Male Sprague Dawley rats (280–320 g) were obtained from Beijing HFK Bioscience Co. Ltd. (Beijing, China). The rats were kept in an animal house with a 12-h light/dark cycle. The study was approved by the Animal Ethics Committee of Hubei University of Medicine (Shiyan, China).

PEP-1-SOD1 fusion protein was isolated and purified in accordance with the method described in one of our previous studies (13,14). Briefly, two prokaryotic expression plasmids for SOD1 and PEP-1-SOD1 (Novogen, Billerica, MA, USA) were constructed using the TA-cloning method. Six histidine residues (His-tag; Promega, Madison, WI, USA) were used to tag the two recombinant at the amino terminus. The two proteins were expressed and purified separately, as previously described (14).

The rats were systemically heparinized [1,000 U/kg, intraperitoneal (i.p.)] and anesthetized with sodium pentobarbital (60 mg/kg, i.p.; Shanghai No.1 Biochemical and Pharmaceutical Co., Ltd., Shanghai, China). The heart was rapidly excised and a modified Langendorff apparatus was used to retrogradely perfuse the heart via the aorta under constant pressure (80 mmHg) (95% O₂ and 5% CO₂) with Krebs-Henseleit buffer (118 mmol/l NaCl, 24.5 mmol/l NaHCO₃, 4.7 mmol/l KCl, 1.2 mmol/l KH₂PO₄, 3.4 mmol/l MgSO₄·7H₂O, 2.5 mmol/l CaCl₂ and 5.5 mmol/l glucose; Shanhhai Zongze Biotechnology, Co., Ltd., Shanghai, China) at 37°C, as previously described (15). A Kreb's solution-filled latex balloon was inserted into the left ventricle in order to monitor the left ventricular hemodynamic parameters using a digital acquisition and analysis system (Chengdu Taimeng Technology Co., Ltd., Chengdu, China). The hemodynamic parameters included heart rate (HR), left ventricular systolic pressure (LVSP) and the maximal rate of pressure rise (+dp/dtmax). Coronary flow (CF) was monitored by collecting pulmonary artery effluent. Following the recording of baseline data, each heart was subjected to 30 min of global ischemia, followed by 60 min of reperfusion. The elevation of the S-T segment on the electrocardiogram was indicative of the successful induction of ischemia.

The rats were assigned to one of six groups (n=10/group), as follows: Sham (no global ischemia), control (subjected to global ischemia), PEP-1-SOD1-treated (25, 50 and 100 µmol/l) and SOD1-treated (100 µmol/l). In the drug-treated groups, PEP-1-SOD1 and SOD1 were administered as components of the perfusion medium 15 min prior to ischemia. The hearts of the rats in the untreated group were perfused for an additional 15 min with perfusion medium only.

Coronary effluent was collected 60 min after reperfusion in order to measure the activity of lactate dehydrogenase (LDH) and serum creatine kinase-MB (CK-MB) using assay kits obtained from the Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Enzyme activity was determined spectrophotometrically.

Myocardial infarct size was assessed with Evans Blue dye and 2,3,5-triphenyltetrazolium chloride (TTC) (Sigma-Aldrich, St. Louis, MO, USA). Following the excision of the heart, the left ventricle was cut into 2-mm transverse slices, from apex to base. Following incubation in 1% TTC (pH 7.4) at 37°C for 15 min, the slices were placed in 4% formaldehyde for 1 day. The infarcted myocardium was not stained by the TTC and thus appeared white, while the non-ischemic myocardium was stained brick-red by the TTC. Infarct area analyses were performed using ImageJ software (National Institutes of Health, Bethesda, MD, USA), and the results are expressed as the percentage of left ventricular volume for each heart.

Arrhythmias were evaluated based on the guidelines of the Lambeth Conventions (16). Ventricular tachycardia (VT) was defined as the occurrence of ≥4 consecutive premature ventricular contractions, while ventricular fibrillation (VF) was defined as an inability to distinguish individual QRS deflections. The prevalence and total duration of VT and VF during the reperfusion period were measured.

ROS generation was measured with the sensitive fluorescent probe 2′,7′-dichlorofluorescein diacetate (DCFH-DA; Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Cardiomyocytes were dispersed from the hearts using digestion buffer, washed with KB solution and then incubated with 5 µmol DCFH-DA for 20 min, according to the instructions of commercial kits. The fluorescence intensity was measured using a fluorospectrophotometer with 488-nm excitation and 525-nm emission filters.

All experiments were performed in triplicate, and the data are presented as the mean ± standard deviation. Statistical significance was determined using one-way analysis of variance. P<0.05 was considered to indicate a statistically significant difference.

The LVSP, +dp/dtmax, CF and HR in the control group were observed to have decreased markedly after 60 min of reperfusion (P<0.01) (Table I). Compared with the control group, a significant recovery of LVSP, +dp/dtmax and CF was apparent in the PEP-1-SOD1-treated groups, and this effect was concentration-dependent (P<0.05). The rats in the SOD1 pretreated group had less obvious improvements in LVSP, +dp/dtmax and CF. No significant difference in HR was found between the control and pretreated groups.

The activity of the cardiac biomarkers in the coronary effluent was measured as an index of myocardial cellular injury following I/R and found to be elevated during reperfusion. As shown in Fig. 1, the LDH (Fig. 1A) and CK-MB (Fig. 1B) activities were significantly increased in the control group compared with those in the sham group. Pretreatment with PEP-1-SOD1 markedly decreased the LDH activity after 60 min of reperfusion compared with the control (P<0.05). Similar to the LDH activity, the CK-MB activity was also significantly reduced by PEP-1-SOD1 pretreatment (P<0.05).

As shown in Fig. 2A and B, infarct size was significantly decreased in the PEP-1-SOD1-pretreated groups compared with that in the control group. The effect of PEP-1-SOD1 was concentration-dependent (P<0.05).

To assess the I/R-induced arrhythmia, all ventricular arrhythmias that occurred during the 60-min reperfusion period were recorded. In the sham group, no obvious ventricular arrhythmias were noted. Compared with the control group, PEP-1-SOD1 significantly reduced both the duration and incidence of VT/VF (Fig. 3A and B). I/R-induced arrhythmias were additionally evaluated using a scoring system. The I/R-induced arrhythmia score was 4.3 in the control group; however, PEP-1-SOD1 administration was shown to significantly decrease this score (Fig. 3C, P<0.05).

It is generally accepted that ROS are one of the major contributors to I/R-induced myocardial damage; therefore, the effect of PEP-1-SOD1 on I/R-induced ROS generation was measured. As shown in Fig. 4, the intracellular ROS level in the control group increased to 303% of the level of the sham group. Significant reductions in ROS levels were observed in the PEP-1-SOD1-treated groups compared with the control group (P<0.05).