Langendorff-perfused mouse heart preparation, IPC, and I/R injury.

Adult (12- to 14-week-old) male C57BL/6 mice (The Jackson Laboratory, 000664) were injected with heparin (100 IU, i.p.) 20–30 minutes prior to the Langendorff procedure (71, 72). Mice were anesthetized with isoflurane, cervical dislocation was performed, and hearts were removed quickly by a midsternal incision and placed into ice-cold modified pH 7.4 Krebs-Henseleit (K-H) solution. The heart was attached to a Langendorff apparatus (Radnoti) and perfused through the aorta at a constant rate of 2.5 ml/min with the K-H buffer. The K-H solution was constantly gassed with 95% O₂/5% CO₂. Perfusion medium was passed through water-jacketed tubing and cylinders, and the temperature was maintained at 37°C with a temperature-controlled circulating water bath. The hearts were allowed to equilibrate for 20 minutes to achieve a steady state before they were subjected to 30 minutes of global ischemia, followed by 60 minutes of reperfusion. Control hearts were perfused continuously throughout the protocol. During no-flow ischemia, the heart was immersed in warm K-H buffer in order to maintain warmth and moisture.

For the IPC experiments, the isolated mouse hearts were allowed to equilibrate for 20 minutes to achieve a steady state before they were subjected to 4 repeated cycles of 3.5-minute ischemia followed by 5-minute reperfusion (31). After preconditioning, the hearts were subjected to prolonged I/R of 30-minute ischemia and 60-minute reperfusion.

For ECG recordings, electrodes (140155-M, Radnoti) were used to record the electrocardiogram and heart rate throughout the experiment using labchart software (ADInstruments) with Power lab and Bio Amp hardware (ADInstruments). A Millar MIKRO-TIP (SPR-671) pressure catheter (Millar Instruments) was inserted into the left ventricle from the left atrium to measure left ventricular pressure throughout the experiment using a Bridge Amp (ADInstruments). LVEDP, LVDP, and heart rate were monitored and recorded continuously using PowerLab system (ADInstruments). Hearts were paced at 360 beats per minute using a pacing electrode (140157M, Radnoti) and a stimulus delivered from a stimulator (stimulus isolator, ADInstruments). After the initial 20-minute stabilization, hearts were excluded from further study if they exhibited one or more of the following exclusion criteria: LVEDP higher than 20 mmHg; LVDP less than 50 mmHg; intrinsic heart rate less than 280 bpm; or irregular heart rate; or aortic regurgitation. Hearts were then subjected to 30 minutes of global ischemia followed by 1 hour of reperfusion. Pacing was initiated at 2 minutes after reperfusion.