Surgical procedure

Rats were weighed before the experiments. Heart surgery was performed following the methods of our previous studies [12, 14, 15], with minor modifications (Fig. 1). In brief, rats were anesthetized with sodium pentobarbital (50 mg/kg, i.p.). The adequacy of anesthesia was controlled by monitoring the lack of response to toe-pinching, the absence of corneal reflex, and no response to surgical manipulation. Rats were artificially ventilated throughout the experiments. To maintain anesthesia, additional sodium pentobarbital (20 mg/kg) was administered every 30 min. All rats were subjected to 10 min stabilization after surgical preparation and instrumentation. The left chest of each rat was opened to identify the main left coronary artery, which was tied with a 6-0 silk ligature (Ethicon, Somerville, NJ, USA). Five minutes after ligation, the suture was released and then 20 min of reperfusion was performed. In the ischemic zone, a valid coronary artery occlusion was confirmed by the presence of regional dyskinesia and epicardial cyanosis. Reperfusion was confirmed by visualizing an epicardial hyperemic response. Cardiac activity was continuously recorded using a standard limb lead II configuration electrocardiographic system (Powerlab/8sp system, AD Instruments, Colorado Springs, CO, USA) throughout the experiment. RLIPC was achieved by clamping the portal vein, hepatic arterial and venous trunk for four cycles of 5 min of liver ischemia with 5 min intermittent reperfusions (liver I/R cycles).

Experimental time-courses. Time courses for protocols in Experiments 1 and 2. Black liver ischemia cycles; gray with white O, left main coronary artery occlusion; R cardiac reperfusion, CON control, RLIPC remote liver ischemia preconditioning, DM STZ-induced diabetes