的内肌浆网钙光学标测²⁺和的Langendorff灌注兔心脏跨膜电位

Summary

本文介绍了详细的协议和必要的跨膜电位_（V M）双光学测绘和免费内部肌浆网（SR）设备^钙在的Langendorff灌注兔心脏。此方法允许直接观察和第V _米量化和SR ^的 Ca 2+动力学在完整心脏。

Abstract

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 (V_m) 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 V_m, allowing for investigations into the bidirectional relationship between V_m and SR Ca²⁺ and the role of SR Ca²⁺ in arrhythmogenic phenomena.

Introduction

Dual optical mapping of intracellular Ca²⁺ and transmembrane potential (V_m) in the intact Langendorff-perfused heart has become a mainstay of investigations in cardiac electrophysiology, including mechanisms of arrhythmia and excitation-contraction coupling^1-4. This approach has provided unprecedented knowledge into normal and abnormal electrophysiology and, importantly, into the bidirectional relationship between V_m and intracellular Ca²⁺. However, optical mapping of intracellular Ca²⁺ with high-affinity fluorescent indicators (such as Rhod-2 and Fluo-4) only reports on bulk changes in intracellular Ca²⁺ and is unable to distinguish whether these changes are due to transmembrane Ca²⁺ flux, release and reuptake into intracellular stores, or in most instances, some combination of both. Furthermore, high-affinity Ca²⁺ indicators have slow on-off kinetics and may not accurately report rapid changes in Ca²⁺ concentration⁵.

Each action potential triggers a rise in intracellular Ca²⁺, known as the intracellular Ca²⁺ transient (CaT). In the mammalian heart, approximately 70 – 90% of the total CaT is due to release of Ca²⁺ from the sarcoplasmic reticulum (SR) via opening of ryanodine receptors (RyRs)⁶. Within the SR, approximately half of the total Ca²⁺ is bound to calsequestrin (CSQ) and other intra-SR buffers⁷, which play an important role in SR Ca²⁺ homeostasis^8,9. The amount of free SR Ca²⁺ dictates the driving force for SR Ca²⁺ release as well as gating of RyR, and therefore has a significant impact on the intracellular CaT. Furthermore, alterations in SR Ca²⁺ release or reuptake can, in turn, impact V_m via the electrogenic Na⁺-Ca²⁺ exchange, which may have arrhythmogenic consequences. Therefore, in addition to the CaT, monitoring of free SR Ca²⁺ can provide important insights into contractile and electrophysiological dysfunction.

Over the past several years, investigators have made significant advances in the monitoring of SR Ca²⁺ in isolated cardiac myocytes and from a single location on the intact heart. One such method requires rapid pulses of caffeine to open RyRs and the SR Ca²⁺ content is then inferred or calculated from the immediate rise in intracellular Ca²⁺¹⁰. Another intriguing approach uses low-affinity Ca²⁺ indicators, such as Fluo-5N¹¹ or Mag-Fluo4¹², which bind to free SR Ca²⁺. These indicators have dissociation constants (K_d) in the range of 10 – 400 μM and therefore exhibit minimal fluorescence in the cytosol compared to the SR lumen, where the Ca²⁺ concentration ([Ca²⁺]_SR) is in the millimolar range. Using low-affinity Ca²⁺ indicators, several aspects of SR Ca²⁺ cycling have been investigated at the level of the isolated myocyte, including fractional SR Ca²⁺ release and the mechanisms of Ca²⁺ alternans^13,14. However, in order to fully understand the heterogeneous nature of SR Ca²⁺ cycling in the intact heart and the role of SR Ca²⁺ in spatially distinct arrhythmic phenomena, methods for imaging SR Ca²⁺ across the epicardial surface of the intact heart are required¹⁵.

This article describes methodology for dual optical mapping of free SR Ca²⁺ and V_m in the intact Langendorff-perfused rabbit heart with the low-affinity Ca²⁺ indicator Fluo-5N. In addition to revealing SR Ca²⁺ characteristics spatially across the epicardial surface of the heart, this approach has the advantage of simultaneous monitoring of V_m, allowing for investigations into the bidirectional relationship between V_m and SR Ca²⁺.

Protocol

所有涉及动物的程序由加州大学戴维斯分校的动物护理和使用委员会批准，并坚持以指南实验动物卫生的国家机构发布的管理和使用。 1.准备准备提前和存储改良台氏液2浓缩（25X）的股票在4℃下：（1）股票我（在MM：氯化钠3205， 氯化钙 32.5，氯化钾117.5，氢化钠2 PO 4 29.75， 氯化镁 26.25）和（2）股票II（毫米： 碳酸氢钠 500）?…

Representative Results

图1A示出了用于双重V M和SR 的 Ca 2+映射的光学配置的示意图。与此设置，有一个在V 米和SR 的 Ca 2+信号（图1B）的完整光谱分离。用于将Fluo-5N染料加载的双回路灌注系统的示意图示于图1C。图1D示出了心脏的灌注菜水平方向。代表性V M和过程中从对心脏的心外膜表面的不同位置连续起搏的SR 的 Ca 2+?…

Discussion

成功的关键的Fluo-5N染料加载是小容积循环灌流设置，其允许高的Fluo-5N浓度，而不需要大量的染料，加载时间（1小时）的长度，并执行装载在RT。如果载入在生理温度，细胞的酶活性迅速裂解当染料穿过细胞膜，捕集染料分子在细胞质中，而不是使它们穿过的SR膜的-AM标签进行。在RT，然而，酶活性被减慢和染料的足够量可以跨越两个细胞膜和SR膜-AM标记之前被裂解，捕捉在SR中的染料。

<p class="…

Materials

NaCl	Fisher Scientific	S271-1	Component of Tyrode's solution
CaCl2 (2H2O)	Fisher Scientific	C79-500	Component of Tyrode's solution
KCl	Fisher Scientific	S217-500	Component of Tyrode's solution
MgCl2 (6H2O)	Fisher Scientific	M33-500	Component of Tyrode's solution
NaH2PO4 (H2O)	Fisher Scientific	S369-500	Component of Tyrode's solution
NaHCO3	Fisher Scientific	S233-3	Component of Tyrode's solution
D-Glucose	Fisher Scientific	D16-1	Component of Tyrode's solution
95% O2 5% CO2	AirGas	carbogen	For oxygenation and pH of Tyrode's solution
Blebbistatin	Tocris Bioscience	1760	Excitation-contraction uncoupler
RH237	Biotium	61018	Voltage-sensitive dye
Fluo-5N AM	Invitrogen	F-26915	Low-affinity Ca2+ indicator; Alternative: Invitrogen F-14204; Loading must be performed at room temperature
Pluronic F127	Biotium	59004	For Ca2+ indicator loading; Warm until the solutiion is clear before use
Dimethyl sulphoxide (DMSO)	Sigma-Aldrich	D2650	For dissolving blebbistatin and dyes
Filter	EMD Millipore	NY1104700	11um in-line filter
Pressure Transducer	WPI	BLPR2	For measuring perfusion pressure
Transbridge Transducer Amplifier	WPI	SYS-TBM4M	For transducing/amplifing pressure signal; PowerLab may also be used with appropriate BioAmp
PowerLab 26T	ADInstruments		For continuous recording of pressure and ECG signals
THT Macroscope	SciMedia		Macroscopic optical setup. Details: 0.63x objective (NA=0.31), 2x condensing objective, resultant field of view = 3.1×3.1 cm, depth of focus = ~1.5mm
MiCam Ultima-L CMOS	SciMedia		Optical mapping cameras
Precision LED Spot Light	Mightex	PLS-0470-030-15-S	LED light source