A total of 24 healthy male C57BL/6 mice at the age of eight weeks, ~25–30 g in weight, were obtained from the experimental Animal Center of the Harbin Medical University (Harbin, China). The mice were randomly divided into the sham group, myocardial ischemia (MI) group, MI+EPI group and MI+EPI+LY294002 group, and n=6 in each group. Subsequently, according to the different groups, the mice were subjected to a sham or MI operation and the mice that underwent MI surgery were administered EPI or EPI+LY294002. The animals were kept under standard animal room conditions (atmosphere 0.03% CO₂; temperature 21±1°C; humidity 55–60%; 12-h light-dark cycle) with laboratory pellet food and autoclaved water freely available. All experimental procedures were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals set by the US National Institutes of Health (Bethesda, MD, USA), which was approved by the Institutional Animal Care and Use Committee of the Harbin Medical University.

To induce myocardial ischemia, mice were anesthetized with Avertin (160 mg/kg; intraperitoneal injection; Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) and their chests were opened to expose the hearts. The left descending coronary artery was ligated with a 7/0 nylon suture at 2 mm below the border between the left atrium and ventricle to induce myocardial ischemia. Ischemia was confirmed by visual observation (cyanosis) and by observing ST segment elevation and QRS widen on electrocardiogram. The sham group mice underwent the same experimental procedures as the MI group but without ligation of left descending coronary artery. Prior to the myocardial ischemia operation, the mice in the MI+EPI (cat. no. 68097; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) and MI+EPI+LY294002 (cat. no. L9908; Sigma-Aldrich, Merck KGaA) groups were treated with 1 mg EPI per kg body weight by oral gavage every day for 10 days. Additionally, the mice in the MI+EPI+LY294002 group were treated with LY294002 at 10 mg/kg body weight by intraperitoneal injection 30 min before treatment of EPI every day for 10 days. The mice in the sham group were administered with an equal dosage of saline. All mice were sacrificed at day 7 after the myocardial ischemia operation.

Cardiomyocytes were isolated from neonatal C57BL/6 mice (aged 1–2 days, weight 1–3 g, a random mix of male or female; purchased from the experimental Animal Center of Harbin Medical University) as previously described, from mice that were sacrificed following purchase (18). Briefly, the heart was digested by pancreatin (cat no. C0201; Beyotime Institute of Biotechnology, Haimen, China) and maintained in Dulbecco's modified Eagle's medium (DMEM; cat no. 11965084; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 100 U/ml penicillin and 10% fetal bovine serum (FBS; cat no. 04-001-1A; Biological Industries, Kibbutz Beit Haemek, Israel) (18). Cardiomyocytes were purified by differential adherence and 0.1 mM 5-bromo-2-deoxyuridine (cat no. B23151; Thermo Fisher Scientific, Inc.) was added to suppress cardiac fibroblasts for 48 h in a 5% CO₂ and 37°C humidified atmosphere. Then, as the anoxia (ANO) group, ANO+EPI group or ANO+EPI+LY group, the purified cardiomyocytes were treated with an equal amount of DMEM or EPI (5 µM) or EPI (5 µM) + LY294002 (20 µM) separately in a 5% CO₂ and 37°C humidified atmosphere for 1 h and then incubated under anoxia condition at 37°C for 12 h. As the control group, the purified cells were treated with an equal amount of DMEM in a 5% CO₂ and 37°C humidified atmosphere for 13 h.

Transthoracic echocardiography was performed to measure alterations to the left ventricular function using a Vevo2100 system (VisualSonics, Inc., Toronto, ON, Canada) equipped with a 10-MH2 phased-array transducer with the M-mode recordings. Functional parameters were evaluated and analyzed including heart rate (HR), left ventricular end-diastolic dimensions (LVEDd), left ventricular end-systolic dimensions (LVESd), left ventricular ejection fraction (EF) and fractional shortening (FS).

Hearts were collected and fixed with 4% paraformaldehyde at 4°C for 48 h, embedded in paraffin, sliced into 5-µm sections and subjected to standard hematoxylin and eosin (H&E) staining and Masson staining at room temperature for 4 h. The sections were visualized using a light microscope (80i; Nikon Corporation, Tokyo, Japan). A total of 10 fields of view each were analyzed. Image J software (version 1.46r; National Institutes of Health, USA) was used for quantitative analysis of the % area of fibrosis..

Cell viability was measured by the mitochondrial dependent reduction of MTT reagent (cat no. C0009; Beyotime Biotechnology, Shanghai, China), as previously described (19). Neonatal mouse cardiomyocytes (NMCMs) were plated onto a 96-well plate and treated with an equal amount of DMEM or EPI (5 µM) or EPI (5 µM) + LY294002 (20 µM) in a 5% CO₂ and 37°C humidified atmosphere for 1 h and then incubated under anoxia condition at 37°C for 12 h. As control group, NMCMs plated onto a 96-well plate treated with an equal amount of DMEM in a 5% CO₂ and 37°C humidified atmosphere for 13 h, as designated. A total of 20 µl MTT solution (Sigma-Aldrich, Merck KGaA) and 180 µl DMEM was added into each well and the plates were incubated at 37°C for 4 h. Then, the medium of each well was carefully removed and 200 µl DMSO was added into each well to dissolve formazan. The absorbance values were read at a wavelength of 490 nm using a microplate reader (Tecan Group, Ltd., Mannedorf, Switzerland).

For Western blot analysis, protein samples extracted from the left ventricles of mice or cultured NMCMs were used. Briefly, protein was lysed with radioimmunoprecipitation assay lysis buffer containing a complete protease inhibitor cocktail (cat. no. 4693116001; Roche Molecular Diagnostics, Pleasanton, CA, USA). The protein concentration was determined by BCA Protein Assay kit (cat no. P0012S; Beyotime Institute of Biotechnology, Shanghai, China). A total of 10 µg protein was separated by 12% SDS-PAGE and transferred onto polyvinylidene difluoride membranes. The blots were probed with primary antibodies overnight at 4°C. The primary antibodies included Bcl-2 (1:1,000; cat no. 3498), Bax (1:1,000; cat no. 14796), caspase-3 (1:500; cat no. 9665), cleaved caspase-3 (1:500; cat no. 9661), PTEN (1:1,000; cat no. 9559; all Cell Signaling Technology, Inc., Danvers, MA, USA), total AKT (T-AKT; 1:200; cat no. sc-8312; Santa Cruz Biotechnology, Inc., Dallas TX, USA), phosphorylated-AKT (p-AKT; 1:1,000; cat no. 13038) and GAPDH (1:1,000; cat no. 2118; both Cell Signaling Technology, Inc.). After washing, the membranes were incubated with a horse radish peroxidase-conjugated goat-anti-rabbit immunoglobulin G (1:1,000; cat no. A0208; Beyotime Institute of Biotechnology) for 1 h at room temperature. The signal was detected by an Enhanced Chemiluminescence reagent (cat no. 29050; Engreen Biosystem New Zealand Ltd., Auckland, New Zealand), quantified by using Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA) and analyzed by Odyssey Infrared Imaging System matched application software (version 3.0.16; LI-COR Biosciences, Lincoln, NE, USA).

Apoptosis was assessed by a TUNEL assay (Roche Molecular Diagnostics), as previously described (20). Then, 6-mm frozen sections or cells were also co-stained with DAPI (1:1,000; cat no. D3571; Invitrogen; Thermo Fisher Scientific, Inc.) at room temperature for 5 mins to visualize nuclei, and mouse monoclonal anti-sarcomeric α-actinin antibody (1:1,000; cat no. A7811; Sigma-Aldrich; Merck KGaA) at 4°C overnight. Then the sections or cells were incubated with goat-anti-mouse IgG-Alexa Fluor 594 at 37°C for 2 h (1:300; cat no. A-11032; Invitrogen; Thermo Fisher Scientific, Inc.). The average of TUNEL-positive cell ratio in at least five representative microscopic fields was calculated with ZEN2012 (version 1.1.2.0; Carl Zeiss AG, Oberkochen, Germany) to compare the apoptosis ratio within different groups.

All data was expressed as the mean ± standard error. GraphPad Prism version 5.0 software (GraphPad Software, Inc., La Jolla, CA, USA) was used for statistical analysis. For two group comparisons, an unpaired Student t-test was used. For multiple group comparisons, a one-way analysis of variance with Bonferroni post-test was used for comparisons between selected two groups as well as Dunnett post-test for comparisons among all other treatment groups to the corresponding control. P<0.05 was considered to indicate a statistically significant difference.

The present study confirmed the appropriate dosage of EPI. In a previously published study, EPI was administered at 1 mg/kg/day via oral gavage for 10 days (21) to maintain a sufficient drug level. Therefore, the present study administered EPI (1 mg/kg daily) to 25–30 g male C57BL/6 mice for 10 days before left descending coronary artery ligation for in vivo experiments. By paying monitoring the overall survival of the mice, it was confirmed that the drug dosage (1 mg/kg daily) was not lethal (Fig. 1A). Additionally, the eating behavior of the mice was observed daily and documented the weight of them over the 10 days. However, there was no significant difference between the two group and therefore it was concluded that the dose was safe (data not shown). According to previous studies for in vitro experiments (21–23), the present study pretreated NMCMs with 5 µM EPI for 1 h in 5% CO₂ and 37°C and then incubated cells under anoxia conditions for 12 h. The possible toxicity effect of EPI on NMCMs was determined by an MTT assay. As shown in Fig. 1B, the dosage of EPI was safe for cardiomyocytes.

C57BL/6 mice underwent left descending coronary artery occlusion operation to induce myocardial ischemic, or sham operation for negative control. H&E and Masson staining analysis demonstrated that the areas of cardiomyocytes and cardiac fibrosis of the MI group mice were significantly increased 1 week following MI operation, when compared with the hearts of the sham group mice. EPI treatment suppressed MI-induced cardiac hypertrophy and fibrosis, as evidenced by decreased areas of cardiomyocytes and cardiac fibrosis in the MI+EPI group mice when compared with the MI group (Fig. 2A). TUNEL assay revealed that cardiomyocyte apoptosis increased in the MI group when compared with the sham group, which was significantly inhibited by EPI administration in the MI+EPI group mice (Fig. 2B). Considering the crucial role of EPI in myocardial ischemia, the underlying molecular mechanism through which EPI exerts its protective effect was further investigated. The PI3K/AKT signaling pathway mediates cell apoptosis, which may be a potential treatment target for ameliorating myocardial ischemia. Therefore, this study used LY294002, a specific PI3K inhibitor, to confirm whether EPI inhibits the myocardial ischemia-induced cardiac injury through the PI3K/AKT pathway. The protective effect of EPI for myocardial ischemia was abolished by LY294002 (Fig. 2A and B). Therefore, these results illustrated that the inhibition of the PI3K/AKT pathway offsets the cardiac protective effect of EPI.

The present study used two-dimensional echocardiography and M-mode tracings to examine left ventricular functions at day 7 of myocardial ischemic surgery. The left ventricular wall thickness was increased in MI group, which was suppressed by EPI. The effect of EPI on left ventricular wall thickness was attenuated by LY294002 (Fig. 3A). HR, LVEDd, LVESd, EF and fractional shortening FS were detected by transthoracic echocardiography. There was no significant difference of HR between any group (Fig. 3B). EPI significantly inhibited the increase of LVEDd and LVESd induced by the myocardial ischemic operation (Fig. 3C, D). In addition, EPI administration increased FS and EF levels in the MI+EPI group compared with the MI group (Fig. 3E and F). As shown in Fig. 3C and D, LVEDd and the LVESd in the MI+LY294002+EPI group were significantly increased when compared with EPI-treated myocardial ischemic mice. FS and EF were decreased in the MI+LY294002+EPI group after co-treatment with EPI and LY294002 (Fig. 3E, F), compared with the MI+EPI group. These results demonstrated that EPI protects left ventricular function from myocardial ischemic injury via the AKT pathway.

Next, the present study further identified the underlying molecular mechanism by which EPI safeguards cardiac functions. As earlier work demonstrated, PTEN is a primary negative regulator of the PI3K/AKT signaling pathway (24). To examine whether PTEN mediates the cardiac protective action of EPI, this study used western blot analysis to compare protein levels of components of the PTEN/AKT pathway in each group. As shown in Fig. 4A, compared with the sham group, myocardial ischemia significantly induced the decrease of Bcl-2 and the increase of Bax and cleaved caspase-3 in MI group, which were rescued by the administration of EPI. However, in the MI+EPI+LY294002 group, LY294002 significantly abolished the rescue effect of EPI. Furthermore, Fig. 4B indicated that PTEN and the p-AKT/T-AKT ratio were upregulated and downregulated, respectively, in the MI group compared with the sham group. PTEN and p-AKT/T-AKT ratio were decreased and increased, respectively, in the MI+EPI group compared with the MI group, which were both significantly reversed by LY294002 co-treatment in the MI+EPI+LY294002 group. Taken together, these results demonstrated that EPI exerts an anti-apoptotic function during myocardial ischemia in a PTEN/AKT pathway dependent manner.

The above results indicated that EPI protects myocardial ischemia-induced heart injury through the PTEN/AKT signaling pathway in vivo. To further confirm the effect of EPI on cardiomyocytes, NMCMs were incubated under normal or hypoxic condition in vitro. As shown in Fig. 5A, cell viability was decreased in ANO group when compared with the control group. Treatment with EPI rescued the decrease of cell viability induced by hypoxia, which was significantly counteracted by LY294002 co-treatment (Fig. 5A). In agreement with the previous results in vivo, hypoxia induced the decrease of Bcl-2 and the increase of Bax and cleaved caspase-3 in the NMCMs of ANO group in vitro. EPI treatment significantly rescued the decrease of Bcl-2 and suppressed the increase of Bax and cleaved caspase-3 in hypoxia-treated NMCMs when compared to the level of the control group, which was abolished by the addition of LY294002 (Fig. 5B). In addition, treatment with EPI significantly attenuated hypoxia-induced upregulation of PTEN and downregulation of p-AKT when compared with the MI group. Similarly, LY294002 treatment reversed the effect of EPI on the PTEN and the p-AKT. The level of T-AKT was not affected in each group (Fig. 5C). Taken together, these results indicated that EPI protects cardiomyocytes from hypoxia-induced apoptosis through the PTEN/AKT pathway in vitro.