The present study was conducted with the approval of the Institutional Animal Care and Use Committee at West China Hospital of Sichuan University. A total of fifty 10-week-old male Wistar rats weighing 220–250 g were obtained from Hua Fu Kang Experimental Animal Center (Beijing, China). The rats were housed and acclimated in a specific pathogen-free facility with a 12 h light:dark cycle and fed with chow food and water ad libitum.

As previously described (11), the rats were anesthetized with pentobarbital sodium (100 mg/kg, intraperitoneally). Rats were subsequently placed in a supine position and secured on an electric heating pad to maintain constant body heat at 37°C. Catheters were cannulated into the carotid artery and jugular vein for arterial blood pressure monitoring and drug administration. The electrocardiogram was monitored with subcutaneous stainless steel electrodes in the chest. A left thoracotomy through a left parasternal incision was performed to expose the anterior wall of the left ventricle. Following a stabilization period of 20 min, myocardial ischemia was induced by 4-0 silk suture slipknot at the level of the proximal left anterior descending coronary artery for 30 min. Reperfusion was started by releasing the slipknot and the heart was harvested following a 2 h reperfusion.

The Wistar rats were randomly allocated into five groups (n=10 in each group; Fig. 1): i) Sham-operated control (SO) group (rats were subjected to surgical manipulation without any induction of I/R injury); ii) I/R group (rats were subjected to the coronary artery occlusion for 30 min followed by 2 h reperfusion); iii) EGCG group (EGCG 10 mg/kg, administered intravenously at 5 min prior to the onset of reperfusion); iv) EGCG + wortmannin (WOR) group (WOR, a PI3K inhibitor, 0.6 mg/kg + EGCG 10 mg/kg administered intravenously at 10 min prior to the reperfusion); and v) WOR group (WOR alone intravenously administered at 10 min prior to the reperfusion). The dosage of EGCG and WOR was determined according to previous studies (11–13).

At the end of reperfusion, blood samples were collected and centrifuged at 500 × g for 15 min at 4°C to assess the serum levels of lactate dehydrogenase (LDH) and creatine kinase (CK) by using commercial assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The hearts were subsequently harvested and the left anterior descending artery was occluded again. Diluted fluorescent polymer microspheres (Duke Scientific Corporation, Palo Alto, CA, USA) were infused into the aorta to demarcate the infarct area. Subsequent to being frozen at −20°C, the hearts were cut into 2 mm transverse sections and incubated in 1% 2,3,5-triphenyltetrazolium chloride (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) at 37°C for 20 min. The infarct regions and the area at risk were quantified using Image-Pro Plus (version 3.0; Media Cybernetics, Silver Spring, MD, USA). The infarct regions and area at risk were converted into volumes by multiplying them by section thickness.

Western blot analysis of the protein levels for phosphorylated (p)-p85, total (t)-Akt and p-Akt in myocardial tissues was carried out as previously reported (14). Antibodies against p-p85 (1:1,000; catalog no. ab182651; Abcam, Cambridge, MA, USA), p-Akt (1:500; catalog no. 4051; Cell Signaling Technology, Inc., Danvers, MA, USA) and t-Akt (1:1,000; catalog no. 2920; Cell Signaling Technology, Inc.) were used to probe the membranes, followed by incubation with a horseradish peroxidase-conjugated secondary antibody (1:500; catalog no. 111-035-003; Jackson ImmunoResearch Laboratories, West Grove, PA, USA). β-actin (1:300; catalog no. A0A068F1Y2; Abmart (Shanghai) Co., Ltd., Shanghai, China,) was used for normalization. The reactive bands were visualized using the ECL-Plus reagent (Amersham Biosciences Corp., Piscataway, NJ, USA). The density of each reactive band was quantified using the Labworks image acquisition platform and its related analytic software (GDS8000; UVP, Inc., Upland, CA, USA).

As previously reported (16), the whole plasma DNA was isolated from plasma using the DNeasy Blood and Tissue kit (69504; Qiagen Inc., Valencia, CA, USA). A total of 50 µl plasma samples were added to 50 µl PBS and centrifuged at 700 × g at 4°C for 5 min to obtain the supernatant. All procedures were performed according to the manufacturer's protocols. Plasma mtDNA levels were measured by SYBR-Green dye-based qPCR assay (45 cycles of 95°C for 15 sec and 60°C for 1 min) using a PRISM 7300 sequence detection system. The primer sequences were rat NADH dehydrogenase 1 gene (mtDNA): Forward CGC CTG ACC AAT AGC CAT AA and reverse ATT CGA CGT TAA AGC CTG AGA. All samples were measured with standards at the same time and standard mtDNA was diluted in 10-fold serial dilutions. All measurements were conducted three times for quality control. Concentration of plasma mtDNA was converted to copy number via a DNA copy number calculator (cels.uri.edu/gsc/cndna.html; University of Rhode Island Genomics and Sequencing Center). Plasma mtDNA levels were shown in copies per µl plasma according to the following formula: c=QxV_DNA/V_PCRx1/V_ext, where c is the concentration of plasma mtDNA (copies/µl), Q is the quantity of DNA measured by RT-PCR, V_DNA is the total volume of plasma DNA solution obtained from the extraction (200 µl), V_PCR is the volume of plasma DNA solution for RT-PCR (1 µl) and V_ext is the volume of plasma used for the extraction (50 µl).

Expression levels of TNF-α and IL-6 and −8 in myocardial tissue supernatants were quantified by using specific ELISA kits (BioSource International; Thermo Fisher Scientific, Inc., Waltham, MA, USA), according to the manufacturer's protocols. Additionally, total RNA was extracted from myocardial tissues with Trizol, according to the manufacturer's protocol (Takara Bio, Inc., Otsu, Japan). The mRNA from each tissue sample was reverse transcribed with PrimeScript RT Master Mix (Takara Bio, Inc.). qPCR amplifications (45 cycles of 95°C for 15 sec and 55°C for 1 min) were carried out using the ABI 7500 system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The primers used were as follows: TNF-α forward 5′-CTGAACTTCGGGGTGATCGG-3′ and reverse 5′-GGCTTGTCACTCGAATTTTGAGA-3′; IL-6 forward 5′-CTTCCAGCCAGTTGCCTTCT-3′ and reverse 5′-GAGAGCATTGGAAGTTGGGG-3′; IL-8 forward 5′-AGCTTCCTTGTGCAAGTGTCT-3′ and reverse 5′-GACAGCCCAGGTCAAAGGTT-3′; and β-actin forward 5′-AGAGGGAAATCGTGCGTGAC-3′ and reverse 5′-CAATAGTGATGACCTGGCCGT-3′. The amount of mRNA for each gene was normalized by β-actin and the relative expression levels were calculated using the 2^−ΔΔCq method (17).

Results are expressed as the mean ± standard deviation. Differences in hemodynamic parameters and infarct measurements between groups were determined by one-way analysis of variance with Tukey's honest significant difference post hoc test. χ² analysis was used to test the differences in the incidence of ventricular arrhythmias. The Kruskal-Wallis test was used to compare the means for all nonparametric scores between groups. The Pearson correlation coefficient test was performed. All analyses were performed using SPSS (version 19.0; IBM SPSS, Armonk, NY, USA) and P<0.05 was considered to indicate a statistically significant difference.

A total of 49 rat hearts were successfully harvested for the experiment. One rat in the WOR group was excluded due to an irreversible ventricular fibrillation during the stabilization period. No significant difference in body or heart weight, areas at risk or hemodynamic parameters between groups was observed (Tables I and II).

The episodes and cumulative duration of the premature ventricular contraction, ventricular tachycardia and ventricular fibrillation were recorded. The arrhythmia scoring system suggested by Miller et al (18) was used to assess the severity of I/R-induced ventricular arrhythmia. As presented in Table III, ventricular arrhythmia scores in the I/R, EGCG+WOR and WOR groups were significantly increased compared with those in the SO and EGCG groups (P<0.05, respectively).

The infarct size induced by an I/R injury was significantly reduced by EGCG compared with the I/R group (22.5±4.2 vs. 50.0±3.2%, P<0.05). Administration of PI3K inhibitor (WOR) eliminated the cardioprotective effects of EGCG on the infarct size compared with the EGCG group (40.2±4.4 vs. 22.5±4.2%, P<0.05). However, WOR alone did not affect the infarct size compared with the I/R group (43.2±6.0 vs. 50.0±3.2%, P>0.05), suggesting that the infarct reducing effects of EGCG involves the activation of the PI3K-Akt signaling pathway (Table II). Following a 2 h reperfusion, LDH and CK levels in the I/R group were significantly increased compared with the SO group (P<0.05, respectively). However, a notable decrease of these kinases was observed in the EGCG group (Fig. 2). No significant differences in LDH and CK levels were observed between the WOR and EGCG+WOR group and those in the I/R group.

mtDNA, as a pro-inflammatory agent, is released following I/R injury to cause inflammatory damage to the heart. In order to study the role of EGCG on mtDNA release, blood samples were collected from all rats. As demonstrated in Fig. 3, EGCG significantly reduced the plasma levels of mtDNA following I/R injury; 2.13-fold lower compared with the I/R group (P<0.05). Additional treatment with WOR is able to eliminate the mtDNA reducing effects of EGCG, implying that EGCG may exert its inhibitory effect on mtDNA release by activating PI3K-mediated signaling pathways.

To investigate the role of the PI3K/Akt signaling pathway in the effect of EGCG, the expression of the p-p85 (an activated regulatory subunit of PI3K), p-Akt and t-Akt were examined. As demonstrated in Fig. 4A and B, EGCG significantly increased the expression levels of p-p85 (62.3±4.3% of the I/R group, P<0.05) and p-Akt (36.4±6.3% of the I/R group, P<0.05). However, the upregulating effects of EGCG on p-p85 and p-Akt expression were abolished by WOR.

As demonstrated in Fig. 5A and B, the protein levels of TNF-α, IL-6 and IL-8 in heart tissues were significantly higher in the I/R group than those in the SO group. Consistently, an I/R injury increased the mRNA expression of TNF-α by 3.68-fold, IL-6 by 3.75-fold and IL-8 by 2.89-fold compared with those in the SO group. It is noted that EGCG markedly reduced the protein and mRNA levels of these cytokines. However, the anti-inflammatory effect of EGCG could be inhibited by WOR (P<0.05). Similarly, I/R-stimulated TNF-α, IL-6 and IL-8 overproduction remained unaffected in rat hearts treated with WOR alone, suggesting that the EGCG-activated PI3K/Akt signaling pathway may serve as a negative feedback mechanism in the setting of I/R-triggered inflammatory responses.

To further analyze the association between mtDNA and inflammatory cytokines, a bivariate correlation study was performed to examine these parameters in all groups with the exception of the control group. As hypothesized, positive correlations were reached between mtDNA level and TNF-α (r=0.765, P<0.01), IL-6 (r=0.665, P<0.01) and IL-8 (r=0.652, P<0.01) expression levels (Fig. 6A-C), implying that increasing mtDNA level may contribute to inflammatory cytokine expression levels.