Langendorff-Perfused Heart Preparation

A constant pressure mode of 80 mmHg was used in the Langendorff isolated heart perfusion as previously described [3, 9-11]. Briefly, sEH^−/−, A_2AAR^−/−, and wild-type (WT) mice (16–18 wks.) were euthanized using 100 mg/kg body weight sodium pentobarbital intra-peritoneally. Hearts were excised and instantly placed into ice-cold Krebs-Henseleit buffer containing (in mM) 1.2 KH₂PO₄, 11.0 glucose, 4.7 KCl, 119.0 NaCl, 1.2 MgSO₄, 2.5 CaCl₂, 2.0 pyruvate, 22.0 NaHCO₃, 0.5 EDTA, and heparin (5 U/mL). The lungs and surrounding tissues around the heart were then removed, the aorta cannulated, and the heart continuously perfused with warm (37°C) buffer bubbled with 95% O₂. Measurement of left ventricular developed pressure (LVDP) and heart rate (HR) was provided by a water-filled balloon placed into the left ventricle and connected to a pressure transducer. Coronary flow (CF) was measured by a flow transducer with an ultrasonic flow probe (Transonic Systems, Ithaca, NY). Data acquisition and recording was done by a Power–Lab Chart (AD Instruments, Colorado Springs, CO). Hearts were left to stabilize for 30–40 min before experimentation. Hearts with CF increase by less than two-fold in response to a 15-second total occlusion, as well as hearts with LVDP <80 mmHg or persistent arrhythmias were excluded from analysis.