Isolated Perfused Rat Heart Protocol

Isolated perfused rat hearts were prepared as previously described [53 (link)]. In brief, rats were anesthetized by a subcutaneous injection of Hypnorm-Dormicum (fentanyl citrate, 0.158 mg/kg body weight; fluanisone, 0.5 mg/kg body weight; midazolam, 0.5 mg/kg body weight) and mechanically ventilated through a tracheostomy. A bolus of 500 IU heparin for anticoagulation was administered through the femoral vein. Then, the ascending aorta was cannulated via a thoracoabdominal incision and retrogradely perfused at a constant pressure of 80 mmHg with 37 °C KH buffer continuously aerated with 5% CO₂/balance O₂. Following transfer to the Langendorff system, a balloon was inserted in the left ventricle through the left atrial appendage and pressurized to 5–8 mmHg to simulate preload. After the hearts had stabilized in KH buffer for 45 minutes, we tested the influence of adding 3 or 10 mM racemic Na-3-OHB or NaCl. We report changes from baseline (measured 10 or 20 minutes after buffer change) for Na-3-OHB after subtraction of the response to equimolar NaCl. We excluded hearts that during stabilization did not fulfill the following inclusion criteria adjusted for rat size [14 (link)]: Coronary flow: 10–35 mL/min, arrhythmias: <10 ectopic beats and no sustained ventricular tachycardia or fibrillation, heart rate: 150–400 min^–1, left ventricular systolic pressure: >120 mmHg.