Animals
Every animal experiment used in this study received approval from the Capital Medical University Animal Care and Use Committee (AEEI-2015-193) (NIH Publication No. 8523, revised 2011) and conducted in accordance with the "Guide for the Care and Use of Laboratory Animals" adopted by the Beijing Municipal People's Government. Animals were kept in a temperature-controlled room at the Capital Medical University's Center for Experimental Animals (grade SPF), where they were given 12-hour light-dark cycles.
Langendorff perfusion system for Murine SAM isolation
Buffers and materials
Tyrode’s solution (Table 1)
Enzyme solution (Table 1).
Low Ca2+/Mg2+ solution (Table 1).
Low Ca2+/Mg2+ enzyme solution (Table 1).
Kraftbruhe (KB) solution (Table 1).
Tyrode-BSA solution (Table 1).
Ca2+-free Tyrode’s solution (Table 1).
Collagenase Type II (Worthington Biochemical Corp, LS004176, Lakewood, Colorado, USA)
Protease (Sigma, P51476, Darmstadt, Hessen, Germany)
BSA (Sigma, V900933, Darmstadt, Hessen, Germany)
Assemble tools required for dissection (Fine Vannas scissors, Fine forceps, Dissecting scissors, Tissue forceps) (Figure 1a).
10 cm Petri dishes containing ~25 mL cured SYLGARD™ 186 Silicone Elastomer Kit (Dow, 1673939, Midland, MI, USA) (Figure 1b).
Langendorff-perfusion system (Figure 1c).
Solution and perfusion apparatus preparation
1. Sterilize the surgical tools given in Figure 1.
2. Prepare the solutions according to the reagents given in Table 1.
NOTE: The Tyrode’s solution must be saturated with carbogen (95% O2/5% CO2) before adjusting the pH.
3. Clean the perfusion system by circulating deionized water. Turn on the circulating water bath and adjust a suitable input temperature to ensure that the perfusate circulation system's outflow from the cannula reaches 37°C. Set the flow rate of the perfusate perfusion system at 2.5 mL/min.
Langendorff-perfused heart
1. Male C57BL/6 mice (age 2-3 months) were intraperitoneally injected with heparin (concentration 200 units) to avoid blood clots.
2. After 10 min heparin injection. Mice were anesthetized with isoflurane (1.5%) and placed in the supine position on the surgical platform. The chest was disinfected using 75% alcohol (Figure 2a, i).
3. The thoracic cavity was opened by cutting along the diaphragm with dissection scissors. The xiphoid process was grasped with tissue forceps to expose the heart (Figure 2a, ii). A pin was used to fix the xiphoid process (Figure 2a, iii). The aortic arch was clamped with tissue forceps, and the intact heart was then excised and placed in the (pre-warmed) Tyrode’s solution (Figure 2a, iv; 2b, i).
4. The blood was cleared by gentle perfusion with cold Tyrode’s solution. Excess tissue including lungs, thymus and fat from the heart were removed by using fine Vannas scissors and fine forceps. Be careful not to damage the auricular area of the heart.
5. The heart was cannulated through the aorta and retrograde perfused on a Langendorff apparatus with a drop velocity of 1 drop per second (2.5 mL/min) (Figure 2b, ii-iv) (Video 1).
NOTE: Make sure the atria are sufficiently perfused. The atrial appendages will expand during perfusion.
6. Tyrode’s solution was first perfused into the heart for 5 min, followed by 50 mL of enzyme solution for approximately 15 min at 37 °C (Video 2). Be careful to avoid any air bubbles entering the heart during perfusion.
NOTE: Once the enzyme buffer enters the heart, the droplet flow rate would suddenly slow down. The droplet flow rate gradually increased as the cardiac extracellular matrix began hydrolyses (usually a few minutes). Langendorff-perfusion could be stopped as the droplet flow rate increase to around 4-5 drops per second.
Isolation of SAN
1. After Langendorff perfusion, the hearts were placed in the pre-warmed low Ca2+/Mg2+ solution. Carefully make a horizontal incision at the junction between the ventricles and atria by using fine Vannas scissors.
2. The atrial/conduction tissue was transferred in a clean dish containing low Ca2+/Mg2+ solution, and then pinned onto silicone dissection dishes by using fine insect pins. The animal's right atrium was on the experimenter's left, and the left atrium was on the right to expose the superior vena cava to the pulmonary artery (Figure 2c).
3. The SAN tissue (around 2.0 mm × 1.5 mm × 1.0 mm) was dissected from the region bordered by the crista terminalis and the superior and inferior vena cava under a stereo microscope.
Digestion of SAN
The SAN tissue was cut into small pieces and washed twice with low Ca2+/Mg2+ solution. The pieces of SAN tissue were then digested at 35 °C for 5 min in low Ca2+/Mg2+ enzyme solution with gentle agitation (Figure 2d).
Dissociation of SAMs
1. The digestion was stopped by gently transferring the pieces to a round-bottomed tubule with 2.5 mL of KB solution at 35 °C for a total of three times.
2. Transfer the tissue to the large, round-bottomed tube containing 2.5 mL KB solution. The tissue was dissociated in the large round-bottomed tube by constant trituration at approximately 0.5-1 Hz for 5 min. Be careful to keep the dissociation tube submerged in the 35 °C water bath and to avoid introducing bubbles into the solution.
Ca2+ re-adaptation SAMs
1. The isolated SAMs were stored in KB solution at room temperature for 20 min before Ca2+ re-adaptation. Then, the premixed working solution contains 0.1mM CaCl2 (55 μL of Tyrode-BSA, 945 μL of Ca2+-free Tyrode’s solution) was added to a tube containing SAMs. After further incubation for 5 min, the supernatant was discarded. Repeat the above steps to add CaCl2 solution with the following concentrations: 0.3, 0.6, 0.9 and 1.8 mM CaCl2 solution. Finally, the resulting supernatant was discarded, and the cell pellet was resuspended and centrifuged at 2000 g for 2 min.
2. The resulting supernatant was discarded, and the cell pellet was resuspended in Tyrode-BSA solution, followed by cell counting. The cell suspension was transferred to a carbon dioxide cell incubator (37 °C and 5% CO2) and allowed to stand still for at least 30-45 min (Figure 2e).
Isolation of adult cardiomyocytes
Isolation of adult murine cardiomyocytes was performed as previously described15. Briefly,
Mice were heparinized and anaesthetized with 1.5-2% isoflurane inhalation. Heart was excised and mounted on Langendorff system and perfused with enzyme solution for 18-20 min at 37 °C. After sufficient digestion, the atria and ventricles were removed, dissociated using tissue forceps and transfer pipettes, and resuspended in stopping buffer (KB solution). The isolated cardiomyocytes were then preserved in Tyrode-BSA solution.
Immunofluorescence and histology
Acutely isolated myocytes were fixed with 4% PFA in PBS for 20 min at 4 °C and permeabilized using PBS containing 0.3% TritonX-100 (Sigma, T9284, Darmstadt, Hessen, Germany), 200 mM glycerin (Sigma, G2289, Darmstadt, Hessen, Germany) for 20 min at room temperature. Samples were blocked for 30 min in blocking buffer: PBS containing 5% BSA.
For immunostaining, HCN4 (Santa, sc-58622, 1:50, Dallas, Texas, USA) was incubated overnight at 4 °C. Subsequently, the secondary antibody Alexa Fluor 647-labeled goat anti-rat (Invitrogen, A21247, Waltham, MA, USA) was added and incubated for 1 h at room temperature. The nucleus was labeled with DAPI at the final concentration of 1.0 μg/mL in tissue and cell samples. Leica SP5 laser confocal microscope was used to capture and analyze fluorescence pictures.
Western blot
Protein samples were denatured by incubation in a loading buffer at 95 °C for 5 minutes. Protein was electrophoresed on 10% Tris-tricine-SDS-PAGE and transferred to PVDF membranes. The membranes were then blocked in 5% non-fat milk with Tris-buffered saline/Tween-20 (1 TBST, pH 7.4) for 1 h at room temperature. Then the membranes were incubated overnight at 4 °C with anti-HCN4 (Santa, sc-58622, 1:1000, Dallas, Texas, USA), anti-CX40 (Santa, sc- 20466, 1:1000, Dallas, Texas, USA), anti-CX43 (CST, 3512, 1:1000, Danvers, MA, USA), or anti-GAPDH (ZSGB, TA-08, 1:1000, Beijing, China) primary antibodies. Membranes were washed three times with TBST before being subjected to HRP-conjugated secondary antibodies at room temperature for an hour. Fluor Chem FC3 ECL chemiluminescence was then used to illuminate the membranes.