Isolation and Culture of Resident Cardiac Macrophages from the Murine Sinoatrial and Atrioventricular Node

Summary

The protocol presented here provides a step-by-step approach for the isolation of cardiac resident macrophages from the sinoatrial node (SAN) and atrioventricular node (AVN) region of mouse hearts.

Abstract

Resident cardiac macrophages have been demonstrated to facilitate the electrical conduction in the heart. The physiologic heart rhythm is initiated by electrical impulses generated in sinoatrial node (SAN) and then conducted to ventricles via atrioventricular node (AVN). To further study the role of resident macrophages in cardiac conduction system, a proper isolation of resident macrophages from SAN and AVN is necessary, but it remains challenging. Here, we provide a protocol for the reliable microdissection of the SAN and AVN in murine hearts followed by the isolation and culture of resident macrophages.

Both, SAN which is located at the junction of the crista terminalis with the superior vena cava, and AVN which is located at the apex of the triangle of Koch, are identified and microdissected. Correct location is confirmed by histologic analysis of the tissue performed with Masson's trichrome stain and by anti-HCN4.

Microdissected tissues are then enzymatically digested to obtain single cell suspensions followed by the incubation with a specific panel of antibodies directed against cell-type specific surface markers. This allows to identify, count, or isolate different cell populations by fluorescent activated cell sorting. To differentiate cardiac resident macrophages from other immune cells in the myocardium, especially recruited monocyte-derived macrophages, a delicate devised gating strategy is needed. First, lymphoid lineage cells are detected and excluded from further analysis. Then, myeloid cells are identified with resident macrophages being determined by high expression of both CD45 and CD11b, and low expression of Ly6C. With cell sorting, isolated cardiac macrophages can then be cultivated in vitro over several days for further investigation. We, therefore, describe a protocol to isolate cardiac resident macrophages located within the cardiac conduction system. We discuss pitfalls in microdissecting and digesting SAN and AVN, and provide a gating strategy to reliably identify, count and sort cardiac macrophages by fluorescence-activated cell sorting.

Introduction

The sinoatrial node (SAN) physiologically initiates the electrical impulse and is, therefore, the primary pacemaker of the heart. The atrioventricular node (AVN) conducts the electrical impulse from the atria to the ventricles and also acts as a subsidiary pacemaker¹. In general, generation and conduction of electrical impulses is a complex process that can be modulated by various factors², including resident macrophage in SAN/AVN regions. A recent study by Hulsmans et al. demonstrates a specific population of cardiac resident macrophages which are enriched in the AVN and function as key players in keeping a steady heartbeat³. They found that macrophages are electrically coupled to the cardiomyocytes and could change the electrical properties of coupled cardiomyocytes. The authors also note that such conducting cells interleaving with macrophages are also present in other components of the cardiac conduction system, such as the SAN.

Currently, it is not fully known if the phenotype of resident cardiac macrophages differs between the cardiac regions. However, it has been shown that the tissue microenvironment can affect transcription and proliferative renewal of tissue macrophages⁴. Furthermore, since the cardiomyocyte phenotype has been demonstrated to be different between regions, the functional effects of macrophages on cardiomyocytes may also be region-specific, even if the macrophage phenotype itself may be the same. Therefore, further studies on specific cardiac regions are needed.

Recent studies have demonstrated that, at steady state, the tissue resident macrophages are established prenatally, arising independently of definitive hematopoiesis, and persist into adulthood⁵. However, after macrophage depletion or during cardiac inflammation, Ly6c^hi monocytes contribute to replenish cardiac macrophage population⁶. Studies involving genetic lineage tracing, parabiosis, fate mapping, and cell tracking showed the coexistence of a variety of tissue resident macrophages populations in organs and tissues, and, also, different cellular behavior of macrophage subsets that are potentially associated with their ontogeny^7,8,9.

Characterization of resident cardiac macrophages has benefited from the use of magnetic activated cell sorting (MACS) and fluorescent activated cell sorting. These methods are particularly useful for isolating specific cell populations from multiple tissue fractions by labeling them with their cell surface markers. This not only leads to a higher purity of the isolated immune cell type, but also allows for phenotypic analysis. Here, we present a protocol including magnetic beads-coated cells followed with fluorescent activated cell sorting for the enrichment of cardiac resident macrophages specifically isolated from the SAN and AVN region.

To explore the characteristics of cardiac resident macrophages in conduction system and their function for cardiac conduction and arrhythmogenesis, precise localization and dissection of SAN and AVN are critical. For microdissection of SAN and AVN, anatomical landmarks are used for the region identification¹⁰. In brief, SAN is located at the junction of the superior vena cava and right atrium. AVN is located within the triangle of Koch, which is anteriorly bordered by the septal leaflet of the tricuspid valve, and posteriorly by the tendon of Todaro¹¹. We also provide an accurate microdissection procedure of SAN and AVN in mice which is confirmed by histology and immunofluorescence staining.

Isolated resident macrophages could be used for further experiments such as RNA sequencing or could be recovered and cultivated for more than two weeks allowing various in vitro experiments. Therefore, our protocol describes a highly valuable procedure for the immuno-rhythmologist. Table 1 shows the composition of all the solutions needed, Figure 1 shows the microdissection landmarks for SAN and AVN. Figure 2 is schematic illustration of SAN and AVN localization. Figure 3 shows the histological staining of SAN and AVN (Masson's trichrome and immunofluorescence staining). Figure 4 shows a step-by-step gating strategy to isolate cardiac resident macrophages by fluorescence-activated cell sorting.

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. C57BL6/J mice were commercially obtained.

1. Preparations

1. Prepare Cell sorting buffer (Table 1) and store at 4 °C.
   NOTE: During the whole experimental procedure, the cell sorting buffer should always be on ice.
2. Prepare Digestion buffer (Table 1) shortly before the digestion as the activity of collagenase could only be detected for few hours at room temperature.
3. Refer to the previously published protocol for the preparation of dissection dish¹⁰. In brief, add 30 mL of agarose gel (3%-4%) into a 100 mm diameter Petri dish and cool down at room temperature.

2. Animal sacrifice and heart excision

1. Anesthetize the mouse with isoflurane by placing it into an incubation chamber connected with an isoflurane vaporizer and flushed with 5% isoflurane/95% oxygen.
2. After the injection of fentanyl for analgesia, open the rib cage and perfuse the heart by injecting 5-10 mL of ice-cold 1x PBS directly into the left ventricle (LV). Extract the mice heart and put it on the dissection dish. Experimental details have been described in detail previously¹⁰.

3. Microdissection of SAN and AVN

1. After isolating the heart, perform the following microdissection procedures in the dissection dish with ice-cold 1x PBS under the dissecting microscope.
2. Use the cardiac anatomical landmarks, i.e., aorta, pulmonary artery, coronary sinus, left/right ventricle, etc. to determine the left/right (left: LV; right: RV) and anterior/posterior (anterior: aorta; posterior: coronary sinus) of the heart. After the orientation is determined, turn around the heart with the front of it at the bottom of the dish (to expose the large veins that are located posterior).
3. Microdissection of SAN
   NOTE: Microdissection of the SAN have been previously described¹⁰. The process is described in brief below.
   1. Expose the inter-caval region by pining the right atrial appendages (RAA) and the tissue adjacent to superior vena cava (SVC) and inferior vena cava (IVC) on the microdissection dish using insect pins.
   2. Cut the heart along the interatrial septum parallel to the crista terminalis (CT) to separate the inter-caval region and to obtain the SAN sample (Figure 1A, Figure 2A). Put the sample in an empty 1.5 mL microcentrifuge tube on ice.
4. Microdissection of AVN
   1. After collection of the SAN sample ensure that the RAA and parts of the right atrium (RA) have already been cut away leaving only the interatrial septum (IAS) and, interventricular septum (IVS).
   2. Pin the remaining parts of the heart through the tissue adjacent to the IAS and IVS using insect pins to make the right atrial side of IAS facing up.
   3. Look at the right atrium on the endocardial surface for the triangle of Koch. It will be bordered anteriorly by the hinge-line of the septal leaflet of the tricuspid valve (TV), and posteriorly by the tendon of Todaro. The orifice of the coronary sinus is observed at the base. (Figure 1B, Figure 2B).
   4. Cut the triangle of Koch, which contains the AVN, and directly put it in an empty 1.5 mL microcentrifuge tube on ice.

4. Digestion

1. Prepare the Digestion buffer (Table 1) shortly before use.
2. Mince the SAN and AVN tissue well with scalpels.
   NOTE: Mincing the tissue well will increase the digestion efficiency and help to get good cell suspension for sorting. As the SAN and AVN samples are quite small, mincing the tissue directly inside the 1.5 mL microcentrifuge tube is recommended to reduce the loss of sample.
3. Add 500 µL of digestion buffer per sample and wash down all minced tissue from the wall of the 1.5 mL microcentrifuge tube. Gentle pipetting helps digesting the sample.
4. Homogenize the tube on a vortex machine (settings: 37 °C, 750 rpm for 1 h).
5. After digestion, transfer the tissue suspension to a fresh 15 mL centrifuge tube by passing through a 40 µm cell strainer. Rinse the cell strainer with an additional 5 mL of cell sorting buffer to stop the digestion.
6. Centrifuge the 15 mL tube at 350 x g for 7 min at 4 °C. Then remove the supernatant completely using the pipette. Resuspend the cell pellet with 90 µL cell sorting buffer.
   ​NOTE: Before magnetic separation, pipette the cell suspension gently a few times or pass through a 30 µm cell strainer to remove cell clumps if necessary, to obtain a single cell suspension for optimal performance of magnetic enrichment of interesting cell populations.

5. Magnetic enrichment of CD45 and sample staining

NOTE: To isolate the cardiac macrophages with high sorting efficiency, exclusion of undesired cells including lymphocytes was performed with CD45 microbeads according to the manufacturer's protocol. Based on the sorting panel, cardiac resident macrophages were identified as CD45^highCD11b^highCD64^high Ly6C^low/int F4/80^high.

1. Add 10 µL of CD45 microbeads per 10⁷ total cells to the cell suspension in the 15 mL centrifuge tube. Mix the samples well and incubate them for 15 min at 4 °C.
   NOTE: The cell counting using a hemocytometer should be briefly done to make sure that each tube contains no more than 10⁷ total cells. When working with higher cell numbers, the volume of magnetic beads needs to be scaled up.
2. Prepare the antibody mixture by diluting the following antibodies in cell sorting buffer (1:100 dilution for each antibody): CD45-PE, CD11b-APC-Cy7, CD64-APC, F4/80-PE-Cy7, Ly6C-FITC. DAPI will be added later to the staining for live/dead discrimination.
3. After 15 min of magnetic bead incubation, add 100 µL of antibody mixture directly into the cell suspension in the 15 mL tube (then all the antibodies' final concentration is 1:200) and incubate for 20 min at 4 °C.
4. After 20 min of antibody incubation, wash the cell suspension by adding 1-2 mL of cell sorting buffer per 10⁷ cells and centrifuge at 350 x g for 10 min. Completely remove the supernatant by pipetting.
5. Resuspend up to 10⁸ cells in 500 µL of cell sorting buffer.
   NOTE: The maximum cell number for magnetic separation should be determined according to the manufacturer's protocol.
6. Prepare the magnetic separation set.
   1. Attach the magnetic column to a suitable magnetic separator and place a collection tube under the magnetic column.
   2. Prepare the magnetic columns by rinsing with cell sorting buffer: add 500 µL of cell sorting buffer at the top of the column and let the buffer pass through.
7. Apply the cell suspension immediately onto the column while the cell sorting buffer is passing through.
   NOTE: Avoid the formation of air bubbles in the column. As per the manufacture's protocol, although the column filling time might change from storage conditions, it has no influence on the quality of the separation.
8. Wash the column with 3 x 500 µL cell sorting buffer. The flow-through in step 5.7 and step 5.8 contain unlabeled cells, which can be discarded if no further experiment is needed.
   ​NOTE: Add the cell sorting buffer immediately when the column reservoir is nearly empty.
9. Remove the column from the magnetic separator and place it on a new collection tube.
10. Add 1 mL of cell sorting buffer onto the column. Immediately flush the column by firmly applying the plunger supplied with the column. The flow-through contains the magnetically labeled cells.
11. Add DAPI solution into all the collected magnetically labeled cell suspensions shortly before running them on the cell sorter. Adjust the final concentration of DAPI to 0.3-0.5 µg/mL.
12. Perform FACS analysis.

6. Samples for compensation

1. Prepare 6 brown 1.5 mL microcentrifuge tubes labeled as "PE", "APC-Cy7", "APC", "PE-Cy7", "FITC", and "DAPI" respectively to protect antibodies from light. Prepare one more 1.5 mL microcentrifuge tube labeled as "unstained".
   NOTE: This could be done at the same time as incubating the cell suspension with the microbeads and antibodies. The unstained sample could be the cardiomyocyte tissue collected randomly from the spared heart tissue and also treated according to step 4.
2. Dilute each single fluorescence-conjugated antibody with cell sorting buffer into 1:50 in the 1.5 mL brown microcentrifuge tubes that are marked accordingly.
3. Add one drop of compensation beads solution and incubate for 20 min at 4 °C.
4. Add 2 mL of cell sorting buffer into each 1.5 mL brown microcentrifuge tube and centrifuge at 450 x g for 5 min. Completely discard the supernatant and resuspend the bead containing pellet with 300 µL cell sorting buffer and transfer them into cell sorter tubes that are also marked accordingly.

7. Running on the cell sorter and gating strategy

1. Apply the unstained sample and compensation tubes first and adjust the voltages of each channel to align both the positive and negative peak to the proper position of the axis. Save the compensation settings and apply it to the following samples.
2. Apply the samples on the cell sorter. Set the gating strategy as described in Figure 4. Cardiac resident macrophages are identified as CD45^highCD11b^highCD64^highLy6C^low/int F4/80^high. DAPI is used as a cell viability marker.
3. Check the flow cytometry charts to confirm that the cell population of interest is properly shown on the charts. If not, adjust the voltage of each channel to the center view of each chart.
4. If the voltage settings are satisfactory, start the sorting procedure. Collect the sorted cell population into culture medium composed of DMEM containing 10% fetal bovine serum, supplemented with 100 µg/mL streptomycin and 100 U/mL penicillin.

8. Resident macrophages culture

1. After gathering the sorted macrophages, transfer the cells immediately either to 35 mm tissue culture dishes or 24 well plate, or directly use them for subsequent experiments.
2. To culture sorted macrophages, incubate the cells at 37 °C, 5% CO₂ incubator.
3. Change the culture medium every 48-72 h. Floating dead cells can be easily removed by medium change. Use live macrophages attaching to the bottom of culture dish for subsequent experiment.

Results

We describe a practical procedure for the isolation of cardiac resident macrophages specifically from the SAN and AVN region. To confirm a correct dissection, Masson's Trichrome staining and immunofluorescent HCN4-staining is performed (Figure 3)¹². With this protocol, we could collect approximately 60,000 macrophages from one whole heart. Figure 4 shows the gating strategy for sorting cardiac macrophages. Live resident cardiac macrop...

Discussion

In this manuscript, we describe a protocol for the enrichment of cardiac resident macrophages specifically from the SAN and AVN regions at high purity.

Macrophages are divided into subpopulations based on their anatomical location and functional phenotype. They can also switch from one functional phenotype to another in response to variable microenvironmental signals¹³. Compared to other organs such as bone marrow and liver, cardiac tissue contains a lower percentage of...

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
Pipette,volume 10ul, 100ul, 1000ul	Eppendorf	Z683884-1EA
Magnetic stirrer	IKA	RH basic
Microscopes
Dissection stereo- zoom microscope	vwr	10836-004
Leica microscope	Leica microsystems	Leica DM6
Flow cytometry machine
Beckman Coulter	Beckman coulter	MoFlo Astrios
Software
FlowJo v10	FlowJo
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
Cover slips	Thermo scientific	7632160
Eppendorf Tubes	Eppendorf	30121872
5ml Syringe	Braun	4606108V
Chemicals
0.5 M EDTA	Sigma	20-158
Acetic acid	Merck	100063	Component of TEA
Agarose	Biozym	850070
Bovine Serum Albumin	Sigma	A2153-100G
Collagenase I	Worthington Biochemical	LS004196
Collagenase XI	Sigma	C7657
DNase I	Sigma	D4527
Hyaluronidase	Sigma	H3506
HEPES buffer	Sigma	H4034
Bovine Serum Albumin	Sigma	A2153-100G
DPBS (1X) Dulbecco's Phosphate Buffered Saline	Gibco	14190-094
Fetal bovine serum	Sigma	F2442-500ml
Penicillin − Streptomycin	Sigma	P4083
DMEM	Gibco	41966029
Drugs
Fentanyl 0.5 mg/10 ml	Braun Melsungen
Isoflurane 1 ml/ml	Cp-pharma	31303
Oxygen 5L	Linde	2020175	Includes a pressure regulator
Antibodies
Anti-mouse Ly6C FITC (clone HK1.4)	BioLegend	Cat# 128006	diluted to 1:100
Anti-mouse F4/80 PE/Cy7(clone BM8)	BioLegend	Cat# 123114	diluted to 1:100
Anti-mouse CD64 APC (clone X54-5/7.1)	BioLegend	Cat# 139306	diluted to 1:100
Anti-mouse CD11b APC/Cy7(clone M1/70)	BioLegend	Cat# 101226	diluted to 1:100
Anti-mouse CD45 PE (clone 30-F11)	BioLegend	Cat# 103106	diluted to 1:100
Hoechst 33342, Trihydrochloride, Trihydrate (DAPI)	Invitrogen	H3570	diluted to 1:1000
Animals
Mouse, C57BL/6	Charles River Laboratories