Q293T was obtained from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China) and used for lentivirus amplification. Rat H9c2 cell lines were obtained from the ATCC (American Type Culture Collection, Manassas, VA, USA), cultured in high glucose Dulbecco's modified Eagle's medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and supplemented with 10% (v/v) fetal bovine serum (FBS; Gibco/Invitrogen; Thermo Fisher Scientific, Inc.), 100 µg/ml penicillin, 100 µg/ml streptomycin at 37°C in a humidified incubator with 5% CO₂.

When the H9c2 cells were cultured to 70–80% confluence, the medium was changed to Krebs-Ringer Bicarbonate buffer (115 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl₂, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄, 24 mM NaHCO₃, 10 mM HEPES; pH 7.4) and cultured at 95% N₂ and 5% CO_2, creating an anoxic environment to stimulate hypoxia (8).

When the cells were cultured to 70–80% confluence, SIRT1 or UCP2 shRNA were transfected into the medium to overexpress SIRT1 or decrease expression of UCP2, using Lipofectamine 2000 (Thermo Fisher Scientific, Inc.). 24 h following transfection, cells underwent 2% O₂ hypoxia treatment for 2, 4, 6, 8, 12 or 24 h. Cell number was then counted under a light microscope following trypan blue staining. All counts were performed on triplicate wells and repeated in three independent experiments at ×100 magnification with Image J (version, 1.6.0.24; National Institutes of Health, Bethesda, MD, USA).

In vitro cell survival was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cells were treated with 50 µM MTT for 4 h at 37°C, and the formazan crystals were dissolved with 200 µl dimethyl sulfoxide for 10 min at room temperature. Absorbance was measured at a wavelength of 570 nm.

Total RNA from heart tissue treated with or without I/R and H9c2 cells were extracted using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). RNA concentration was determined by UV spectrophotometer (NanoDrop 2000; Thermo Fisher Scientific, Inc., Wilmington, DE, USA), reverse-transcribed using M-MMLV reverse transcriptase with RNasin Ribonuclease Inhibitors (Promega Corporation, Madison, WI, USA) at 42°C for 30 min, 95°C for 5 min. qPCR amplifications were carried out using the StepOnePlus™ Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.) and Thunderbird SYBR Master Mix (Toyobo Co., Ltd, Osaka, Japan). Primer sequences were: UCP-2, forward 5′-TGTGGTAAAGGTCCGCTTCC-3′ and reverse 5′-CCCGAAGGCAGAAGTGAAGT-3′; Sirt-1, forward 5′-TGAGGTAGGGTCCCCGTTTA-3′ and reverse 5′-TTGAGGCCGGTTTGGCTTAT-3′; and GAPDH, forward 5′-GATCCCGCTAACATCAAATG-3′ and reverse 5′-GAGGGAGTTGTCATATTTCTC-3′. qPCR was performed with the following cycles: 95°C for 10 min, followed by 95°C for 15 sec, 60°C for 30 sec, and 72°C for 30 sec for 40 cycles. GAPDH expression was used as an internal control. 2^−ΔΔCq was calculated for every sample. The mRNA expression levels were indicated with 2^−ΔΔCq and normalized to GAPDH (9).

Lentivirus containing Rat UCP-2 shRNA (cat. no. V3SR11242238929649; Dharmacon, Inc.; GE Healthcare Life Sciences, Chalfont, UK) was used for UCP-2 gene silencing, and Lentivirus containing a scrambled sequence (shScramble, VSH11660, Thermo Scientific Open Biosystems) was used as the negative control. Lentivirus was produced in transfected 293T packaging cells as described previously (10).

Q293A cells were transfected with retroviral vectors expressing rat SIRT1 (cat. no. 4331182 Thermo Fisher Scientific, Inc.) using Lipofectamine 2000 (Thermo Fisher Scientific, Inc.), and changed to fresh medium on the following day. Following 30 h further incubation, retroviral particle-containing supernatant was harvested and filtered through a 0.45 µm filter (EMD Millipore, Billerica, MA, USA). H9c2 cells were transduced with virus supernatant. Transduced cells were used for RT-qPCR or western blot assays 48 h later.

To determine whether the entire rat UCP-2 3′-untranslated region (UTR) segment was amplified by PCR, using mouse genomic DNA as a template. The PCR products were inserted into the p-MIR-reporter plasmid (Ambion; Thermo Fisher Scientific, Inc.). 2×10⁵ cells were seeded in 6-well plates and transfected with 1 µg of firefly luciferase reporter plasmid and 0.5 µg of Renilla expression vector as a transfection control (Ambion; Thermo Fisher Scientific, Inc.) for luciferase assays. Cells were collected and measured 48 h following transfection according to the manufacturer's instructions (Dual-Luciferase Assay System; Promega Corporation, Madison, WI, USA).

ATP levels were measured with a Molecular Probes ATP Determination kit (A22066; Invitrogen; Thermo Fisher Scientific, Inc.). 100 µl lysis buffer was added into H9c2 cells to prepare the total lysate at room temperature and was centrifuged at 2380 × g at 4°C for 15 min, and the supernatant was retrieved for use in the ATP assay according to the manufacturer's instructions. Protein concentration was measured via bicinchoninic acid (BCA) assay. The pH of the extracellular medium was measured using pH meter (pB-10, Sartorius AG, Göttingen, Germany).

A total of 30 adult male C57BL/6 mice, 8 weeks old and weighing 30.2±3.5 g were supplied by the Model Animal Research Center of Nanjing University (Nanjing, China). All animal experiments were conducted under the guidelines on humane use and care of laboratory animals for biomedical research published by the U.S. National Institutes of Health (NIH Publication No. 85–23, revised 1996). All experimental preparations and protocols involving animals were reviewed and approved by the Animal Care and Use Committee of Nanjing University (Nanjing, China).

Resveratrol stock solution was dissolved in DMSO (Sigma-Aldrich; Merck Millipore, Darmstadt, Germany) and then diluted into 5 mg/ml with saline, and the DMSO was less than 5%. Resveratrol (5 mg/ml) or saline was injected intraperitoneally (i.p.) 24 h prior to subjection to 1 h of myocardial ischemia and 24 h of reperfusion. Mice were anesthetized with pentobarbital sodium (60 mg/kg). Ischemia was achieved through ligation of the left anterior descending coronary artery (LAD) using an 8-0 silk suture with a section of PE-10 tubing placed over the LAD, 1 mm from the tip of the normally positioned left atrium. Following ischemia for 1 h, reperfusion was initiated by releasing the ligature. Sham-operated control mice underwent the same surgical procedures except that the suture placed under the left coronary artery was not tied. A total of 30 animals were included and randomly distributed into the sham, I/R and RES+IR groups, with 10 animals in each group.

Hearts were harvested 24 h subsequent to this directly for measurement of infarct size and stored in liquid nitrogen immediately, and was processed using TRIzol (Thermo Fisher Scientific, Inc.) for RT-qPCR assays or radioimmunoprecipitation assay buffer (cat. no. 89900; Thermo Fisher Scientific, Inc.) for western blot assays.

Following reperfusion, myocardial infarct size was evaluated by tetrazolium chloride (TTC) staining. The hearts were immediately removed and sectioned into 5 mm thick short-axis sections from the apex towards the base of the heart, then incubated in 1% TTC for 20 min at 37°C. Following this, the sections were photographed with a Nikon D810 digital camera (Nikon Corporation, Tokyo, Japan). Areas of infarct size were measured digitally using Image Pro Plus software 6.0 (Media Cybernetics, Inc., Rockville, MD, USA).

Protein concentrations of the complete lysate were analyzed with the Bradford protein assay. Protein extracts were incubated for 10 min at 100°C in sample buffer (10% (v/v) glycerol, 5% (w/v) sodium dodecyl sulfate (SDS), 0.25% (w/v) bromophenol blue, 5% (v/v) 2-mercaptoethanol, and 0.0625 M Tris-HCl, pH 6.8) prior to loading onto the gels. A total of 30 µg of homogenate protein was loaded into each lane of a 12% SDS-PAGE mini-gel (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Electrophoresis was conducted at 80 V in running buffer (0.025 M Tris-HCl, 0.2 M glycine, 1 mM EDTA and 3.5 mM SDS). Proteins were subsequently transferred to a polyvinylidene fluoride membrane (GE Healthcare Life Sciences, Chalfont, UK) and blocked at room temperature with 5% milk in TBS with 5% Tween-20 (TBST) for 1 h. Western blotting was performed with mouse monoclonal to GAPDH primary antibody (1:2000; ab8245; Abcam, Cambridge, UK), polyclonal mouse anti-UCP2 primary antibody (1:1,000; ab67241; Abcam) or mouse anti-SIRT1 primary antibody (1:1,000; ab104833; Abcam), followed by goat anti-mouse horseradish peroxidase-conjugated secondary antibody (sc-3791; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at a dilution of 1:5,000. Following washing, the bands were detected by an enhanced chemiluminescence reagent (EMD Millipore). The band intensities were quantified using the Quantity One 1-D software (version, 4.6.6) in ChemiDoc™ MP System (Bio-Rad Laboratories, Inc.).

All results were expressed as the mean + standard error from at least three independent experiments. Student's t-test was used for normally distributed data, and the Mann-Whitney U test was used for abnormally distributed data. P<0.05 and P<0.01 were considered to indicate a statistically significant and a highly significant difference, respectively.

Cell viability was detected by MTT assays and cell counting to evaluate whether H9c2 cells were suffering from hypoxic injury, and determine which hypoxia condition should be used in subsequent experiments. Hypoxia markedly decreased H9c2 cell viability and number as time increased, and following 6 h hypoxia treatment, cell viability decreased to 61.25±5.14% compared with the control group (P=0.021 Fig. 1A). Therefore, this condition was used in subsequent experiments. SIRT1 and UCP-2 mRNA and protein levels in H9c2 under normoxic and hypoxic conditions were evaluated by qPCR and western blot assays. Decreased expression of SIRT1 mRNA and protein was observed in hypoxic H9c2 cells compared with normoxic H9c2 cells (P=0.0001 and P=0.0004, respectively; Fig. 1B and C, respectively). UCP-2 mRNA and protein levels were significantly increased in hypoxic H9c2 cells compared with normoxic H9c2 cells (P=0.0008 and P=0.0007, respectively; Fig. 1B and C, respectively). This result demonstrates that SIRT1 and UCP-2 may be involved in the development of hypoxia-induced injury in cardiomyocytes.

The effect of SIRT1 in hypoxia-induced cardiomyocyte injury was explored using gain-of-function methods in H9c2 cells. UCP-2 mRNA and protein expression was significantly downregulated in H9c2 cells overexpressing SIRT1, compared with scramble control cells (P=0.0003 and P=0.0002; Fig. 2A and B, respectively). Furthermore, the dual luciferase assay revealed that overexpression of SIRT1 in H9c2 cells repressed UCP-2 (P=0.0004; Fig. 2C). This evidence demonstrates that SIRT1 directly regulates the expression of UCP-2 by binding directly to the UCP-2 promoter. The viability of H9c2 cells overexpressing SIRT1 also increased significantly compared with the parental strain and empty vector control, demonstrated by MTT assay and cell counting (P<0.01; Fig. 2D and E).

shRNA against UCP-2 was used to block the expression of UCP-2 to study the effects of absence of UCP-2 on cell survival under hypoxic conditions. UCP-2 specific shRNA significantly reduced UCP-2 protein and mRNA expression levels compared with scramble control cells (P=0.0008 and P=0.0009 respectively; Fig. 3A and B, respectively). Increased cell viability was observed in H9c2 cells transfected with UCP-2 shRNA compared with scramble control cells from 4 h (P<0.01; Fig. 3C) demonstrating that UCP-2 knockdown improves cell resistance to hypoxia.

This enhanced cell survival in response to hypoxia treatment, induced by the decreased expression of UCP2, may have been mediated by the regulation of ATP biosynthesis. Under normoxia, there was no significant difference between the UCP-2 shRNA and scramble shRNA groups (11). However, increased ATP accumulation under hypoxic conditions was revealed in the UCP-2 knockdown group compared with the scramble and parental controls (P=0.0006 and P=0.0002 Fig. 3D). In addition, the pH of the extracellular medium was increased compared to the scramble and the parental controls (P=0.0006 and P=0.0003; Fig. 3D).

Based on the finding that exogenous expression of SIRT1 in H9c2 cells improved resistance to ischemia injury in vitro, potentially through reducing the expression of UCP-2 and therefore increasing ATP levels, the effect of a putative activator of SIRT1, resveratrol, on ischemia/reperfusion injury was explored in vivo. The results demonstrated that treatment with resveratrol significantly reduced infarct size compared with the saline control (P=0.003 Fig. 4A), suggesting a protective effect against myocardial ischemia/reperfusion injury. Higher mRNA and protein expression levels of SIRT1 in resveratrol-treated myocardial tissue compared with saline controls were validated by RT-qPCR and western blot, respectively, and were concomitant with decreased expression of UCP-2 mRNA and protein (P<0.05; Fig. 4B and C, respectively). These findings demonstrate that the SIRT1/UCP2 axis may represent a novel therapeutic target for myocardial ischemia/reperfusion injury treatment.