A total of 105 male Wistar rats purchased from the Center of Experimental Animals, China Medical University (Shenyang, Liaoning, China), weighing 200±10 g were used in the present study. All of the animals used in this study were treated in accordance with the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health (NIH). The study procedure was approved by the institutional Ethics Committee of China Medical University (Shenyang, China).

Fasudil was purchased from Chase Sun Pharmaceutical Company (Tianjin, China). Wortmannin, L-NAME and 2,3,5-triphenyl tetrazoliumchloride (TTC) were purchased from Sigma (St. Louis, MO, USA).

Prior to the initiation of the 8-week feeding period, the blood samples were obtained from the rats’ vena caudalis for determination of the plasma levels of total cholesterol (TC), low density lipoprotein (LDL) and high density lipoprotein (HDL) using commercial kits (Total Cholesterol Assay kit - Fluoro Cholesterol; Cell Technology, Mountain View, CA, USA; Cholesterol LDL direct; Biosystems S.A., Barcelona, Spain; and Cholesterol HDL direct, Biosystems S.A). All of the animals were fed with a diet enriched with 1.5% cholesterol, 5% egg yolk powder, 10% lard, 0.5% sodium cholate, 3% sugar and 80% normal feed for 8 weeks, and this formula was modified as previously described (19). Following the 8-week feeding period, the blood samples were obtained from the rats’ vena caudalis again for determination of serum lipid in order to judge the success of hypercholesterolemic models.

The rats were anesthetized with an intraperitoneal injection of pentobarbital sodium (100 mg/kg). Heparin (1,500 IU/kg) was administered intravenously to prevent intracoronary clot formation. The heart was rapidly excised and immediately immersed in ice-cold heparinized modified Krebs-Henseleit buffer containing (in per mmol) 127 NaCl, 17.7 NaHCO₃, 5.1 KCl, 1.5 CaCl₂, 1.26 MgCl₂ and 11D-glucose (pH 7.4). The heart was mounted on a Langendorff-perfusion apparatus and retrogradely perfused through the aorta with recirculating buffer saturated with 95% O₂-5% CO₂ at 37°C. The heart was maintained in a thermostatic chamber at 37°C. Perfusion was maintained at a constant pressure of 75 mmHg. The fluid-filled latex balloon was inserted in the left ventricle (LV) via the left atrium for pressure measurement. The balloon was connected to a pressure transducer and inflated to an initial LV end-diastolic pressure between 8 and 10 mmHg.

The rats were further divided into seven groups with 15 animals/group. In all of the groups, the isolated rat hearts were perfused with a K-H solution (127 mM NaCl, 17.7 mM NaHCO₃, 5.1 mM KCl, 1.5 mM CaCl₂, 1.26 mM MgCl₂ and 11mM D-glucose, pH 7.4) and allowed 10 min of stabilization. Next, all of the isolated rat hearts were subjected to 30 min global ischemia and 120 min reperfusion.

In the control group, the isolated rat hearts were subjected to 30 min global ischemia and 120 min reperfusion. For the ischemic postconditioning group (IPoC group): the isolated rat hearts were subject to ischemia for 30 min, followed by six cycles of reperfusion and ischemia, both with equal lengths of 10 sec and reperfusion for 120 min. In the fasudil preconditioning group (FD group), the isolated rat hearts were perfused with K-H solution containing 1 μM dose fasudil 15 min prior to ischemia. In the ischemic postconditioning+fasudil group (IPoC+FD group), 1 μM fasudil was added to the perfusate for 15 min prior to ischemia, then 30 min ischemia was followed by six cycles of reperfusion and ischemia, both with equal lengths of 10 sec and reperfusion for 120 min.

To investigate whether a higher concentration of fasudil alone was able to restore cardioprotection in hypercholesterolemic rat heart, an additional group with 10 μM fasudil treatment prior ischemia was also established. In the high dose fasudil preconditioning group (FD2 group), the isolated rat hearts were perfused with K-H solution containing 10 μM fasudil 15 min prior to ischemia

To further determine whether the PI3K/Akt/eNOS pathway participated in regulating fasudil restored cardioprotection of IPoC in the state of hypercholesterolemia, two more groups were added to the experimental studies, with PI3K specific inhibitor wortmannin and eNOS specific inhibitor L-NAME treatment. In the ischemic postconditioning+fasudil+wortmannin group (IPoC+FD+wortmannin group), wortmannin at the dose of 30 μM was administrated immediately following IPoC. The ischemic postconditioning and fasudil preconditioning were performed as described above. In the ischemic postconditioning+fasudil+L-NAME group (IPoC+FD+L-NAME group), L-NAME at the dose of 30 μM was administrated immediately following IPoC. Ischemic postconditioning and fasudil preconditioning were conducted as described above.

The homodynamic assessment included heart rate (HR), left ventricular developed pressure (LVDP), positive first order derivative of ventricular pressure (+dp/dt) and negative first order derivative of ventricular pressure (−dp/dt). These parameters were continuously monitored throughout the experimental procedures. The HR, LVDP, +dp/dt and −dp/dt were sampled and digitally processed via a homodynamic system (BIOPAC MP150; BIOPAC systems, Goleta, CA, USA).

The infarct size was determined as previously described (20). Briefly, following 2 h reperfusion, the hearts were harvested and the LVs were sectioned from the apex to the base into 2–3 mm sections, following incubation for 20 min at 37°C in 1% triphenyltetrazolium chloride (TTC). The unstained tissue was carefully separated from the stained tissue by an independent observer. While the unstained tissue represented the dead cells, the stained tissue represented the viable cells. The unstained mass was expressed as a percentage of total LV mass. The total LV mass also corresponded to the risk area because a global ischemia was induced.

At the end of 2 h reperfusion, the heart was removed as described above. Cardiomyocyte apoptosis was detected using an In Situ Cell Death Detection kit (Roche, South San Francisco, CA, USA) according to the manufacturer’s instructions. Briefly, the tissue sections were washed in PBS and then fixed in 4% paraformaldehyde solution prior to incubation in 20 μg/ml proteinase K for 10 min. Following being washed in PBS for three times, the tissue sections were incubated with terminal deoxynucleotidyl transferase enzyme (TUNEL) in a humidified chamber at 37°C for 60 min for incorporation of the biotinylated nucleotides at the 3′-OH DNA ends. The reaction was terminated by transferring the slides to a 2× sodium citrate saline solution. Endogenous peroxidase activity was quenched by incubation in 0.3% hydrogen peroxide. Finally, streptavidin horseradish peroxidase was bound to the biotinylated nucleotides and the peroxidase activity was demonstrated in each section by the application of a stable chromogen diaminobenzidine. In this technique, the apoptotic nuclei are stained dark brown. The sections were counter stained with hematoxylin for total nuclei. Three sections from each myocardial sample were randomly selected and ten microscopic fields (Olympus BX51 microscope; Olympus, Tokyo, Japan) per section were evaluated by two independent blind observers. In each field, the nuclei were counted and the percentage of TUNEL-positive nuclei was calculated.

After 30 min of global ischemia followed by 120 min of reperfusion, the hearts were removed rapidly from the Langendorff apparatus and homogenized. The content of NO was measured using Nitric Oxide assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instructions.

At the end of 30 min reperfusion, the LVs were homogenized in a lysis buffer [(in mmol/l) 10 Tris-HCl, 20 ortho-phosphate, 1 EGTA, 1 EDTA, 2 Na₃VO₄, 1 phenylmethylsulfonyl fluoride; pH 7.4]. Following sonication, the lysates were centrifuged, the proteins were separated by electrophoresis on SDS-PAGE and transferred onto polyvinylidenedifluoride-plus membranes. The membranes were blocked with 5% skimmed milk followed by incubation overnight at 4°C with the antibodies: MYPT-1 (1:300; Abcam); phospho-MYPT-1 (at Ser853,1:300; Abcam, Hong Kong, China); Akt (1:200; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA); phospho-Akt (at Ser473, 1:200; Santa Cruz Biotechnology, Inc.); eNOS (1:200; Santa Cruz Biotechnology Inc.); phospho-eNOS (at Ser1177, 1:200; Santa Cruz Biotechnology Inc.). Following incubation, the membranes were washed three times with 0.1% Tween-20 for 15 min and incubated with horseradish peroxidase for 2 h. The levels of phosphorylated proteins were normalized to their total protein levels. The relative densitometry was performed using a computerized software package (NIH Image 1.63 software).

The data are expressed as the mean ± standard deviation values. The statistical analysis was performed using Sigma Stat software version 3.5 (Systat software). The differences between the groups were evaluated using one-way analysis of variance, followed by Student-Newman-Keuls post hoc test. P<0.05 were considered to indicate a statistically significant difference.

Table I summarizes the average values for TC, HDL and LDL in the plasma of animals prior to and following the 8 week cholesterol-enriched diet. A significant increase (P<0.05) in the average TC and LDL values were observed in the animals fed with the cholesterol-enriched diet as compared with the control group. There was no significant difference in the value of HDL between the cholesterol-enriched diet group and the control group (P>0.05).

Table II demonstrates the values of HR, LVDP, +dp/dt and −dp/dt at baseline and different times of reperfusion. There were no significant differences among the groups in HR, LVDP, +dp/dt and −dp/dt at baseline. No significant differences in HR, LVDP, +dp/dt and −dp/dt were observed in the IPoC and FD groups compared with the control group (P>0.05). However, IPoC+FD group significantly increased the values of HR, LVDP, +dp/dt and −dp/dt at different times of reperfusion compared with the control group (P<0.05). Furthermore, high doses of fasudil treatment alone (FD2 group) also improved the values of HR, LVDP, +dp/dt and −dp/dt at different times of reperfusion compared with the control group (P<0.05).

As demonstrated in Fig. 1, no significant decrease in infarct size was observed in IPoC group and FD group as compared with the control group (42.3±6.5 and 40.9±8.9%, respectively, vs. 44.4±7.9%, P>0.05). However, the IPoC+FD group had a significantly reduced infarct size as compared with the control group (26.3±8.5 vs. 44.4±7.9%, P<0.05), suggesting that low dose fasudil preconditioning restored the cardioprotection of IPoC in hypercholesterolemic conditions. In addition, a high dose of fasudil treatment alone (FD2 group) also reduced infarct size as compared with the control group (30.8±11.3 vs. 44.4±7.9%, P<0.05).

The percentage of apoptotic cardiomyocytes of all of the experimental groups is demonstrated in Fig. 2. The IPoC+FD group and FD2 group significantly reduced cardiomyocyte apoptosis as compared with the control group (24.7±4.5 and 29.6±9.8%, respectively, vs. 40.6±5.0%, P<0.05). However, IPoC and low dose of fasudil treatment alone failed to exert the anti-apoptosis effect, as evidenced by no significant decrease in the percentage of cardiomyocyte apoptosis in IPoC and FD groups, as compared with the control group (39.1±6.4 and 38.5±6.4%, respectively, vs. 40.6±5.0%, P>0.05).

As revealed in Fig. 3, the content of NO in rat myocardium was significantly increased in the IPoC+FD group, as compared with the control group (6.15±1.60 vs. 2.60±0.78, P<0.05), but the content of NO was not increased in the IPoC and FD groups (2.77±1.20 and 2.65±2.31, respectively, vs. 2.60±0.78, P>0.05). High dose fasudil preconditioning also markedly enhanced the content of NO in the rat myocardium as compared with the control group (4.12±1.43 vs. 2.60±0.78, P<0.05).

The Rho-kinase activity was assessed by examining the phosphorylation of MYPT-1, a well-established Rho-kinase specific substrate. As demonstrated in Fig. 4A and B, IPoC+FD treatment resulted in a significant reduction in MYPT-1 phosphorylation as compared with the control group (0.126±0.006 vs. 0.310±0.024, P<0.05), whereas no significant decrease in MYPT-1 phosphorylation was revealed in the IPoC and FD groups (0.289±0.039 and 0.323±0.019, respectively, vs. 0.310±0.024, P>0.05).

As demonstrated in Fig. 5A and B, the Akt phosphorylation was enhanced in IPoC+FD group, as compared with the control group (0.951±0.146 vs. 0.402±0.166, P<0.05), but there is no significant change in Akt phosphorylation in the IPoC and FD groups, as compared with the control group (0.445±0.170 and 0.390±0.024, respectively, vs. 0.402±0.166, P>0.05). Furthermore, as revealed in Fig. 5C and D, the eNOS phosphorylation was also enhanced in the IPoC+FD group as compared with the control group (1.043±0.124 vs. 0.516±0.037, P<0.05), whereas no significant change in eNOS phosphorylation was demonstrated in the IPoC and FD groups as compared with the control group (0.577±0.023 and 0.575±0.026, respectively, vs. 0.516±0.037, P>0.05).

To further assess the role of PI3K/Akt/eNOS pathway in the restoration of cardioprotection of IPoC in hypercholesterolemic myocardium by fasudil, the PI3K/Akt specific inhibitor, wortmannin and eNOS specific inhibitor, L-NAME in fasudil combined with IPoC treatment group were administered. The results demonstrated that the cardioprotection of fasudil combined with IPoC treatment in hypercholesterolemic myocardium was abrogated by treatment with wortmannin and L-NAME alone, as evidenced by no significant differences in hemodynamic parameters, myocardial infarct size, the percentage of cardiomyocyte apoptosis and the content of NO were demonstrated in IPoC+FD group, as compared with the control group.