High-resolution Optical Mapping of the Mouse Sino-atrial Node

Summary

Here, we present a protocol for optical mapping of electrical activity from the mouse right atrium and especially the sino-atrial node, at a high spatial and temporal resolution.

Abstract

Sino-atrial node (SAN) dysfunctions and associated complications constitute important causes of morbidity in patients with cardiac diseases. The development of novel pharmacological therapies to cure these patients relies on the thorough understanding of both normal physiology and pathophysiology of the SAN. Among the studies of cardiac pacemaking, the mouse SAN is widely used due to its feasibility for modifications in the expression of different genes that encode SAN ion channels or calcium handling proteins. Emerging evidence from electrophysiological and histological studies has also proved the representativeness and similarity of the mouse SAN structure and functions to larger mammals, including the presence of specialized conduction pathways from the SAN to the atrium and a complex pacemakers' hierarchy within the SAN. Recently, the technique of optical mapping has greatly facilitated the exploration and investigation of the origin of excitation and conduction within and from the mouse SAN, which in turn has extended the understanding of the SAN and benefited clinical treatments of SAN dysfunction associated diseases. In this manuscript, we have described in detail how to perform the optical mapping of the mouse SAN from the intact, Langendorff-perfused heart and from the isolated atrial preparation. This protocol is a useful tool to enhance the understanding of mouse SAN physiology and pathophysiology.

Introduction

Novel scientific breakthroughs that lead to leaps in the understanding of human physiology are often preceded by technological advances. Fluorescent optical mapping, for example, enables investigation of multiple physiological parameters in both cells and tissues.^1,2 It significantly improved our understanding of how the anatomical structure is associated with electrophysiological functions and dysfunctions. In the heart, the natural pacemaker and conduction systems such as the sinoatrial node (SAN) and the atrioventricular junction consist of nodal myocytes that are insulated by the surrounding atrial myocytes.^3-5 Such organization creates complex three-dimensional structures with specialized electrical properties. Both structural and functional remodeling of these pacemaker structures has been recognized to form significant electrophysiological heterogeneities.^6,7 Understanding the mechanism underlying how such heterogeneities result in SAN dysfunctions and atrial arrhythmogenesis will dramatically benefit the clinical treatment of these diseases. It requires a technique to visualize the propagation of electrical signals at the tissue level, such as optical mapping.

Recently, accumulating evidence has proven the advance of optical mapping in studies of atrial electrophysiology and pathology.² However, novel and rigorous studies are dependent on the accurate interpretation of experimental data, whose validity and stability rely on careful experiment protocols. Genetically modified mice are extensively used for research as animal models of human diseases including sick sinus syndrome, pacemaker abnormalities and atrial arrhythmias.^6,8-11 Thus, a combination of fluorescent optical mapping with transgenic mouse models provides a powerful tool to study cardiac electrical abnormalities associated with various pathologies. In this paper, we present a protocol for high-resolution optical mapping of the mouse SAN and atrium. Specifically, we discuss and compare different dye loading approaches, time effects on dye bleaching and heart rate stability during the experiment.

Protocol

All experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Pub. No. 80-23). All methods and protocols used in these studies have been approved by The University of Wisconsin Animal Care and Use Protocol Committee following the Guidelines for Care and Use of Laboratory Animals published by NIH (publication No. 85-23, revised 1996). All animals used in this study received humane care in compliance with the Guide for the Care and Use of Laboratory Animals.

1. Heart Removal and Langendorff Perfusion

1. Prior to heart isolation, warm Tyrode solution to 37 °C by using a water bath and a water jacket.
2. Anesthetize the mouse with 5% isoflurane/95% O₂.
3. Ensure appropriate level of anesthesia by checking the loss of pain reflex.
4. Perform thoracotomy. Open the chest by using curved 5.5" Mayo scissors and 5.5" Kelly hemostatic forceps to make a 1 cm cut on the front of the thorax.
   1. Quickly extract the heart from the chest (within 30 sec). Use 4" curved Iris forceps to grab the lung tissue and cut out the lung, thymus and heart together with the pericardium using 4.3" Iris scissors. Do not grab the heart directly.
   2. Wash it in oxygenated (95% O₂ / 5% CO₂), constant-temperature (36.8 ± 0.4°C) modified Tyrode's solution. Use the following solution composition (in mM): 128.2 NaCl, 4.7 KCl, 1.19 NaH₂PO₄, 1.05 MgCl₂, 1.3 CaCl₂, 20.0 NaHCO₃, and 11.1 glucose (pH 7.35 ± 0.05).
5. While bathed in the same solution, identify the lung, thymus, and fat tissue. Carefully dissect them from the heart. Use curved 3" Vannas-Tubingen scissors and 4.3" #5 forceps.
6. Then identify the aorta and cannulate it onto a custom-made 21 G cannula. Use two curved 4.3" #5B forceps.
7. After cannulation, simultaneously perfuse (through the cannula at a flow rate of ~5 ml/min; this should be adjusted based on the aortic pressure, see below) and superfuse (in the bath at a flow rate of ~50 ml/min) the heart with warmed and filtered Tyrode solution throughout the entire experiment. Pass the perfusion solution through an in-line 11 µm nylon filter to prevent clogging of coronary circulation.
8. Using a pressure transducer (or a pressure gauge) connected via a 3-way stopcock Luer lock to the perfusion line, monitor the aortic pressure and maintain it between 60 and 80 mmHg. Adjust perfusion speed if required to keep the aortic pressure within this range.

2. Optical Mapping of the SAN from the Langendorff-perfused Whole Heart

1. Instrumentation
   1. Place the heart horizontally and pin the ventricular apex to the silicon-coated bottom of the chamber using 0.1 mm diameter pins to prevent stream-induced movement during the experiment.¹²
   2. Insert a small (.012" I.D. x .005") silicon tube into the left ventricle (LV) via the pulmonary vein, the left atria (LA) and the mitral valve (MV). Fix the tube by a silk suture (4-0) to the sounding connective tissue.
   3. Position the heart with its posterior side facing up (Figure 1A, left panel).
   4. Pin the edge of the RA appendage (RAA) to the silicon bottom of the chamber using 0.1 mm diameter minutien pins. Adjust its level in order to make the posterior surface of the RA flat and allow it to be located the camera's focal plane. This will enable optical measurements from the maximal surface area of the atrium.
   5. Noose superior (SVC) and inferior (IVC) vena cava by a silk suture (4-0), stretch and pin the other end of the suture to the bottom of a silicon-coated chamber (see Figure 3A). Make sure the sutures do not block the optical field of view.
   6. Place the custom-made pacing electrode on the edge of the RAA. To make electrodes, use silicon coated 0.25 mm diameter silver wires, with 0.5 mm inter-electrode distance and devoid of silicon for a length of 1 mm at the pacing end. Then place two ECG 12 mm needle (29-gauge) monopolar electrodes near the base of the right and left ventricles. Place the ground ECG electrode near the apex of the ventricles .
2. Staining
   1. Since both fluorescent dyes and electro-mechanical uncoupler blebbistatin are light-sensitive, perform all the procedures described below in a dark room. First prepare voltage-sensitive dye RH-237 or di-4-ANNEPS as a stock solution, 1.25 mg/ml in dimethyl sulfoxide (2.5 mM), aliquot it (30 µl each) and store the aliquots at -20 °C.
   2. Dilute 5-10 µl of dye stock solution in 1 ml of warmed (37 °C) Tyrode solution and then inject it into the coronary perfusion line over a period of 5-7 min using an in-line Luer injection port.
   3. Prepare blebbistatin as a stock solution (2 mg/ml in dimethyl sulfoxide, 6.8 mM) in advance and store it at 4°C.
   4. After 20 min of stabilization, add 0.5 ml of warmed (37 °C) blebbistatin in the perfusate and dilute 0.1 ml of blebbistatin in 1 ml of warmed (37 °C) Tyrode solution. Then inject into the coronary perfusion line over a period of 5-7 min using an in-line Luer injection port.

3. Optical mapping of the SAN from the Isolated Atrial Preparation

1. Instrumentation
   1. For the isolated atrial preparation, isolate and cannulate the heart as described above in steps 1.1-1.5 for whole-heart preparation.
   2. Dissect the ventricles away from the anterior side. See Figure 1A, right panel, cut #1 for details.
   3. Open the RA by cutting through the tricuspid valve (TV) along the TV-SVC axis. See Figure 1A, right panel, cut #2 for details.
   4. Cut the medial limb of the crista terminalis to open the RAA. See Figure 1A, right panel, cut #3.
   5. Open the anterior atrial free wall by performing a cut from the midline of the previous cut#3 to the edge of the right bottom corner of the RAA, flatten and pin the atrial free wall to the bottom of a silicon-coated chamber. See the direction shown by the hollow arrow in Figure 1A, cut #4. Preserve a rim of ventricular tissue for pinning the preparation to prevent damage to the atria.
   6. Similarly, open LA by cutting through the MV along the MV-upper corner of the LA appendage (LAA).
   7. To open the LAA, cut from the middle of the opened LAA, through the anterior atrial free wall until near a middle rim of the LAA.
   8. Open the anterior atrial free wall along the same direction, then flatten and pin it to the bottom of a Sylgard-coated chamber.
   9. Partially remove the interatrial septal tissue. This will reduce scattering of the optical signal from tissue that is not in focus. Therefore, both the LA and RA as well as the SAN and atrio-ventricular junction (AVJ) are accessible in this preparation (Figure 1B).
   10. Lift up the final preparation by about 0.5 mm from the bottom of the silicon-coated chamber in order to allow superfusion from both epicardial and endocardial surfaces.
   11. Superfuse the preparation with warmed (37 °C) Tyrode solution at a constant rate of ~12 ml/min for a focused flow located near the SVC and ~30 ml/min for a bath superfusion.
   12. Place the custom-made pacing Ag/AgCl₂ electrode (0.25 mm diameter) on the edge of the dissected RAA. Then place two ECG 12 mm needle (29G) electrodes (monopolar) near the RAA and LAA, respectively. Place the ground ECG electrode near the AVJ.
2. Staining
   1. Perform a direct application of the voltage-sensitive dye (RH-237 or di-4-ANNEPS). For this, dilute 1-2 µl of the dye stock solution in 1 mm of warmed (37 °C) Tyrode solution and slowly release the diluted dye on the surface of the preparation by using a 1 mm pipette.
   2. Alternatively, perform atrial staining with the voltage-sensitive dye through a coronary perfusion of the Langendorff-perfused heart similar to that described above for the whole-heart preparation (steps 2.2.1-2.2.2).
      1. After the coronary perfusion staining, isolate the atrial preparation as described. Perform additional surface staining when needed to reach a satisfactory fluorescence level. Quick atrial isolation does not bleach the dye and does not affect the quality of staining.
   3. Immobilize the preparation with the electro-mechanical uncoupler, blebbistatin. Dilute 3-5 µl blebbistatin stock solution in the warmed (37 °C) Tyrode solution and slowly release the diluted blebbistatin on the surface of the preparation and surrounding solution. Since blebbistatin has been shown to be light sensitive,^13,14 avoid long exposure to light during preparation and staining.
   4. Perform additional blebbistatin application as much as needed to suppress contraction. Normally up to 3 additional applications (3-5 µl per each application) can be used to suppress the motion artifact sufficiently in order to accurately reconstruct SAN and atrial activation.
   5. Add additional 0.5 ml of warmed blebbistatin in the perfusate. During this procedure, pause the superfusion for 30 sec to allow the dye/blebbistatin to stain the atrial tissue.

4. Optical Mapping Set Up

NOTE: A detailed description of the optical mapping system is provided elsewhere.¹²

1. Use a camera with a temporal resolution of 2,000 frames/sec or higher, and a series of condenser lenses to reach spatial resolution of 100 µm/pixel or higher. This is required to reconstruct SAN activation and intranodal propagation.
2. To reduce motion artifact from the vibrating solution, fix a small cover glass on the surface of the solution over the heart/isolated atrial preparation.
3. Use excitation light (520 ± 45 nm wavelength) provided by a constant-current, low-noise halogen lamp. Direct the filtered light beam onto the preparation by using a flexible bifurcated light guide.
4. Filter the emitted fluorescence by a long-pass filter (>715 nm). Collect, digitize and save the acquired fluorescent signal on a computer using software provided by the camera manufacturer.

5. Data Processing

NOTE: Optical mapping data is collected and stored as a series of matrices of fluorescent intensity at different time points. Each pixel represents a measure of fluorescent intensity collected from a specific location on the tissue preparation at a specific time point. Background fluorescence is automatically removed per pixel to allow for better visualization of fluorescence changes produced by membrane voltage changes and inverted to correspond with action potentials (APs) measured by microelectrode systems. Details of the different steps in processing optical imaging data, including image segmentation, spatial filtering, temporal filtering, and baseline drift removal, are provided in the focused review.¹⁵

1. Manually or by using the special algorithm (for example, a thresholding or edge detection)¹⁵ to determine tissue boundaries, create a binary mask of 1 (included pixels) and 0 (excluded pixels) and apply the mask to each frame in the sequence. This process creates a binary image that highlights areas of interest for further processing.
2. To improve signal-to-noise ratio of the optical data, filter the signals within the selected areas of interest. For this, use spatial (i.e. averaging of neighboring fluorescent pixels as defined by a desired convolution bin or kernel, for instance 3 x 3 or, for noisy data, 5 x 5) and/or temporal filtering (for example, Butterworth, Chebyshev type 1, Chebyshev type 2, Elliptic etc.) (Figure 2). Keep in mind the possibility of filtered-induced artifacts when interpreting the data. For more details, see review.¹⁵
3. Remove drift of baseline in optical recordings when needed (by using high-pass filtering or polynomial fitting to the original signal) and then normalize each pixel signal from 0 (minimum fluorescence) to 1 (maximum fluorescence).
4. Select a time window that encompasses the activation times of all the pixels for a single AP propagation. Assign each pixel its activation time as the time of maximum upstroke derivative (dF/dt_max, where F is fluorescence intensity). Using all pixel activation times, reconstruct the isochronal activation map. Each isochron will thus show the pixels activated at the same time.
5. To reconstruct the AP duration (APD) distribution map, for each pixel calculate the duration between the activation time and the time at a specific level of repolarization (for example, at 80% of repolarization, APD₈₀). Using all pixel APD values, reconstruct the APD distribution isochronal map. Each isochron will thus show the pixels with the same APD.
6. To calculate conduction velocity for AP propagation, fit a surface of the preparation to the activation time data calculated in 5.4. For this, use polynomial surface fitting or local kernel surface smoothing and then calculate local conduction velocity vectors from the gradient of the fitted surface.
7. To create the repolarization map, assign each pixel its repolarization time, defined as the maximum second derivative (d²F/dt²) of the optical signal at the end of the OAP, or the time of 90% repolarization.

Results

Optical Mapping of the Intact SAN from the Langendorff-perfused Heart
A typical example of an RA activation contour map reconstructed for spontaneous sinus rhythm is shown in Figure 3 for a Langendorff-perfused mouse heart. The early activation point is located within the intercaval region near the SVC where the SAN is anatomically defined.^3,16 Two RA activation contour maps acquired at 1.0 and 0.5 msec sampling rate are shown in ...

Discussion

Here, we presented two types of mouse SAN preparations: 1) intact SAN in the Langendorff-perfused whole heart, and 2) SAN in the isolated, opened atrial preparation. These two types of preparation serve different experimental purposes. In the Langendorff-perfused whole heart preparation, the intact atrial structure is preserved which makes it possible to study complex atrial arrhythmias such as atrial fibrillation as well as interactions between the SAN and atrium during reentrant tachyarrhythmias.⁶ In contras...

Materials

Name	Company	Catalog Number	Comments
water jacket	Radnoti	1660 Series Tissue Bath for Large Organ or Single Cell Isolation Procedures
water bath / circulator	Fisher Scientific	1016S
pressure amplifier	AD Instruments	MLT0670
EMD Millipore Nylon Net Filters	Fisher Scientific	NY1102500
Pressure transducer	AD Instruments	MLT0670
Stainless Steel Minutien Pins - 0.1mm Diameter	Fine Science Tools	26002-10
Perfusion pump	World Precision Instruments	PERIPRO-4LS
Superfusion pump	World Precision Instruments	PERIPRO-4HS
Vannas Tubingen scissors	World Precision Instruments	503379
Dumont forceps	World Precision Instruments	501201, 500085
Mayo scissors	World Precision Instruments	501750
Kelly hemostatic forceps	World Precision Instruments	501241
Iris forceps	World Precision Instruments	15917
Iris scissors	World Precision Instruments	501263
ECG 12 mm needle (29-gauge) electrodes (monopolar)	AD Instruments	MLA1203
in-line Luer injection port	Ibidi	10820
Ultima-L CMOS camera	SciMedia	MiCAM-05
halogen lamp	Moritex USA Inc	MHAB-150W
NaCl	Fisher Scientific	S271-1
CaCl2 (2H2O)	Fisher Scientific	C79-500
KCl	Fisher Scientific	S217-500
MgCl2 (6H2O)	Fisher Scientific	M33-500
NaH2PO4 (H2O)	Fisher Scientific	S369-500
NaHCO3	Fisher Scientific	S233-3
D-Glucose	Fisher Scientific	D16-1
Blebbistatin	Tocris Bioscience	1760
RH237	ThermoFisher Scientific	S1109
Dimethyl sulphoxide (DMSO)	Sigma-Aldrich	D2650