Pur (Fig. 1) was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Rapamycin (Ra), 3-methyladenine (3-MA), Akt signaling inhibitor (API-2) and 5-bromo-2′-deoxyuridine (5-BrdU) were purchased from Sigma-Aldrich Merck KGaA (Darmstadt, Germany). 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl) −2H-tetrazolium inner salt (MTS) was purchased from Promega Corporation (Madison, WI, USA). Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were purchased from Thermo Fisher Scientific, Inc. (Beijing, China). All antibodies for immunoblotting were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA), with the exception of the GAPDH antibody (KangChen Bio-tech, Inc., Shanghai, China) and goat anti-rabbit secondary antibody (Boster Systems, Inc., Pleasanton, CA, USA). Adenovirus-monomeric red fluorescent protein-green fluorescent protein-light chain 3 (Ad-mRFP-GFP-LC3) was purchased from HanBio Biotechnology Co., Ltd. (Shanghai, China). Neonatal Sprague Dawley rats (~220; 1–2 days; 5–6 g) were obtained from the Experimental Animal Center of Southern Medical University (Guangzhou, China; license no. scxk-Guangdong-2006-0015) where they had been housed with female rats under standard conditions with a light/dark cycle of 12 h and fed by female rats.

Neonatal rat cardiomyocytes (NRCs) were isolated from the ventricular heart of 1–2 day old Sprague Dawley rats as previously described (15), following which the animals were immediately sacrificed by decapitation. The isolation procedure was performed in accordance with the Guide for the Care and Use of Laboratory Animals, published by the US National Institutes of Health (publication no. 85-23, revised 1996) and was approved by the Southern Medical University Experimental Animal Ethics Committee (Guangzhou, China). NRCs were plated at a density of 5×10⁵ cells/60 mm diameter plate, and cultured in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin (the complete medium) in a 5% CO₂ humidified atmosphere at 37°C. 5-BrdU (0.1 mM) was added to the culture medium to inhibit fibroblast proliferation during the first 48 h. The cells were subsequently cultured with the complete medium for a further 48 h. The culture medium was changed daily.

Following culture for 96 h, NRCs were randomly divided into the following groups: The control group, in which cells were incubated under normal conditions; the hypoxia/reoxygenation (H/R) group, in which cells were exposed to 3 h hypoxia followed by 3 h reoxygenation; the H/R + Pur group, in which cells were pretreated with Pur and were then exposed to H/R; H/R + 3-MA/Ra/API-2 group, in which cells were pretreated with 3-MA, Ra or API-2 and were then exposed to H/R; and the H/R + Pur + 3-MA/Ra/API-2 group, in which cells were pretreated with Pur plus 3-MA, Ra or API-2 and were then exposed to H/R. Pur (50, 100 or 200 µM) (16), 3-MA (5 mM), Ra (0.1 µM) and API-2 (10 µM) were added to the culture medium 2 h prior to H/R.

The myocardial H/R model was used to simulate I/R injury. For experiments under hypoxic conditions, NRCs were incubated with phosphate-buffered saline (PBS) in a Modular Hypoxic Chamber (Billups-Rothenberg, Inc., Del Mar, CA, USA) saturated with <0.1% O₂, 95% N₂ and 5% CO₂ at 37°C for 3 h. Cells were subsequently subjected to reoxygenation by replacing PBS with the culture medium, and were returned to normoxic conditions for 3 h (17).

To assess cell viability, the MTS assay was used according to the manufacturer's protocol. NRCs were seeded in 96-well plates at a density of 5,000 cells/well. Following the indicated treatments, 20 µl MTS solution was added to each well, and plates were subsequently incubated at 37°C for 3 h. The optical density in each well was measured at 490 nm using a microplate reader (ELx800; BioTek Instruments, Inc., Winooski, VT, USA).

Western blotting was performed to determine LC3-II/LC3-I, p62, phosphorylated (p-)Akt (Ser473), Akt, P-5′ adenosine monophosphate-activated protein kinase (p-AMPK) (Thr172), AMPK and GAPDH protein expression levels. Following the indicated treatments, cells were lysed using a total protein extraction kit (KangChen Bio-tech, Inc.) and a protease inhibitor cocktail (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Lysates were subsequently centrifuged at 18,506 × g at 4°C for 15 min. Protein concentration was determined using an enhanced bicinchoninic acid protein assay kit (Thermo Fisher Scientific, Inc.). The loading buffer was added and the protein samples were boiled in a water bath for 10 min. Western blotting was performed according to standard procedures. Equal amounts of protein were resolved via SDS/PAGE (10 or 12%) and transferred to PVDF membranes (Merck KGaA, Darmstadt, Germany). After blocking in 5% non-fat milk at room temperature for 2 h, membranes were incubated with the following primary antibodies overnight at 4°C: anti-LC3B (3868S), anti-p62 (8025S), anti-p-Akt (4060S), anti-Akt (4691P), anti-p-AMPK (2531S), anti-AMPK (2532S; all from Cell Signaling Technology, Inc.) and anti-GAPDH (KC-5G4; KangChen Bio-tech, Inc.). All dilutions were 1:1,000. After incubation for 2 h at room temperature with goat anti-rabbit secondary antibodies (1:8,000; BA1054; Boster Systems, Inc.), the bands were visualized using enhanced chemiluminescence (Merck KGaA). The blots were quantified by densitometry using Image-Pro Plus software version 6.0 (Media Cybernetics, Inc., Rockville, MD, USA) and the relative protein expression was compared with GAPDH.

Following the indicated treatments, cells were washed, scraped, collected in a 1.5 ml tube and centrifuged at 106 × g at 4°C for 5 min. The cell pellets were subsequently fixed with 2.5% glutaraldehyde at 4°C overnight. Following fixation, cell pellets were dehydrated with a graded series of ethanol (50, 70, 90 and 100%), propylene oxide and then infiltrated with a 1:1 mixture of propylene oxide and EMbed 812. The samples were sliced into ultrathin sections (75–80 nm), which were stained and examined using a transmission electron microscope (JEM1200-EX; JEOL Ltd., Tokyo, Japan). In the samples, vesicles containing membranous structures (autophagosomes) in each cell were counted.

Adenoviral transfection was performed according to the manufacturer's protocol. NRCs were seeded in 15 mm diameter glass-bottomed dishes at a density of 150 cells/mm² and 36 h later were incubated in DMEM containing 2% FBS and adenovirus (multiplicity of infection of 15) at 37°C for 10 h. The transfection medium was then replaced with the complete medium, and cells were further cultured at 37°C for 50 h. Following the H/R treatments, cells were fixed with 4% paraformaldehyde and nuclei were stained with 2 µg/ml Hoechst 33342 (Sigma-Aldrich; Merck KGaA) for 5 min. Images of the cells were captured using a confocal fluorescence microscope (Olympus FV-1,000; Olympus Corporation, Tokyo, Japan). Autophagic flux was determined by evaluating the number of mRFP and GFP puncta/cell using Image-Pro Plus software version 6.0 (Media Cybernetics).

All experiments were performed ≥3 times. SPSS 20.0 software (IBM SPSS, Armonk, NY, USA) was used for statistical analysis. The data were expressed as the mean ± standard deviation. Statistical significance was analyzed by one-way analysis of variance followed by Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

To confirm the protective effect of Pur on myocardial I/R injury, an MTS assay was performed. Pur (50, 100 and 200 µM) was applied to NRCs 2 h prior to H/R. NRC viability was markedly decreased to 38.4±2.8% in the H/R group compared with the control group (Fig. 2A). However, NRC viability was significantly increased by 15.2–22.5% following 50, 100 and 200 µM Pur pretreatment compared with the H/R group (Fig. 2A), with the largest increase observed in the 200 µM group, where NRC viability was significantly increased compared with the 50 µM Pur pretreatment group (Fig. 2A). Accordingly, 200 µM was selected as the concentration of Pur used in subsequent experiments.

3-MA is a classic autophagy inhibitor, which blocks autophagosome formation via the inhibition of type III PI3K, and was also tested in the myocardial H/R model. Treatment with 3-MA significantly increased NRC viability compared with the H/R group (P<0.05; Fig. 2B), which was similar to the effect observed following Pur pretreatment. These results indicated that autophagy during myocardial H/R may contribute to cell death.

To investigate the effects of Pur on H/R-induced autophagy in cardiomyocytes, western blot analysis was performed (Fig. 3A). As presented in Fig. 3B and C, H/R induced and increased the ratio of LC3-II/LC3-I, and decreased the expression levels of p62; however, Pur pretreatment (50, 100 and 200 µM) decreased LC3-II/LC3-I ratio and increased p62 expression (P<0.01). These results suggested that Pur may inhibit H/R-induced autophagy in cardiomyocytes, which was confirmed by the results of TEM. As shown in Fig. 3E-F, large vesicles containing membranous structures (autophagosomes) appeared in the cytoplasm of cells in the H/R group. Conversely, autophagosomes were scarce in the control group, which was characterized by normal cytoplasm, mitochondria, endoplasmic reticulum and nuclei. In addition, the number of autophagosomes induced by myocardial H/R was significantly decreased by Pur pretreatment (P<0.01; Fig. 3G).

To further investigate the inhibitory effects of Pur on autophagy, Ad-mRFP-GFP-LC3, a specific marker for autophagosomes and autolysosomes, was transfected into NSCs. The yellow puncta in merged pictures, as a result of merged m-RFP and GFP, represent autophagosomes, whereas the red puncta represent autolysosomes (Fig. 4A). Only a few yellow puncta appeared in the control group, whereas the number of yellow and red puncta visibly increased in the H/R group (Fig. 4A). Following pretreatment with Pur, the number of yellow puncta (autophagosomes) significantly decreased by 60.1% compared with the H/R group (P<0.01; Fig. 4B). Conversely, there was no significant difference in the number of red puncta (autolysosomes) between the Pur group and H/R group (Fig. 4B).

Ra is one of the most commonly used drugs to stimulate autophagy. As aforementioned, Ra-mediated autophagy induction was performed in NRCs exposed to H/R, and western blotting was performed (Fig. 5A) The LC3-II/LC3-I ratio was significantly increased in the Ra group compared with the H/R group (P<0.01; Fig. 5B). However, following pretreatment with Ra + Pur, the LC3-II/LC3-I ratio was significantly decreased compared with the Ra group (P<0.01; Fig. 5B) and the expression levels of p62 were elevated compared with the Ra group (P<0.01; Fig. 5C). These results suggested that the increased autophagy induced by Ra during myocardial H/R was inhibited by Pur pretreatment.

To investigate the potential molecular mechanisms involved in the autophagy-inhibiting effect of Pur during myocardial H/R, the PI3K/Akt and AMPK signaling pathways were assessed. The expression levels of p-Akt, Akt, p-AMPK and AMPK in groups were determined by western blotting. There was no significant difference in the ratios of p-Akt/Akt and p-AMPK/AMPK between the H/R group and the control group (P>0.05; Fig. 5D and E). Pur pretreatment increased the phosphorylation of Akt compared with the H/R group (P<0.01; Fig. 5D), but it did not significantly affect the phosphorylation of AMPK (Fig. 5E).

To further elucidate the involvement of the PI3K/Akt signaling pathway in the autophagy-inhibiting effects of Pur pretreatment during myocardial H/R, API-2 was applied to cells with or without Pur pretreatment and the effects were measured by western blotting (Fig. 6A). The LC3-II/LC3-I ratio was significantly increased in the H/R +API-2 + Pur group compared with the H/R + Pur group (P<0.01; Fig. 6B) and p62 protein expression levels were significantly decreased in the H/R + API-2 + Pur group compared with the H/R + Pur group (P<0.01; Fig. 6C). Akt phosphorylation was significantly increased and autophagy was inhibited in the Pur group compared with the H/R group (P<0.01; Fig. 6B and D); however, following pretreatment with Pur + API-2, Akt phosphorylation was significantly decreased and LC3-II/LC3-I ratio was increased compared with the H/R + Pur group (P<0.01; Fig. 6D). These findings indicated that the autophagy-inhibiting effects of Pur were abolished by API-2.