener, alle involveret i sarkoplasmatisk retikulær (SR) lagerbutik Ca 2+ frigivelse i denne tilstand: den hyppigst rapporterede RYR2-kodning af ryanodinreceptor 2 (RyR2) af Ca2+ frigivelseskanaler 5,6, FKBP12.67, CASQ2-kodning af hjertekalsekvestrin8, TRDN kodning af det junctionale SR-proteintriadin 9 og CALM1 9, CALM2 10 og CALM3 identisk kodende calmodulin11,12. Disse genotypiske mønstre tilskriver de arytmiske hændelser til den uregulerede patologiske frigivelse af SR-lager Ca2+12.
Spontan Ca 2+ frigivelse fra SR kan detekteres som Ca 2+ gnister eller Ca 2+ bølger, som aktiverer Na+/Ca 2+ veksleren (NCX). Veksleren af en Ca2+ for tre Na+ genererer en indadgående strøm, som fremskynder den diastoliske depolarisering og driver membranspændingen til tærsklen for handlingspotentiale (AP). Hos RyR2-knock-in-mus fører den øgede aktivitet afRyR2 R4496C i sinoatriale knude (SAN) til et uventet fald i SAN-automaticitet ved Ca 2+-afhængigt fald i I Ca,L og SR Ca2+ udtømning under diastol, hvilket identificerer subcellulære patofysiologiske ændringer, der bidrager til SAN-dysfunktionen hos CPVT-patienter13,14. Forekomst af de relaterede kardiomyocytcytosoliske Ca2+-bølger er mere sandsynlig efter stigninger i baggrundscytosolisk [Ca2+] efter RyR-sensibilisering af catecholamin, herunder isoproterenol (ISO), udfordring.
Detaljerede kinetiske ændringer i Ca 2+ signalering efter RyR2-medieret Ca2+ frigivelse som reaktion på aktivering af handlingspotentiale (AP), der kan være årsagen til de observerede ventrikulære arytmier i intakte hjerte-CPVT-modeller, mangler at blive bestemt for hele spektret af rapporterede RyR2-genotyper12. Dette papir præsenterer instrumenteringsopsætningen og den eksperimentelle procedure til kortlægning med høj kapacitet af Ca2+ signaler og transmembranpotentialer (Vm) i murine vildtype (WT) og heterozygote RyR2-R2474S / + hjerter kombineret med programmeret elektrisk pacing før og efter isoproterenoludfordring. Denne protokol giver en metode til mekanistisk undersøgelse af CPVT-sygdom i isolerede musehjerter.
Protocol
Representative Results
Discussion
Baseret på vores erfaring inkluderer nøglerne til en vellykket dobbeltfarvet optisk kortlægning af et musehjerte en velforberedt løsning og hjerte, farvestofbelastning, opnåelse af det bedste signal-støj-forhold og reduktion af bevægelsesartefakten.
Fremstilling af opløsning
Krebs-løsningen er afgørende for et vellykket hjerteeksperiment. MgCl2- ogCaCl2-stamopløsninger (1 mol/l) fremstilles på forhånd under hensyntagen til deres vandabsorption og til…
Declarações
The authors have nothing to disclose.
Acknowledgements
Denne undersøgelse støttes af National Natural Science Foundation of China (81700308 til XO og 31871181 til ML og 82270334 til XT), Sichuan Province Science and Technology Support Program (CN) (2021YJ0206 til XO, 23ZYZYTS0433 og 2022YFS0607 til XT og 2022NSFSC1602 til TC) og State Key Laboratory for Chemistry and Molecular Engineering of Medicinal Resources (Guangxi Normal University) (CMEMR2017-B08 til XO), MRC (G10031871181 til ML02647, G1002082, ML), BHF (PG/14/80/31106, PG/16/67/32340, PG/12/21/29473, PG/11/59/29004 ML), BHF CRE i Oxford (ML) tilskud.
Materials
| 0.2 μm syringe filter | Medical equipment factory of Shanghai Medical Instruments Co., Ltd., Shanghai, China | N/A | To filter solution |
| 15 mL centrifuge tube | Guangzhou Jet Bio-Filtration Co., Ltd. China | CFT011150 | |
| 1 mL Pasteur pipette | Beijing Labgic Technology Co., Ltd. China | 00900026 | |
| 1 mL Syringe | B. Braun Medical Inc. | YZB/GER-5474-2014 | |
| 200 μL PCR tube | Sangon Biotech Co., Ltd. Shanghai. China | F611541-0010 | Aliquote the stock solutions to avoid repeated freezing and thawing |
| 50 mL centrifuge tube | Guangzhou Jet Bio-Filtration Co., Ltd. China | CFT011500 | Store Tyrode's solution at 4 °C for follow-up heart isolation |
| 585/40 nm filter | Chroma Technology | N/A | Filter for calcium signal |
| 630 nm long-pass filter | Chroma Technology | G15604AJ | Filter for voltage signal |
| Avertin (2,2,2-tribromoethanol) | Sigma-Aldrich Poole, Dorset, United Kingdom | T48402-100G | To minimize suffering and pain reflex |
| Blebbistatin | Tocris Bioscience, Minneapolis, MN, United States | SLBV5564 | Excitation-contraction uncoupler to eliminate motion artifact during mapping |
| CaCl2 | Sigma-Aldrich, St. Louis, MO, United States | SLBK1794V | For Tyrode's solution |
| Custom-made thermostatic bath | MappingLab, United Kingdom | TBC-2.1 | To keep temperature of perfusion solution |
| Dimethyl sulfoxide (DMSO) | Sigma-Aldrich | (RNBT7442) | Solvent for dyes |
| Dumont forceps | Medical equipment factory of Shanghai Medical Instruments Co.,Ltd.,Shanghai, China | YAF030 | |
| ElectroMap software | University of Birmingham | N/A | Quantification of electrical parameters |
| EMCCD camera | Evolve 512 Delta, Photometrics, Tucson, AZ, United States | A18G150001 | Acquire images for optical signals |
| ET525/36 sputter coated filter | Chroma Technology | 319106 | Excitation filter |
| Glucose | Sigma-Aldrich, St. Louis, MO, United States | SLBT4811V | For Tyrode's solution |
| Heparin Sodium | Chengdu Haitong Pharmaceutical Co., Ltd., Chengdu, China | (H51021209) | To prevent blood clots in the coronary artery |
| Iris forceps | Medical equipment factory of Shanghai Medical Instruments Co.,Ltd.,Shanghai, China | YAA010 | |
| Isoproterenol | MedChemExpress, Carlsbad, CA, United States | HY-B0468/CS-2582 | |
| KCl | Sigma-Aldrich, St. Louis, MO, United States | SLBS5003 | For Tyrode's solution |
| MacroLED | Cairn Research, Faversham, United Kingdom | 7355/7356 | The excitation light of fluorescence probes |
| MacroLED light source | Cairn Research, Faversham, United Kingdom | 7352 | Control the LEDs |
| Mayo scissors | Medical equipment factory of Shanghai Medical Instruments Co.,Ltd.,Shanghai, China | YBC010 | |
| MetaMorph | Molecular Devices | N/A | Optical signals sampling |
| MgCl2 | Sigma-Aldrich, St. Louis, MO, United States | BCBS6841V | For Tyrode's solution |
| MICRO3-1401 | Cambridge Electronic Design limited, United Kingdom | M5337 | Connect the electrical stimulator and Spike2 software |
| MyoPacer EP field stimulator | Ion Optix Co, Milton, MA, United States | S006152 | Electric stimulator |
| NaCl | Sigma-Aldrich, St. Louis, MO, United States | SLBS2340V | For Tyrode's solution |
| NaH2PO4 | Sigma-Aldrich, St. Louis, MO, United States | BCBW9042 | For Tyrode's solution |
| NaHCO3 | Sigma-Aldrich, St. Louis, MO, United States | SLBX3605 | For Tyrode's solution |
| NeuroLog System | Digitimer | NL905-229 | For ECG amplifier |
| OmapScope5 | MappingLab, United Kingdom | N/A | Calcium alternans and arrhythmia analysis |
| Ophthalmic scissors | Huaian Teshen Medical Instruments Co., Ltd., Jiang Su, China | T4-3904 | |
| OptoSplit | Cairn Research, Faversham, United Kingdom | 6970 | Split the emission light for detecting Ca2+ and Vm simultaneously |
| Peristalic pump | Longer Precision Pump Co., Ltd., Baoding, China, | BT100-2J | To pump the solution |
| Petri dish | BIOFIL | TCD010060 | |
| Pluronic F127 | Invitrogen, Carlsbad, CA, United States | 1899021 | To enhance the loading with Rhod2AM |
| RH237 | Thermo Fisher Scientifific, Waltham, MA, United States | 1971387 | Voltage-sensitive dye |
| Rhod-2 AM | Invitrogen, Carlsbad, CA, United States | 1890519 | Calcium indicator |
| Silica gel tube | Longer Precision Pump Co., Ltd., Baoding, China, | 96402-16 | Connect with the peristaltic pump |
| Silk suture | Yuankang Medical Instrument Co., Ltd.,Yangzhou, China | 20172650032 | To fix the aorta |
| Spike2 | Cambridge Electronic Design limited, United Kingdom | N/A | To record and analyze ECG data |
| Stimulation electrode | MappingLab, United Kingdom | SE1600-35-2020 | |
| T510lpxr | Chroma Technology | 312461 | For light source |
| T565lpxr | Chroma Technology | 321343 | For light source |
Referências
- Priori, S. G., Chen, S. R. Inherited dysfunction of sarcoplasmic reticulum Ca2+ handling and arrhythmogenesis. Circulation Research. 108 (7), 871-883 (2011).
- Goddard, C. A., et al. Physiological consequences of the P2328S mutation in the ryanodine receptor (RyR2) gene in genetically modified murine hearts. Acta Physiologica. 194 (2), 123-140 (2008).
- Sabir, I. N., et al. Alternans in genetically modified langendorff-perfused murine hearts modeling catecholaminergic polymorphic ventricular tachycardia. Frontiers in Physiology. 1, 126 (2010).
- Zhang, Y., Matthews, G. D., Lei, M., Huang, C. L. Abnormal Ca2+ homeostasis, atrial arrhythmogenesis, and sinus node dysfunction in murine hearts modeling RyR2 modification. Frontiers in Physiology. 4, 150 (2013).
- Leenhardt, A., et al. Catecholaminergic polymorphic ventricular tachycardia in children. A 7-year follow-up of 21 patients. Circulation. 91 (5), 1512-1519 (1995).
- Priori, S. G., et al. Mutations in the cardiac ryanodine receptor gene (hRyR2) underlie catecholaminergic polymorphic ventricular tachycardia. Circulation. 103 (2), 196-200 (2001).
- Wehrens, X. H., et al. FKBP12.6 deficiency and defective calcium release channel (ryanodine receptor) function linked to exercise-induced sudden cardiac death. Cell. 113 (7), 829-840 (2003).
- Novak, A., et al. Functional abnormalities in iPSC-derived cardiomyocytes generated from CPVT1 and CPVT2 patients carrying ryanodine or calsequestrin mutations. Journal of Cellular and Molecular Medicine. 19 (8), 2006-2018 (2015).
- Napolitano, C., Mazzanti, A., Bloise, R., Priori, S. G., Adam, M. P., et al. CACNA1C-related disorders. GeneReviews. , (1993).
- Makita, N., et al. Novel calmodulin mutations associated with congenital arrhythmia susceptibility. Circulation. Cardiovascular Genetics. 7 (4), 466-474 (2014).
- Gomez-Hurtado, N., et al. Novel CPVT-associated calmodulin mutation in CALM3 (CALM3-A103V) activates arrhythmogenic Ca waves and sparks. Circulation, Arrhythmia and Electrophysiology. 9 (8), (2016).
- Wleklinski, M. J., Kannankeril, P. J., Knollmann, B. C. Molecular and tissue mechanisms of catecholaminergic polymorphic ventricular tachycardia. Journal of Physiology. 598 (14), 2817-2834 (2020).
- Neco, P., et al. Paradoxical effect of increased diastolic Ca2+ release and decreased sinoatrial node activity in a mouse model of catecholaminergic polymorphic ventricular tachycardia. Circulation. 126 (4), 392-401 (2012).
- Bogdanov, K. Y., Vinogradova, T. M., Lakatta, E. G. Sinoatrial nodal cell ryanodine receptor and Na(+)-Ca(2+) exchanger: molecular partners in pacemaker regulation. Circulation Research. 88 (12), 1254-1258 (2001).
- O’Shea, C., et al. ElectroMap: High-throughput open-source software for analysis and mapping of cardiac electrophysiology. Scientific Reports. 9 (1), 1389 (2019).
- O’Shea, C., et al. High-throughput analysis of optical mapping data using ElectroMap. Journal of Visualized Experiments: JoVE. (148), e59663 (2019).
- Choi, B. R., Salama, G. Simultaneous maps of optical action potentials and calcium transients in guinea-pig hearts: mechanisms underlying concordant alternans. Journal of Physiology. 529, 171-188 (2000).
- Rybashlykov, D., Brennan, J., Lin, Z., Efimov, I. R., Syunyaev, R. Open-source low-cost cardiac optical mapping system. PLoS One. 17 (3), 0259174 (2022).
- Lucas-Lopez, C., et al. Absolute stereochemical assignment and fluorescence tuning of the small molecule tool, (-)-blebbistatin. European Journal of Organic Chemistry. 2005 (9), 1736-1740 (2005).
- Ponsaerts, R., et al. The myosin II ATPase inhibitor blebbistatin prevents thrombin-induced inhibition of intercellular calcium wave propagation in corneal endothelial cells. Investigative Ophthalmology & Visual Science. 49 (11), 4816-4827 (2008).
- Jou, C., Spitzer, K., Tristani-Firouzi, M. Blebbistatin effectively uncouples the excitation-contraction process in zebrafish embryonic heart. Cellular Physiology & Biochemistry. 25 (4-5), 419-424 (2010).
- Brack, K. E., Narang, R., Winter, J., Ng, G. A. The mechanical uncoupler blebbistatin is associated with significant electrophysiological effects in the isolated rabbit heart. Experimental Physiology. 98 (5), 1009-1027 (2013).
- O’Shea, C., et al. High-throughput analysis of optical mapping data using ElectroMap. Journal of Visualized Experiments: JoVE. (148), e59663 (2019).
- He, S., et al. A dataset of dual calcium and voltage optical mapping in healthy and hypertrophied murine hearts. Scientific Data. 8 (1), 314 (2021).
- Lei, M., Huang, C. L. Cardiac arrhythmogenesis: a tale of two clocks. Cardiovascular Research. 116 (14), e205-e209 (2020).
- Mal Baudot, ., et al. Concomitant genetic ablation of L-type Cav1.3 α1D and T-type Cav3.1 α1G Ca2+ channels disrupts heart automaticity. Scientific Reports. 10 (1), 18906 (2020).
- Dai, W., et al. ZO-1 regulates intercalated disc composition and atrioventricular node conduction. Circulation Research. 127 (2), e28-e43 (2020).
- Glukhov, A. V., et al. Calsequestrin 2 deletion causes sinoatrial node dysfunction and atrial arrhythmias associated with altered sarcoplasmic reticulum calcium cycling and degenerative fibrosis within the mouse atrial pacemaker complex1. European Heart Journal. 36 (11), 686-697 (2015).
- Torrente, A. G., et al. Burst pacemaker activity of the sinoatrial node in sodium-calcium exchanger knockout mice. Proceedings of the National Academy of Sciences of the United States of America. 112 (31), 9769-9774 (2015).
- Yang, B., et al. Ventricular SK2 upregulation following angiotensin II challenge: Modulation by p21-activated kinase-1. Journal of Molecular and Cellular Cardiology. 164, 110-125 (2022).
- Dong, R., et al. A protocol for dual calcium-voltage optical mapping in murine sinoatrial preparation with optogenetic pacing. Frontiers in Physiology. 10, 954 (2019).
- He, S., et al. A protocol for transverse cardiac slicing and optical mapping in murine heart. Frontiers in Physiology. 10, 755 (2019).
- Hoeker, G. S., Katra, R. P., Wilson, L. D., Plummer, B. N., Laurita, K. R. Spontaneous calcium release in tissue from the failing canine heart. American Journal of Physiology. Heart and Circulatory Physiology. 297 (4), H1235-H1242 (2009).
- Laurita, K. R., Singal, A. Mapping action potentials and calcium transients simultaneously from the intact heart. American Journal of Physiology. Heart and Circulatory Physiology. 280 (5), H2053-H2060 (2001).
- Johnson, P. L., Smith, W., Baynham, T. C., Knisley, S. B. Errors caused by combination of Di-4 ANEPPS and Fluo3/4 for simultaneous measurements of transmembrane potentials and intracellular calcium. Annals of Biomedical Engineering. 27 (4), 563-571 (1999).
