Optical Mapping of Intra-Sarcoplasmic Reticulum Ca2+ and Transmembrane Potential in the Langendorff-perfused Rabbit Heart

Summary

This article describes the detailed protocol and equipment necessary for dual optical mapping of transmembrane potential (V_m) and free intra-sarcoplasmic reticulum (SR) Ca²⁺ in the Langendorff-perfused rabbit heart. This method allows for direct observation and quantification of V_m and SR Ca²⁺ dynamics in the intact heart.

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

All procedures involving animals were approved by the Animal Care and Use Committee of the University of California, Davis, and adhered to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health.

1. Preparation

1. Prepare two concentrated (25X) stocks of modified Tyrode’s solution in advance and store at 4 °C: (1) Stock I (in mM: NaCl 3,205, CaCl₂ 32.5, KCl 117.5, NaH₂PO₄ 29.75, MgCl₂ 26.25) and (2) Stock II (in mM: NaHCO₃ 500).
   1. Freshly prepare 2 L Tyrode's solution of the following composition (in mM): NaCl 128.2, CaCl₂ 1.3, KCl 4.7, MgCl₂ 1.05, NaH₂PO₄ 1.19, NaHCO₃ 20 and glucose 11.1 by combining 1840 ml of deionized (DI) water, 80 ml Stock I, 80 ml of Stock II, and 4 g of glucose. Do not directly mix Stock I and Stock II; add them to 1,840 ml DI to avoid precipitation.
2. Prepare stock solution of the excitation-contraction uncoupler blebbistatin at 10 mg/ml in anhydrous di-methylsulfoxide (DMSO) in advance and store the stock solution at 4 °C.
3. Prepare stock solutions of fluorescent dyes: (1) Voltage-sensitive dye RH237 (1 mg/ml in DMSO) and (2) Low-affinity calcium indicator Fluo-5N AM (2 mg/ml in DMSO) according to manufacturer’s recommendations. Use the preparations immediately to avoid decomposition with subsequent loss of loading capacity.
4. Prime the recirculating Langendorff perfusion system with oxygenated (95% O₂, 5% CO₂) Tyrode’s solution. Adjust the flow of O₂/CO₂ to keep the pH of the Tyrode’s solution at 7.4 ± 0.05. Use an in-line nylon woven net filter (pore size: 11 µm) to continuously filter the perfusate.
   1. Prime the perfusion system at RT, as subsequent loading of Fluo-5N AM is performed at RT. For Fluo-5N AM loading, use a small volume of recirculating perfusate (~ 150 ml). After dye loading, use a larger volume of perfusate (1 - 2 L, see Figure 1C).
5. Turn on data acquisition system and prepare for continuous monitoring of ECG and perfusion pressure. Prepare pressure-monitoring system: Connect a pressure transducer to the perfusion line and monitor the perfusion pressure with a transbridge amplifier. Adjust the baseline perfusion pressure to 0 mmHg when the heart is not attached to the perfusion system.
6. Align the optical mapping cameras to ensure spatial alignment between the V_m and SR Ca²⁺ signals.
   1. First, focus the cameras by placing a ruler or an object with text into the perfusion dish. Turn the mapping cameras into a live view mode and adjust the focus until the text is crisp and clear. Acquire a single frame image from each camera.
   2. Overlay these images and adjust the transparency of the top image so that both images are visible. The text in the images should overlap exactly. If not perfectly aligned, adjust the angle of the dichroic mirror or the position of each camera until the two images are aligned.

2. Harvesting, Perfusion, and Dye-loading of Rabbit Heart

1. Secure the rabbit in an approved rabbit restrainer. Deeply anesthetize via an intravenous injection (marginal ear vein) of sodium pentobarbital (50 mg/kg) and heparin (1,000 IU).
   1. When the rabbit displays lack of nociceptive reflexes, make a midline skin incision to expose the sternum and ribs. Cut through the sternum with blunt-tip surgical scissors from the xyphoid to the manubrium. Take care not to damage the heart while opening the sternum. Spread the ribs to expose the heart.
   2. Quickly excise the entire heart-lung block by rapidly cutting all vessels and connective tissue. Immediately place the heart-lung block into ice-cold Tyrode’s solution.
2. Locate the aorta for retrograde perfusion. Cut the ascending aorta just proximal to the three branches of the aortic arch. Cannulate the aorta to an 8 G cannula connected to the perfusion system. Use a piece of USP 0 silk suture to secure the aorta onto the cannula.
3. Carefully dissect the trachea, lungs, and epicardial fat from the heart. With a sharp forceps and dissection scissors, locate the mitral valve and carefully damage or remove one leaflet to prevent solution congestion in the left ventricle.
4. Submerge the heart in the glass-jacketed perfusion chamber horizontally with the anterior surface of the heart facing up for imaging. Secure the cannula to a piece of Sylgard with U-shaped pins onto the silicon bottom of the dish to prevent movement of the heart during mapping (Figure 1D). If desired, insert a small insect pin at the apex of the heart for stabilization.
5. Position electrocardiogram (ECG) electrodes in the bath on the right and left side of the heart. Fully submerge the electrodes in the bath and ensure they are not in contact with the heart surface to provide a volume-conducted ECG analogous to a Lead I configuration. Verify a normal Lead I ECG morphology. Adjust the flow (25 - 35 ml/min) to ensure that the aortic pressure is maintained at 60 - 70 mmHg.
6. Turn off the room lights and add 0.3 - 0.6 ml of blebbistatin stock solution (step 1.2) to the perfusate (final concentration 10 - 20 µM).
   1. Turn off room lights to avoid photoinactivation of blebbistatin. If necessary, a small spotlight or headlamp can be used to provide task lighting.
      Note: Blebbistatin is a myosin ATPase inhibitor and an excitation-contraction uncoupler. Because Fluo-5N loading requires extended (60 min) RT (hypothermia) conditions (see steps 2.8 – 2.10), cardiac energy production may be compromised. Therefore, addition of blebbistatin into the perfusate prior to dye loading may reduce the energy demand¹⁶ and eliminate motion artifacts during subsequent optical recordings¹⁷.
7. When contraction of the heart has ceased (10 - 15 min, verified on the aortic pressure recording), switch to the small-volume recirculating perfusion system (see step 1.4.1).
8. Prepare and load Fluo-5N AM loading solution: Add 0.25 ml of Fluo-5N AM stock solution (step 1.3) to 0.25 ml warm 20% Pluronic F127. Add 0.5 ml warm Tyrode’s solution to the mixture, mix well, and add to the small-volume recirculating perfusion system.
9. Continuously monitor perfusion pressure and ECG and adjust the flow if necessary. Dye loading takes approximately 1 hr at RT. 45 min after loading has begun, turn on the circulating water bath to begin warming the perfusion system and perfusate to 37 °C.
10. After 60 min of Fluo-5N AM loading, switch the perfusion to the larger volume recirculating perfusion system (see step 1.4).
11. Dilute 50 µl of voltage sensitive dye RH237 stock solution (step 1.3) in 1 ml of warm Tyrode's solution and add slowly (over ~ 5 min) into an injection port proximal to the cannula.

3. Optical Mapping

1. During the final moments of dye loading, position the heart and focus the optical mapping cameras to ensure the appropriate field of view for the experiment.
2. If desired, place a bipolar pacing electrode on the epicardial surface of the heart for pacing.
3. Place a plastic or glass cover slip on the surface of the perfusion chamber to reduce motion artifact on the liquid surface that may be present due to recirculation of the perfusate.
   Note: As an alternative to a cover slip, use a clean plastic 50 mm Petri dish cover.
4. Focus the light guides to uniformly illuminate the surface of the heart with excitation light. Use blue light emitting diode (LED) light sources and further filter the light with a 475 - 495 nm bandpass filter (Figure 1A).
5. Collect emitted fluorescence with a macroscope and two complementary metal-oxide-semiconductor (CMOS) optical mapping cameras. Split the emitted light with a dichroic mirror at 545 nm.
   1. Long-pass filter the longer wavelength moiety, containing the V_m signal, at 700 nm. Band-pass filter the shorter wavelength moiety, containing the SR Ca²⁺ signal, from 502 - 534 nm (Figure 1A). Set the frequency of optical data acquisition to 0.5 - 1 kHz.
6. For each optical recording, first initiate the desired pacing protocol, turn on the excitation light, and collect 1 - 4 sec of data. If desired, synchronize the pacing protocols and data acquisition.
   Note: It is not possible to re-load the Fluo-5N AM, therefore the signal-to-noise ratio (SNR) will decrease throughout the experiment due to dye leakage from the SR. Limit experimental protocol to 1 - 2 hr to ensure high SNR values.
7. Following the experiment, wash the perfusion system in the sequence of DI water, 70% reagent alcohol, and again with DI water.
8. Filter each dataset with a spatial Gaussian filter (radius 3 pixels) using commercially available software packages according to the manufacturer’s protocol.
9. If necessary, spatially align the V_m and SR Ca²⁺ data sets. As in Step 1.6, overlay images from each camera and verify that the anatomical structures of the heart (i.e., coronary vasculature, edges of the atria or ventricles) or other items in the field of view (i.e., cannula, pacing electrode) exactly overlap. If not, spatial alignment of the datasets is necessary.
10. Shift the top transparent image in the x- and y-direction until exact overlap is achieved, making note of the number of pixels the image must be shifted in each direction. The entire dataset corresponding to the top image (either V_m or SR Ca²⁺) must then be shifted by the determined number of pixels in each direction to assure precise alignment between the V_m and SR Ca²⁺ datasets.

Results

Figure 1A shows a schematic diagram of the optical configuration for dual V_m and SR Ca²⁺ mapping. With this setup, there is complete spectral separation of the V_m and SR Ca²⁺ signals (Figure 1B). A diagram of the dual-loop perfusion system used for Fluo-5N dye loading is illustrated in Figure 1C. Figure 1D shows the horizontal orientation of the heart in the perfusion dish. Representative V_m and SR C...

Discussion

The keys to successful Fluo-5N dye loading are the small-volume recirculating perfusion setup, which allows for a high Fluo-5N concentration without the need for large amounts of dye, the length of loading time (1 hr), and performing the loading at RT. If loading is performed at physiological temperatures, cellular enzymatic activity quickly cleaves the –AM tag when the dye crosses the cell membrane, trapping the dye molecules in the cytosol and not allowing them to cross the SR membrane. At RT, however, enzymatic ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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 solution is clear before use
Dimethyl sulphoxide (DMSO)	Sigma-Aldrich	D2650	For dissolving blebbistatin and dyes
Filter	EMD Millipore	NY1104700	11 μm 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 cm x 3.1 cm, depth of focus = ~ 1.5 mm
MiCam Ultima-L CMOS	SciMedia		Optical mapping cameras
Precision LED Spot Light	Mightex	PLS-0470-030-15-S	LED light source