A total of 110 male Wistar rats, weighing 100±10 g, were purchased from Changsheng Biotechnology Co., Ltd. All rats were housed in an environment of constant temperature (22-25°C) and relative humidity (50-60%) on a 12/12 h light/dark cycle, and food and water were provided ad libitum. To establish the hypercholesterolemic rat model, the rats were divided in two groups as follows: i) High-cholesterol diet (HCD) group (n=90), rats were consecutively fed a cholesterol-enriched feedstuff, including 1.5% cholesterol, 10% egg yolk powder, 10% lard, 0.5% sodium cholate, 3% sugar and 75% normal feedstuff for 12 weeks; and ii) normal diet (ND) group (n=20), rats were consecutively fed normal feedstuff for the same time period. After 12 weeks, 1 ml blood samples were collected from the tail vein to determine the serum values of total cholesterol (TC), high-density lipoprotein-cholesterol and low-density lipoprotein (LDL)-cholesterol using commercial kits (Nanjing Jiancheng Bioengineering Institute, cat. nos. A111-1, A112-2 and A113-2) according to the manufacturer's instructions. All rats were handled according to the Guide for the Care and Use of Laboratory Animals (National Institutes of Health) (21). The experimental protocol was approved by the Institutional Ethics Committee of China Medical University (Shenyang, China).

Heart preparation was performed as previously described (22). Briefly, the rats were anesthetized with intraperitoneal injection of pentobarbital sodium (30 mg/kg). Each rat heart was rapidly isolated and then placed into an iced heparinized K-H solution (NaCl 0.15 mol/l, KCl 0.006 mol/l, CaCl₂ 0.002 mol/l and NaHCO₃ 0.002 mol/l) for trimming. Subsequently, the isolated rat heart was rapidly mounted to the Langendorff apparatus through the aorta and perfused with K-H solution saturated with 95% O₂ and 5% CO₂ at 75 mmHg and 37°C. A water-filled latex balloon was inserted into the left ventricle through the left atrium and connected to a pressure transducer for pressure measurement. All isolated rat hearts were stabilized for 10 min before ischemia, and then subjected to 30 min global ischemia followed by 60 min reperfusion to create the MIRI model. As for the induction of IPoC, six cycles of brief ischemia/reperfusion [(10/10 sec) x6] were performed at the onset of sustained reperfusion.

Rats in the HCD group (n=90) were randomly divided into six groups with 15 rats per group as follows: i) IR group, isolated rat hearts were subjected to 30 min global ischemia followed by 60 min reperfusion, as previously described; ii) IPoC group, following 30 min of ischemia, six cycles of IPoC were performed at the onset of reperfusion, followed by 58 min of reperfusion; iii) 5 mg/kg/day LP treatment (LP-5) group, LP (CAS no. 502-65-8, purity ≥90%), purchased from Meilun Biological Technology Co. Ltd., was dissolved in corn oil (Sigma-Aldrich; Merck KGaA), rats were treated with LP (5 mg/kg/day) for 5 consecutive days then other experimental procedures were as described for the IR group; iv) IPoC + LP-5 group, rats were treated with LP (5 mg/kg/day) for 5 consecutive days, and then IPoC was performed as described for the IPoC group; v) 20 mg/kg/day LP treatment (LP-20) group, rats were treated with LP (20 mg/kg/d) for 5 consecutive days, then other experimental procedures were as described for the IR group; vi) sodium 4-phenylbutyrate treatment (4-PBA) group, 4-PBA (Sigma-Aldrich; Merck KGaA), an ER stress inhibitor, was intraperitoneally administered at a dose of 20 mg/kg 1 h prior to ischemia, then IR experimental procedures were as described for the IR group.

According to previous studies (23-25), the dose of LP used for intraperitoneal injection in rats ranged between 2.5 and 20 mg/kg/day; therefore, 5 mg/kg/day was selected as the low dose and 20 mg/kg/day as the high dose of LP treatment. Moreover, the period of LP treatment through intraperitoneal injection ranged between 1 and 10 days in previous studies. Therefore, an intermediate treatment period of 5 days was selected for this study. LP was administered intraperitoneally at a dose of 5 mg/kg/day for 5 consecutive days. The dose of 4-PBA was based on a previous study (26).

To assess whether LP has an effect on the level of serum lipids, 45 rats in the LP-5, IPoC + LP-5 and LP-20 group (n=15 per group) were performed lipid profile as described earlier, after received LP administration at a dose of 5 or 20 mg/kg/day for 5 consecutive days.

At the end of the reperfusion period, a 1×1×1-mm piece of cardiac tissue was isolated from the left ventricle and electron microscopic sections were prepared as previously described (27). The morphological changes of the mitochondria were observed by transmission electron microscopy (JEM-1200EX; JEOL, Ltd.).

The hearts were removed from the perfusion apparatus at the end of the reperfusion period. The left ventricular heart tissues were fixed in 4% paraformaldehyde for 24 h at room temperature, dehydrated and embedded in paraffin. The paraffin blocks were then cut into 5-µm sections. The paraffin sections were dewaxed, stained with hematoxylin for 5 min, followed by 1% hydrochloric acid alcohol differentiation for 3 sec and eosin staining for 3 min at room temperature. The pathological changes in heart tissues were observed under a light microscope and imaged. To evaluate myocardial damage quantitatively, the sections were scored by two independent observers blinded to the experimental protocol, as previously described (28): 0, no damage; 1, interstitial edema and focal necrosis; 2, diffuse myocardial cell swelling and necrosis; 3, necrosis with the presence of contraction bands and neutrophil infiltrate; and 4, widespread necrosis with the presence of contraction bands, neutrophil infiltrate and hemorrhage.

Myocardial infarct size was measured by triphenyltetrazolium chloride (TTC) staining. The hearts were harvested at the end of the reperfusion period and the heart samples were frozen at −80°C for 30 min. The frozen hearts were cut into 1-2-mm sections from the apex to the bottom and incubated in 1% TTC solution at 37°C for 30 min. Subsequently, the heart sections were washed with 1X PBS and fixed in 4% paraformaldehyde overnight at room temperature. The stained sections were photographed by a digital camera and analyzed using ImageJ2x analysis software (National Institutes of Health). The severity of myocardial infarction was reflected by the ratio of infarct size to total size.

LDH and CK-MB are released from cardiomyocytes into the coronary effluent during perfusion (29). The activities of LDH and CK-MB in the coronary effluent were determined using the LDH and CK-MB assay kits (Nanjing Jiancheng Bioengineering Institute; cat. nos. A020-1 and E006) according to the manufacturer's protocols. The absorbance value was detected at 450 nm by ultramicro microporous plate spectrophotometer (BioTek Instruments, Inc.).

To evaluate the change in cardiac function, heart rate, left ventricular developed pressure (LVDevP), positive first-order derivative of ventricular pressure (+dp/dt) and negative first-order derivative of ventricular pressure (−dp/dt) were continuously recorded using a hemodynamic system (MP150; Biopac Systems, Inc.) during the IR and IPoC the experimental procedure.

Myocardial cell apoptosis was detected by TUNEL assay using the In Situ Cell Death Detection Kit (Roche Diagnostics) according to the manufacturer's instructions. The apoptotic cells were observed under a light microscope and imaged.

The mitochondria were isolated from heart tissues using the Mitochondrial Extraction Kit (BestBio Science) according to the manufacturer's instructions. The sensitivity of mPTP to calcium was determined using the Purified Mitochondrial Membrane Pore Channel Colorimetric Assay kit (Shanghai Genmed Pharmaceutical Technology Co., Ltd.) according to the manufacturer's instructions.

The heart tissues were minced and homogenized with RIPA buffer (Beyotime Institute of Biotechnology). Following centrifugation, the supernatant was collected and protein concentrations were measured using a bicinchoninic acid protein concentration kit (Beyotime Institute of Biotechnology). After 30 µg protein was denatured at 100°C for 10 min, it was separated by SDS-PAGE on 8 or 10% and transferred to a PVDF membrane. The membrane was blocked using 5% skimmed milk for 1 h at room temperature, and then incubated with specific antibodies, including anti-glucose-regulated protein 78 (GRP78; 1:1,000; Abcam; cat. no. ab108613), anti-C/EBP homologous protein (CHOP; 1:1,000; Abcam; cat. no. ab11419), anti-AKT (1:1,000; Abcam; cat. no. ab179463), anti-phospho (p)-AKT (phospho S473; 1:1,000; Abcam; cat. no. ab81283), anti-ERK1/2 (1:1,000; Abcam; cat. no. ab17942), anti-p-ERK1/2 (phospho T202 + Y204; 1:1,000; Abcam; cat. no. ab214362), anti-glycogen synthase kinase-3β (GSK-3β; 1:1,000; Abcam; cat. no. ab93926), anti-p-GSK-3β (phospho S9; 1:1,000; Abcam; cat. no. ab107166), anti-cytochrome c (1:1,000; Abcam; cat. no. ab133504), anti-cleaved caspase-9 (1:1,000; Abcam; cat. no. ab2324), anti-cleaved caspase-3 (1:1,000; Abcam; cat. no. ab2302) and anti-β-actin (1:1,000; Zhongshan Jinqiao Biotechnology; cat. no. TA-09), at 4°C overnight. On the following day, the PVDF membrane was incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:4,000; EarthOx Life Science; cat. no. E030120-01) or goat anti-mouse IgG (1:4,000; EarthOx Life Science; cat. no. E030110-01) at room temperature for 30 min. Detection of protein bands was performed using an ECL for western blotting kit (Beyotime Institute of Biotechnology) according to the manufacturer's instructions. Relative densitometry was calculated with Image J2x analysis software.

All data are presented as mean ± SD and were analyzed using SPSS version 17.0 (SPSS, Inc., Chicago, IL, USA). Differences among three or more groups were first evaluated using one-way analysis of variance, and if the differences were significant, multiple comparison analysis was further performed with Fisher's least significant difference test. Differences between two groups were evaluated using independent samples t-test. P<0.05 was considered to indicate a statistically significant difference.

The serum levels of TC and LDL were significantly increased in the HCD group as compared with the ND group after 12 weeks (Table I). The result suggested that the hypercholesterolemic rat model was successfully established. Moreover, there was no significant difference in the serum level of TC and LDL after a 5-day LP treatment either at the dose of 5 or 20 mg/kg/day (Table II), suggesting that short-term LP treatment did not affect the serum levels of TC and LDL.

In the IPoC + LP-5 group, the swelling of the mitochondria was reduced and mitochondrial cristae were denser, as observed by electron microscopy (Fig. 1A); myocardial degeneration and inflammatory infiltration were also less extensive, as observed by light microscopy (Fig. 1B and C); myocardial infarct size (Fig. 2A and B) and the release of CK-MB and LDH (Fig. 2C and D) were reduced and the apoptosis rate was decreased (Fig. 3), whereas cardiac function, as reflected by LVDevP and ±dp/dt, was improved (Fig. 4) in the IPoC + LP group compared with the IR group under conditions of hypercholesterolemia, although no beneficial effects were observed in the LP-5 and IPoC only groups. These results suggest that LP pretreatment may preserve or restore the cardioprotective effect of IPoC in hypercholesterolemic rats. Moreover, the LP-20 and 4-PBA groups displayed beneficial effects similar to the effects in the IPoC + LP-5 group, suggesting that high-dose LP treatment or pharmacological ER stress inhibition may protect hypercholesterolemic rats against MIRI.

As shown in Fig. 5, the protein expression of GRP78 and CHOP (markers of ER stress) was markedly decreased in the IPoC + LP-5 and LP-20 groups compared with IR and IPoC groups, which was similar to the effect of the ER stress inhibitor 4-PBA. However, no significant change was observed in the IPoC and LP-5 only groups. Therefore, high-dose LP or the combination of low-dose LP and IPoC were successful in reducing ER stress in hypercholesterolemic rats.

Considering that the RISK pathway is suppressed in hypercholesterolemic rat hearts (12,13), the effect of LP, alone or combined with IPoC, on the activation of AKT, ERK1/2 and GSK-3β was investigated. The results revealed that the phosphorylation of AKT (Fig. 6A), ERK1/2 (Fig. 6B) and GSK-3β (Fig. 6C) were enhanced in the IPoC + LP-5, LP-20 and 4-PBA groups, compared with the IR group. However, no significant change in the phosphorylation levels of AKT, ERK1/2 or GSK-3β was observed in the IPoC and LP-5 only groups. Thus, high-dose LP or the combination of low-dose LP and IPoC successfully activated the RISK pathway in hypercholesterolemic rats.

mPTP is the end effector of IPoC and is regulated by the RISK pathway (30). Thus, the effect of LP, alone or combined with IPoC, on mPTP opening was investigated. The results demonstrated that the sensitivity of mPTP to calcium, an inducer of mPTP opening (31), was markedly decreased in the IPoC + LP-5, LP-20 and 4-PBA groups, compared with the IR group (Fig. 7A and B), but no significant change was observed in the IPoC and LP-5 groups. mPTP opening may lead to the activation of the mitochondrial apoptosis pathway. As expected, the expressions of mitochondrial apoptosis-related proteins, including cytochrome c, cleaved caspase-9 and cleaved caspase-3, were also reduced in the LP-5, LP-20 and 4-PBA groups, compared with the IR group (Fig. 7C-E), but no significant change was observed in the IPoC and LP-5 only groups, which was in accordance with the extent of mPTP opening. Taken together, these results confirmed that high-dose LP or the combination of low-dose LP and IPoC inhibit mPTP opening in hypercholesterolemic rat hearts.