Langendorff heart preparation

We adopted a well-established Langendorff method of retrograde perfusion in murine hearts⁸. Tyrode’s solution⁸ (2 L) was pumped through via coronary artery as well as around the heart (superfusion). The solution was oxygenated by carbogen gas (95%/5% O₂ to CO₂ ratio) and its pH was maintained at 7.35 ± 0.05. Separate water baths (153–1071, Thermo Scientific GmbH, Munich, Germany) were used to maintain the Tyrode’s solution at 37 °C for the perfusion and superfusion circuits. CD1 mice were overdosed with ketamine (150 mg/kg) and xylazine (10 mg/kg) to harvest their hearts. Once the animal exhibits no pain reflex, established by pinching the paw, the thoracic cavity was opened to excise the heart. A 21-gauge cannula filled with perfusion buffer was inserted in the ascending aorta and fixed using 4-0 black-braided silk sutures (Resorba GmbH, Nuremberg, Germany).