A total of 90 male Sprague-Dawley rats (8-week-old), weighing between 250 and 280 g (Experimental Animal Center, Fudan University, Shanghai, China) were used in the present study. All rats were housed at a controlled temperature (25°C) under a 12-h light/dark cycle with ad libitum access to food and water. The animal investigation protocol used was in compliance with the Guide for the Care of Use of Laboratory Animals published by the National Institutes of Health (NIH Publication no. 85-23, revised 1996) (9) and approved by the Animal Care Committee of Shanghai Jiao Tong University Affiliated Sixth People's Hospital, (Shanghai, China). All rats were housed for 2 weeks to provide an acclimatization period prior to the experiments.

The IR model was performed, as previously described (10). In brief, the rats were anesthetized by intraperitoneal injection with 1.2% pentobarbital sodium (Sigma-Aldrich, St. Louis, MO, USA), at a dose of 50 mg/kg. The left coronary artery (LCA) was ligated using a 6-0 Prolene suture immediately distal to its first branch, and cardiac ischemia was confirmed by the formation of a pale area below the suture, which gradually became cyanotic. After 40 min, the suture was released, and reperfusion was characterized by the rapid disappearance of cyanosis, followed by vascular blush. Following 120 min of reperfusion, the rats were sacrificed with an overdose of pentobarbital sodium (150 mg/kg) and the hearts were harvested for further assessment. For rats undergoing sham surgery, a suture was placed in a corresponding location without ligation.

RIPerC and RIPostC were delivered via non-invasive occlusion of both lower limbs using tourniquets, which was validated by the disappearance of Doppler blood flow of the femoral artery (5–10 MHz; M-Turbo System L38X; SonoSite, Inc., Bothell, WA, USA). Both RIPerC and RIPostC consisted of four cycles of 5-min limb ischemia/reperfusion, with RIPerC initiated at the onset of coronary, while RIPostC initiated at the onset of coronary reperfusion, as shown in Fig. 1.

The rats were randomly assigned to the following experimental groups: i) Sham group (n=6; rats underwent sham surgery); ii) IR group (n=6; rats underwent 40 min left anterior descending artery occlusion followed by 2 h reperfusion) iii) RIPerC group (n=6; as in the IR group, in addition to four cycles of 5 min bilateral hindlimb occlusion followed by 5 min reperfusion during myocardial ischemia); iv) RIPostC group (n=6; as in the IR group, with four cycles of 5 min bilateral hindlimb occlusion followed by 5 min reperfusion at the onset of coronary reperfusion); v) RIPerC + RIPostC group (n=6; RIPerC combined with RIPostC. Following 2 h of subsequent reperfusion, all rats were sacrificed for IS quantification (Fig. 1).

An additional 30 Sprague-Dawley rats (six in each group) underwent the procedures described above and were also sacrificed 2 h following reperfusion. In these rats, a cross section of the left ventricular (LV) myocardium (~5 mm thick) at the papillary muscle level was obtained from each rat for terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining. The remaining myocardium from the area at risk (AAR) was collected for quantification of the protein expression levels of B cell lymphoma (Bcl)-2 and Bcl-2-associated X protein (Bax).

In addition, a further six rats from each group underwent the same procedure, were sacrificed following 40 min of reperfusion, and the myocardium from the AAR was obtained for western blot analysis. All tissues were snap-frozen in liquid nitrogen (Shanghai Jiangnan Gas Co., Ltd., Shanghai, China) and stored in a freezer at -80°C.

Following 2 h of reperfusion, the LCA was re-occluded, and 2% Evans blue dye (Sigma-Aldrich) was retrogradely injected into the ascending aorta to delineate the AAR. The heart was removed and sliced transversely from the base to the apex into five sections (2-3 mm), which were incubated for 15 min at 37°C in a phosphate-buffered 1% 2,3,5-triphenyltetrazolium chloride solution (Sigma-Aldrich) to determine the infarcted area. All slices were then fixed in 10% formalin (Goodbio, Shanghai, China), and the extent of the area of necrosis was quantified by computerized planimetry using ImagePro Plus software, version 6.0 (Media Cybernetics, Inc., Rockville, MD, USA) and corrected for the weight of the tissue slices. IS is expressed as the percentage of total weight of the LV AAR.

Subsequent to 2 h of reperfusion, blood samples were collected into tubes containing microscopic silica particles and rested for 30 min. Following centrifugation at 2,500 × g for 10 min at 25°C, the supernatants were collected and stored at -80°C until required for future analysis. The serum levels of cTnI, tumor necrosis factor α (TNF-α) and interleukin 1β (IL-1β) were assessed using Cardiac Troponin-I enzyme-linked immunosorbent assay (ELISA) (cat. no. CTNI-HS), Rat TNF-alpha Platinum ELISA (cat. no. BMS622), and Rat IL-1 beta Platinum ELISA (cat. no. BMS630) kits, according to the manufacturer's instructions (cTnI, Life Diagnostics, Inc., West Chester, PA, USA; TNF-α and IL-1β, eBioscience, Inc., San Diego, CA, USA). Levels of cTnI were expressed as ng/ml, whereas levels of cytokines were expressed as pg/ml.

The right carotid artery was cannulated using a 1.6F Pressure Catheter (Transonic Scisense, Inc., London, ON, Canada) for measuring the hemodynamic parameters. The catheter was passed retrogradely into the LV, and LV pressure tracings were digitized using a PowerLab Physiological Recorder (ADInstruments Pty, Ltd., Bella Vista, Australia) and stored for later analysis. The LV end-diastolic pressure (LVEDP), maximum/minimum first derivative of LV pressure over time (± dP/dt_max) and mean arterial pressure (MAP) were analyzed in a blinded-manner using LabChart software, version 8 (ADInstruments Pty Ltd, Oxford, UK).

TUNEL staining was performed using a commercially available kit (In Situ Cell Death Detection kit; Roche Diagnostics GmbH, Mannheim, Germany), according to the manufacturer's protocol, on heart tissue slices randomly selected from each group (n=6 tissue slices/group). A minimum of 100 cells from the peri-infarct area were counted using a microscope (magnification, ×400; Q500MC; Leica Microsystems GmbH, Wetzlar, Germany) in 10 fields for each sample. The peri-infarct area was predetermined using hematoxylin and eosin (Goodbio) staining, which was performed on the adjacent tissue slide. The percentages of cells positive for TUNEL staining were calculated as follows: Number of apoptotic cells / total number of cells × 100%.

Western blotting was performed on the myocardium from the AAR obtained following 40 min of reperfusion for quantification of the levels of total and phosphorylated (p-) STAT-3, Akt, extracellular signal-related kinase (ERK) 1/2 and glycogen synthase kinase (GSK) 3β, and following 120 min of reperfusion for Bcl-2 and Bax. Briefly, freshly frozen myocardial tissue samples were ground into small pieces (~1×1×1 mm) in liquid nitrogen. The samples were then transferred to microcentrifuge tubes containing radioimmunoprecipitation lysis buffer (~150 μl per 10 mg tissue; Beyotime Institute of Biotechnology, Shanghai, China) and 1 mM phenylmethylsulfonyl fluoride. The samples were thoroughly homogenized and kept on ice for 1 h, vortexing every 10 min. The samples were subsequently centrifuged at 20,000 × g for 30 min at 4°C, and the supernatants were transferred into fresh tubes and kept on ice. Following protein quantification using a Bicinchoninic Acid assay (Beyotime Institute of Biotechnology), equal quantities of protein (50 μg) were separated on 10% Tris-glycine sodium dodecyl sulfate gels (Beyotime Institute of Biotechnology), and transferred onto a polyvinylidene difluoride membrane (EMD Millipore, Billerica, MA, USA). Subsequent to blocking with 5% non-fat milk for 2 h and washing twice with Tris-buffered saline containing 0.05% Tween (TBST; 5 min/wash; Goodbio), the membranes were incubated overnight at 4°C with the following primary antibodies: Rabbit monoclonal anti-Bcl-2 (1:1,000; cat. no. 2870; Cell Signaling Technology, Inc., Danvers, MA, USA) and rabbit polyclonal anti-Bax (1:300; cat. no. sc-493; Santa Cruz Biotechnology, Inc., Dallas TX, USA) to quantify apoptotic signaling, rabbit monoclonal anti-⁷⁰⁵Tyr-p-STAT-3 (1:1,000; cat. no. 9145), rabbit polyclonal anti-total STAT-3 (1:1,000; cat. no. 9132), rabbit monoclonal anti-⁴⁷³Ser-p-Akt (1:1,000; cat. no. 4060), rabbit polyclonal anti-total Akt (1:800; cat. no. 9272), rabbit monoclonal anti-^202/204Tyr-p-ERK1/2 (1:1,000; cat. no. 4370), rabbit polyclonal anti-total ERK1/2 (1:1,000; cat. no. 9102), rabbit monoclonal anti-⁹Ser-p-GSK-3β (1:2,000; cat. no. 5558) and rabbit monoclonal anti-total GSK-3β (1:1,000; cat. no. 9315) (Cell Signaling Technology, Inc.) to quantify salvage signaling pathways. Mouse monoclonal anti-β-actin (1:3,000; cat. no. A1978; Sigma-Aldrich) was used as a loading control. Following primary antibody incubation, the membranes were washed five times with TBST (5 min/wash) and incubated with the horseradish peroxidase-conjugated goat anti-rabbit (1:5,000; cat. no. sc-2004) or goat anti-mouse (1:3,000; cat. no. sc-2005) IgG secondary antibodies (Santa Cruz Biotechnology, Inc.). Subsequently, the membranes were washed five times with TBST (5 min/wash), and the bands were detected using Western Blotting Luminol reagent (cat. no. sc-2048; Santa Cruz Biotechnology, Inc.) and quantified by densitometric analysis of digitized autoradiograms using Quantity One version 4.6.2 software (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Each immunoblotting experiment was repeated three times, and the averages of the results were calculated.

All values are expressed as the mean ± standard deviation. All data analyses were performed using SPSS statistical software, version 17.0 (SPSS, Inc., Chicago, IL, USA). Differences in the hemodynamic indexes were compared within groups using repeated-measures analysis of variance, and between groups using two-way analysis of variance followed by a least significant difference (LSD) corrected multiple comparisons test. Differences in other variables between groups were evaluated using one-way analysis of variance, followed by Fisher's post-hoc LSD-corrected multiple comparisons test. P<0.05 was considered to indicate a statistically significant difference.

The AAR was similar among the groups (data not shown). Compared with the IR group (49.45±6.59%), IS was significantly reduced in the RIPerC (34.36±5.87%) group and RIPostC (36.04±6.16%) group (P<0.05; Fig. 2). However, no further reductions in IS were observed in the RIPerC + RIPostC group, compared with either the group exposed to RIPerC alone or the group exposed to RIPostC alone (31.43±5.43%; Fig. 2).

As shown in Fig. 3A and B, the results of the TUNEL staining demonstrated that the percentages of positively-stained cells were lower in the RIPerC (22.35±4.22%) and RIPostC (24.63±4.44%) groups, compared with the IR group (35.81±5.27%; P<0.05). The combination of RIPerC and RIPostC did not further reduce the percentage of apoptotic cells, compared with the percentages of apoptosis in either of the groups exposed to RIPerC or RIPostC alone (20.33±3.67%). Similarly, the protein expression ratio of Bcl-2/Bax was found to be higher in all the conditioning groups, compared with the IR group (P<0.05), with no significant differences identified among the groups (Fig. 3C and D).

Following 2 h of reperfusion, the serum levels of cTnI were significantly increased in the IR group, compared with the sham group. However, the increase in the levels of cTnI were significantly attenuated in the RIPerC, RIPostC and RIPerC + RIPostC groups (P<0.05, vs. IR group), with no significant differences observed among the three groups (Table I).

Compared with the sham group, the serum levels of TNF-α and IL-1β following 2 h of reperfusion were significantly increased in the IR group (P<0.05, vs. sham group), and were significantly attenuated by RIPerC, RIPostC and RIPerC + RIPostC (P<0.05, vs. IR group). However, no differences were observed between the groups (Table II).

The heart rates were observed to be consistent among the groups at all time points (data not shown). During the periods of ischemia and reperfusion, there were increases in LVEDP, and a significant reduction in MAP, +dP/dt_max and −dP/dt_max in each group, compared with the data at baseline (P<0.05, vs. baseline). However, compared with the IR group, none of the conditioning methods had a significant effect on MAP, LVEDP, +dP/dt_max or −dP/dt_max at any given time point. Individual group data are presented in Table III.

The protein levels of total Akt, ERK1/2, GSK-3β (RISK pathway) and signal transducer and activator of transcription (STAT) 3 (SAFE pathway) were found to be similar among the groups. The levels of p-Akt, p-ERK1/2, p-GSK-3β and p-STAT-3 are expressed as densitometric levels, normalized by levels of total protein.

In the IR group, the phosphorylation levels of Akt, ERK1/2, GSK-3β and STAT-3 were significantly increased following 40 min of reperfusion, compared with the sham group (P<0.05, vs. sham group; Fig. 4). In the RIPerC, RIPostC and RIPerC + RIPostC groups, further increases in the phosphorylation levels of STAT-3, Akt, ERK1/2 and GSK-3β were detected (P<0.05, vs. IR group; Fig. 4), however, no significant differences were observed among the three groups.