Ex vivo Langendorff-perfused heart rate measurements

ECG recording from Langendorff-perfused hearts was performed as described [^{11, 16}]. Briefly, excised hearts were rapidly mounted on a Langendorff apparatus (ADInstrments Inc. Colorado Springs, USA) for retrograde aortic perfusion at a constant flow of 4 ml/min with oxygenated (95% O₂, 5% CO₂) Krebs-Henseleit solution consisting of (mM): 25.0 NaHCO₃, 118.0 NaCl, 4.7 KCl, 1.2 MgSO₄, 1.2 KH₂PO₄, 2.5 CaCl₂, and 11.0 Glucose, with pH equilibrated to 7.4. Each perfused heart was immersed in a water-jacketed bath and the bath temperature was monitored by T-type Implantable Thermocouple Probe (IT-21) (ADInstrments Inc.). ECG measurements from the intact heart were continuously recorded with Isolated Heart MAP Electrode Set (Mouse, ADI) (ADInstrments Inc.). After the heart was allowed to stabilize for at least 20 minutes, the heart rate and bath temperature were recorded by Powerlab (ADInstrments Inc.), bath temperature was controlled or changed by circulating water from bath heater and circulator.