Whole-Mount Immunofluorescence Staining, Confocal Imaging and 3D Reconstruction of the Sinoatrial and Atrioventricular Node in the Mouse

Summary

We provide a step-by-step protocol for whole-mount immunofluorescence staining of the sinoatrial node (SAN) and atrioventricular node (AVN) in murine hearts.

Abstract

The electrical signal physiologically generated by pacemaker cells in the sinoatrial node (SAN) is conducted through the conduction system, which includes the atrioventricular node (AVN), to allow excitation and contraction of the whole heart. Any dysfunction of either SAN or AVN results in arrhythmias, indicating their fundamental role in electrophysiology and arrhythmogenesis. Mouse models are widely used in arrhythmia research, but the specific investigation of SAN and AVN remains challenging.

The SAN is located at the junction of the crista terminalis with the superior vena cava and AVN is located at the apex of the triangle of Koch, formed by the orifice of the coronary sinus, the tricuspid annulus, and the tendon of Todaro. However, due to the small size, visualization by conventional histology remains challenging and it does not allow the study of SAN and AVN within their 3D environment.

Here we describe a whole-mount immunofluorescence approach that allows the local visualization of labelled mouse SAN and AVN. Whole-mount immunofluorescence staining is intended for smaller sections of tissue without the need for manual sectioning. To this purpose, the mouse heart is dissected, with unwanted tissue removed, followed by fixation, permeabilization and blocking. Cells of the conduction system within SAN and AVN are then stained with an anti-HCN4 antibody. Confocal laser scanning microscopy and image processing allow differentiation between nodal cells and working cardiomyocytes, and to clearly localize SAN and AVN. Furthermore, additional antibodies can be combined to label other cell types as well, such as nerve fibers.

Compared to conventional immunohistology, whole-mount immunofluorescence staining preserves the anatomical integrity of the cardiac conduction system, thus allowing the investigation of AVN; especially so into their anatomy and interactions with the surrounding working myocardium and non-myocyte cells.

Introduction

Arrhythmias are common diseases affecting millions of people, and are the cause of significant morbidity and mortality worldwide. Despite enormous advances in treatment and prevention, such as the development of cardiac pacemakers, treatment of arrhythmias remains challenging, primarily due to the very limited knowledge regarding underlying disease mechanisms^1,2,3. A better understanding of both the normal electrophysiology and the pathophysiology of arrhythmias may help to develop novel, innovative and causal treatment strategies in the future. Additionally, to comprehensively study arrhythmogenesis, it is important to localize and visualize the specific cardiac conduction system in animal models such as the mouse, as mice are widely used in electrophysiology research.

The major parts of the cardiac conduction system are the sinoatrial node (SAN), where the electrical impulse is generated in specialized pacemaker cells, and the atrioventricular node (AVN), which is the only electrical connection between the atria and the ventricles⁴. Whenever the electrophysiological properties of SAN and AVN are altered, arrhythmias such as sick sinus syndrome or atrioventricular block can occur which may lead to hemodynamic deterioration, syncope and even death, and thus underline the essential role of both SAN and AVN in electrophysiology and arrhythmogenesis⁵.

Comprehensive studies on SAN or AVN require a precise localization and visualization of both structures, ideally within their physiologic environment. However, due to their small size and location within the working myocardium, without establishing a clear macroscopically visible structure, studying the anatomy and electrophysiology of SAN and AVN is challenging. Anatomical landmarks can be used to roughly identify the region that contains SAN and AVN^6,7,8. In brief, SAN is located in the inter-caval region of the right atrium adjacent to the muscular crista terminalis (CT), AVN is located within the triangle of Koch established by the tricuspid valve, the ostium of the coronary sinus and the tendon of Todaro. Thus far, these anatomical landmarks were mainly used to localize, remove and then study SAN and AVN as individual structures (e.g., by conventional histology). To better understand the complex electrophysiology of SAN and AVN (e.g., regulatory effects of adjacent cells of the working myocardium), however, studying the conduction systems within the physiologic 3D environment is necessary.

Whole-mount immunofluorescence staining is a method that is used to study anatomical structures in situ while preserving the integrity of the surrounding tissue⁹. Taking advantage of confocal microscopy and image analysis software, SAN and AVN can be visualized with fluorescently labeled antibodies targeting ion channels specifically expressed in these regions.

This following protocol explains the necessary steps to perform a well-established whole-mount staining method for SAN and AVN microscope localization and visualization. Specifically, this protocol describes how (1) to localize SAN and AVN by anatomical landmarks to prepare these samples for staining and microscopy analysis (2) to perform whole-mount immunofluorescence staining of the reference markers HCN4 and Cx43 (3) to prepare SAN and AVN samples for confocal microscopy (4) to perform confocal imaging of SAN and AVN. We also describe how this protocol can be modified to include additional staining of surrounding working myocardium or non-myocyte cells such as autonomous nerve fibers which allows a thorough investigation of the cardiac conduction system within the heart.

Access restricted. Please log in or start a trial to view this content.

Protocol

Animal care and all experimental procedures were conducted in accordance with the guidelines of the Animal Care and Ethics committee of the University of Munich, and all the procedures undertaken on mice were approved by the Government of Bavaria, Munich, Germany (ROB-55.2-2532.Vet_02-16-106, ROB-55.2-2532.Vet_02-19-86). C57BL6/J mice were purchased from Jackson Laboratory.

NOTE: Figure 1 shows the instruments needed for the experiment. Figure 2 shows an illustration of the gross cardiac anatomy. Figure 3 shows the location of SAN and AVN in an adult mouse heart. Figure 4 shows the prepared sample loaded on the confocal microscopy.

1. Preparations

1. Prepare a 4% formaldehyde solution (4% PFA) by diluting 10 mL of 16% formaldehyde solution in 30 mL of PBS.
2. Prepare a 15% sucrose solution and 30% sucrose solution by dissolving 15 g and 30 g of sucrose powder into 100 mL PBS, respectively. After the sucrose powder is fully dissolved, filter the solution using 0.2 µm syringe filter before storing at 4 °C.
3. Prepare a 3%-4% agarose gel, add 3 g or 4 g agarose powder and 100 mL of 1x TAE (diluted from 50x TAE stock solution) into a beaker. Place the beaker on a magnetic stirrer and boil it until the agarose is completely dissolved.
4. Prepare the dissection dish by gently pouring 30 mL of agarose gel (3%-4%) in a 100 mm diameter Petri dish. Leave the Petri dish on the bench at room temperature to cool down and harden.
5. Prepare blocking solution and washing solution according to the recipes provided in the Table 1. Prepare all of the solutions prepared at the day of use. Long term storage is not recommended.
6. Prepare the antibody solutions by diluting them in cold (4 °C) 1x PBS, protect the dilution from light (e.g., by wrapping aluminum foil around) and keep dilutions on ice until used. Dilute the antibodies shortly before incubation. Avoid leaving the diluted antibodies at room temperature.
   NOTE: The appropriate antibody dilution should be tested with comparable tissue samples by performing a conventional immunofluorescent staining. Here, we tested the antibodies by staining frozen sections cut at a thickness of 10 µm.

2. Organ harvest and tissue preparation

1. Anesthetize the mouse by placing it into an incubation chamber connected to an isoflurane vaporizer. Set the vaporizer to deliver 4-5% isoflurane (96-95% oxygen, respectively).
   NOTE: Full anesthesia is confirmed by the loss of the postural reaction and righting reflex by gently rolling the chamber until the mouse is placed on its back.
2. Put the mouse in a supine position on the surgical table. Place the mouse nose into an anesthesia mask connected to a modified Bain circuit with its inner tube connected to the isoflurane vaporizer. To maintain anesthesia, use 1-2% isoflurane in oxygen at a flow rate of 1 L/min. Scavenge away excess anesthetic vapor from the mouse via the outer tube of the mask and draw through a canister of activated charcoal, which absorbs the excess anesthetic gas.
3. When full anesthesia is achieved, inject fentanyl for analgesia (0.1 µg/25 g body weight i.p.).
4. When the toe-pinch reflex is undetectable, make a clear cut from the jugulum to the symphysis using iris scissors to remove fur and skin. Make another cut from left to right underneath the ribs using iris scissors to carefully open the abdomen.
5. Life the xiphoid a little bit using curved forceps to allow cutting the diaphragm from left to right without injuring any organs. Cut the rib cage in a medial axillary line on both sides using iris scissors to flip it cranially and to allow access to the heart.
6. Cut the inferior vena cava and descending thoracic aorta at the level of the diaphragm using iris scissors. Puncture the heart with a 27 G needlein the area of the apex and then carefully push the needle into the left ventricle (LV). Gently inject 5-10 mL of ice-cold PBS into the LV to perfuse the heart.
   NOTE: The color of the heart should turn from red to gray indicating successful perfusion with PBS.
7. Carefully lift the apex of the heart using a tweezer allowing to cut the large vessels and to remove the heart.
8. Excise the heart by cutting the large arteries and veins as far away from the heart as possible to avoid any damage to the superior vena cava (SVC). Conserve the SVC since it will serve as important landmark during later processing.
9. After heart removal, turn off the isoflurane vaporizer.
10. Put the heart into a dissection dish filled with ice cold PBS under the dissecting microscope. After determination of the left/right and front/back of the heart, turn the heart around with the front of the heart at the bottom of the dish (to expose the large vessels that are located posterior).
11. Immobilize the heart by putting little pinsthrough the apex and the left atrial appendage (LAA) into the agarose at the bottom of the dissection dish (Figure 2A). Using fine tweezers and scissors, carefully remove non-cardiac tissue around the SVC and inferior vena cava (IVC) (e.g., lungs, fat, pericardium) to expose the inter-caval region (Figure 3A).
12. Remove the majority of the ventricles by cutting parallel the groove between the ventricles and the atria with the micro scissor. Preserve a small part of the ventricular tissue for the later loading on the Plexiglas ring.
13. For fixation and dehydration, put the sample (containing the atria, SVC and IVC) in 4% PFA overnight at 4 °C.
14. The next day, transfer the heart to 15% sucrose solution for 24 hours at 4 °C.
15. The next day, transfer the heart to 30% sucrose solution for 24 hours at 4 °C.
    NOTE: For the fixation and dehydration in step 2.13, 2.14 and 2.15, the samples are left at 4°C without further stirring or rocking.

3. Whole-mount immunofluorescence staining

1. Wash the heart in 1% Triton X-100 diluted in PBS and block and permeabilize in blocking solution (Table 1) overnight at 4 °C.
2. Place the heart in a 1.5 mL tube and incubate with rabbit anti-mouse connexin-43 (dilution of 1:200) and rat anti-mouse HCN4 (dilution of 1:200) antibodies diluted with blocking solution for 7 days at 4 °C.
   NOTE: The optimal concentration of primary antibodies should be tested before using the datasheets as orientation. Other antigens of interest could also be stained in this step, as long as the host species of the primary antigen is different from the other ones. A higher concentration of Triton X-100 may help obtain more efficient antibody staining as demonstrated before¹⁰, but concentration might be determined individually.
3. After 7 days, remove the solution containing primary antibodies using a pipetteand wash the heart with 1%Triton X-100 solution 3 times (each time for 1 h at room temperature on the orbital shaker).
4. After washing, incubate the heart = with Alexa Fluor 488 goat anti-rat IgG (dilution of 1:200) and Alexa Fluor 647 goat anti-rabbit IgG (dilution of 1:200) for 7 days at 4 °C.
5. After 7 days, remove all the solution containing the secondary antibodies using a pipette. Then wash the heart using washing solution (Table 1) 3 times (each time for 1 h at room temperature on the orbital shaker).
6. To stain nuclei, incubate the heart in DAPI solution (10 µg/mL) overnight at 4 °C.
7. On the next day, wash the heart with washing solution 3 times (each time for 1 h at room temperature on the orbital shaker).
   NOTE: For the blocking, permeabilization, antibodies incubation and DAPI staining in step 3.1, 3.2, 3.4 and 3.6, leave the samples at steady state in the 4 °C, no stirring is required. The stained tissue could be preserved fully covered with washing solution at 4 °C and protected from light for a few days until imaging at the confocal microscope.

4. Confocal microscopy

1. Prepare Plexiglas rings and fill with plasticine (Figure 1). In the center of the plasticine a little groove is formed in the center for loading the heart. Make a shallow groove, as this would be easier to acquire a flat imaging plane, and to adjust the space and avoid air bubbles between the sample and the coverslips in step 4.4.
2. Use the same heart sample for the imaging of both SAN and AVN sequentially. For SAN imaging, directly load the heart whereas for AVN imaging, perform microdissection before.
   1. SAN imaging
      1. Identify the SAN by anatomical landmarks: it is located on the dorsal side of the heart within the inter-caval region (the region between the superior and inferior vena cava). The crista terminalis (CT) is the muscle streak between the SAN and RAA.
      2. Place the heart into the plasticine groove with the back of the heart facing up. Add PBS onto the heart to displace all air within the cavity until the heart is fully covered with PBS (usually a few drops of PBS are sufficient).
      3. Under the dissection microscope, gently press the RAA, LAA and remaining parts of the left ventricle into the plasticine to fix the heart. Make sure that the whole inter-caval region and the crista terminalis could be clearly seen (not covered by plasticine). The area that will be imaged is shown in Figure 3A.
   2. AVN imaging
      1. After finish the imaging of SAN, recollect the same sample and subsequently use for the AVN imaging. For AVN microdissection, place the heart = on a dissection dish under the dissection microscope.
      2. Orient the heart with the right side facing up (including the remaining parts of the right ventricle). Put pins through the remaining part of the LV free wall (which is now at the bottom) to immobilize the tissue (Figure 2B).
      3. Cut the remaining RV free wall upwards through the tricuspid valve and superior vena cava. Then flip the RV and RA away to expose interventricular and interatrial septum. (Figure 3B)
         NOTE: Pay attention not to damage the coronary sinus (CS), as it is an important anatomical landmark to find the triangle of Koch which then allows to localize the AVN. The CS runs transversely in the left atrioventricular groove on the posterior side of the heart, and the CS orifice opening is located between IVC and the tricuspid valve at the inferior part of the interatrial septum (Figure 3A and B).
      4. Identify the triangle of Koch that can be found on the endocardial surface of the right atrium, bordered anteriorly by the hinge-line of the septal leaflet of the tricuspid valve (TV), and posteriorly by the tendon of Todaro. The base is formed by the orifice of the coronary sinus (Figure 3B). Since this is the target region for AVN imaging, it needs to be clearly visible.
      5. Transfer the heart into the plasticine groove within the Plexiglas ring with the triangle of Koch clearly being exposed (i.e. not covered by plasticine). Gently press the tissue around the Koch triangle into the plasticine to fix the sample. Add PBS onto the heart to displace all air within the cavity until the heart is fully covered with PBS (usually a few drops of PBS are sufficient).
3. Apply silicone to the edges of the Plexiglas rings to allow covering the hearts loaded within the plasticine-filled Plexiglas rings with cover slips.
4. Gently press the back side of the plasticine to squeeze out parts of the PBS and to attach the heart to the coverslip while avoiding any air bubbles within the imaging area.
   NOTE: Make sure that the regions of interest are not folded and covered during pressing the back side of the plasticine. A flat imaging plane without sample overcompression is necessary for conserving the anatomy and for proper confocal imaging of the samples. For SAN, it is important to make sure the inter-caval region is clearly exposed. For AVN, the triangle of Koch should be fully exposed.
5. Put the whole-mount staining samples up-side down on the platform of the confocal microscope. The exposed SAN/AVN attached to the cover slip is now on the bottom of the microscope platform (Figure 4).
   NOTE: To take images, we use the Carl Zeiss LSM800 with Airyscan Unit and the software ZEN 2.3 SP1 black.
6. Choose plate "BP420-480 + LP605" for the excitation of Alexa Fluor 647 conjugated anti-HCN4. Vary the master gain from 650-750.
7. Take an overview image of the whole SAN and AVN region by using Tile Scan function. Then select the HCN4-positive region by clicking and adding a square on the overview image around the area of interest that will be scanned.
8. Check the parameters, including plates and master gain for the remaining channels (Alexa Fluor 488 and DAPI) of the confocal microscope and set as described in step 4.6.
9. Slowly adjust the focus from top to bottom of the sample to preview the whole sample and to set the First and Last for the Z-stack range. Set an optimal interval for the Z-stack based on the thickness of the optical sample. We use an 20x objective, and 0.8-1 µm as the interval for Z-stack.
10. After all the parameters are properly set, scan the whole area of the SAN and AVN.
11. Perform 3D reconstruction of the images using software (e.g., Imaris version 8.4.2).
    1. Select Images Processing | Baseline Subtraction to remove background staining.
    2. Select surface creation onto setting for selected channel and region of interest for processing.

Access restricted. Please log in or start a trial to view this content.

Results

By using the protocol outlined above, confocal microscopy imaging of both SAN and AVN can be reliably performed. Specific staining of the conduction system using fluorescent antibodies targeting HCN4 and staining of the working myocardium using fluorescent antibodies targeting Cx43 allows the clear identification of SAN (Figure 5, Video 1) and AVN (Figure 6, Video 2) within the intact anatom...

Access restricted. Please log in or start a trial to view this content.

Discussion

Cardiac anatomy has traditionally been studied using thin histological sections¹¹. However, these methods do not preserve the three-dimensional structure of the conduction system and thus, only provides 2D information. The whole-mount immunofluorescence staining protocol described here allows to overcome these limitations and can be routinely used for SAN and AVN imaging.

In comparison to standard methods such as conventional immunohistochemistry that require paraffin-e...

Access restricted. Please log in or start a trial to view this content.

Materials

Name	Company	Catalog Number	Comments
Anesthesia
Isoflurane vaporizer system	Hugo Sachs Elektronik	34-0458, 34-1030, 73-4911, 34-0415, 73-4910	Includes an induction chamber, a gas evacuation unit and charcoal filters
Modified Bain circuit	Hugo Sachs Elektronik	73-4860	Includes an anesthesia mask for mice
Surgical Platform	Kent Scientific	SURGI-M
In vivo instrumentation
Fine forceps	Fine Science Tools	11295-51
Iris scissors	Fine Science Tools	14084-08
Spring scissors	Fine Science Tools	91500-09
Tissue forceps	Fine Science Tools	11051-10
Tissue pins	Fine Science Tools	26007-01	Could use 27G needles as a substitute
General lab instruments
Orbital shaker	Sunlab	D-8040
Magnetic stirrer	IKA	RH basic
Pipette,volume 10 µL, 100 µL, 1000 µL	Eppendorf	Z683884-1EA
Microscopes
Dissection stereo- zoom microscope	VWR	10836-004
Laser Scanning Confocal microscope	Zeiss	LSM 800
Software
Imaris 8.4.2	Oxford instruments
ZEN 2.3 SP1 black	Zeiss
General Lab Material
0.2 µm syringe filter	Sartorius	17597
100 mm petri dish	Falcon	351029
27G needle	BD Microlance 3	300635
50 ml Polypropylene conical Tube	Falcon	352070
5ml Syringe	Braun	4606108V
Cover slips	Thermo Scientific	7632160
Eppendorf Tubes	Eppendorf	30121872
Chemicals
0.5 M EDTA	Sigma	20-158	Components of TEA
16% Formaldehyde Solution	Thermo Scientific	28908	use as a 4% solution
Acetic acid	Merck	100063	Components of TEA
Agarose	Biozym	850070
Bovine Serum Albumin	Sigma	A2153-100G
DPBS (1X) Dulbecco's Phosphate Buffered Saline	Gibco	14190-094
Normal goat serum	Sigma	NS02L
Sucrose	Sigma	S1888-1kg
Tris-base	Roche	TRIS-RO	Components of TEA
Triton X-100	Sigma	T8787-250ml	Diluted to 1% in PBS
Tween 20	Sigma	P2287-500ml
Drugs
Fentanyl 0.5 mg/10 mL	Braun Melsungen
Isoflurane 1 mL/mL	Cp-pharma	31303
Oxygen 5 L	Linde	2020175	Includes a pressure regulator
Antibodies
Goat anti-Rabbit IgG Alexa Fluor 488	Cell Signaling Technology	#4412	diluted to 1:200
Goat anti-Rat IgG Alexa Fluor 647	Invitrogen	#A-21247	diluted to 1:200
Hoechst 33342, Trihydrochloride, Trihydrate (DAPI)	Invitrogen	H3570	diluted to 1:1000
Rabbit Anti-Connexin-43	Sigma	C6219	diluted to 1:200
Rat anti-HCN4 (SHG 1E5)	Invitrogen	MA3-903	diluted to 1:200
Other
Plexiglass ring			Self-designed and 3D printed
Plasticine	Cernit	49655005
Silikonpasten, Baysilone	VWR	291-1220
Animals
Mouse, C57BL/6	The Jackson Laboratory