Sevoflurane postconditioning alleviates hypoxia-reoxygenation injury of cardiomyocytes by promoting mitochondrial autophagy through the HIF-1/BNIP3 signaling pathway

Cell culture and processing

The H9C2 rat embryonic cardiomyocyte cell line was obtained from Kaiji Biological Co., Ltd., China. The cell culture conditions consisted of DMEM (high sugar) medium + 10% FBS at 37 °C, 5% CO₂, and saturated humidity. H9C2 cells with good growth at 90% confluency were used to prepare a 5 × 10⁴ cell/ml single-cell suspension using complete medium. Cells were inoculated in 96-well plates and incubated for 24 h at 37 °C in a 5% CO₂ incubator. When the cells grew to 80% confluency, the supernatant was discarded, the adherent cells were washed twice with PBS, and serum-free DMEM (low sugar) medium was added. The plates were placed in a saturated tri-gas incubator at 37 °C with 95% N₂ and 5% CO₂ for 3 h. After culturing under hypoxic conditions, the cells were removed, the supernatant was discarded, fresh serum-free DMEM (low sugar) medium was added, and cells were re-oxygenated in a CO₂ incubator for 3 h.

Sevoflurane postconditioning (SpostC) of H9C2 cardiomyocyte

According to the previous study (Obal et al., 2001; Yu et al., 2016), a Vapor 2000 sevoflurane vaporizer (Drager, Lubeck, Germany) was used to apply a gas mixture containing 97.6% O₂ and 2.4% sevoflurane. Briefly, an in-line sevoflurane vaporizer fed a supply of gas mixture containing 97.6% O₂ and 2.4% sevoflurane for at least 10 min until the desired sevoflurane concentration (2.4%) was achieved. Concentrations of sevoflurane and O₂ were monitored using an anesthetic analyzer (Drager Vamous, Lubeck, Germany) in the outlet. The gas flow rate was two l/min. After the cells were treated for 15 min, they were taken out immediately and incubated in a 5% CO₂ cell incubator for 165 min at 37 °C.

Hypoxia-inducible factor-1α inhibitor 2-methoxyestradiol (2ME2) treatment steps: H9C2 cells were cultured in medium (containing 2 μM 2ME2 dissolved in 0.02% DMSO) for 15 min at the beginning of reoxygenation after 3 h of hypoxia, and then cultured in medium without 2ME2 in a 5% CO₂ cell incubator for 165 min. Other groups were only cultured in fresh medium for the corresponding times.

Experimental grouping

The experiment was divided into five groups: the control group (C): H9C2 cells were incubated for 6 h in normoxia. Hypoxia-reoxygenation group (H/R): hypoxia for 3 h and reoxygenation for 3 h. Sevoflurane postconditioning group (SpostC): H9C2 cells were exposed to 2.4% sevoflurane for 15 min at the beginning of reoxygenation after 3 h of hypoxia, and then incubated in a 5% CO₂ cell incubator for 165 min. Hypoxia-inducible factor-1α inhibitor 2ME2 group: H9C2 cells were cultured in medium (containing 2 μM 2ME2) for 15 min at the beginning of reoxygenation after 3 h of hypoxia, and then cultured in medium without 2ME2 in a 5% CO₂ cell incubator for 165 min. 2ME2 + SpostC group (MSP): H9C2 cells were cultured in medium (containing 2 μM 2ME2) in a hypoxia-reoxygenation box filled with 2.4% sevoflurane for 15 min at the beginning of reoxygenation after 3 h of hypoxia, and then cultured in medium without 2ME2 in a 5% CO₂ cell incubator for 165 min (Fig. 1).

RNA interference and gene transfection

siRNA oligonucleotides were synthesized by GenePharma Co., Ltd. (Shanghai, China), and the siRNA gene sequences were as follows: BNIP3-FsiRNA: 5′-CCU GGG UAG AAC UGC ACU UTT-3′, BNIP3-RsiRNA: 5′-AAG UGC AGU UCU ACC CAG GTT-3′, NC-FsiRNA: 5′-UUC UCC GAA CGU GUC ACG UTT-3′, NC-RsiRNA: 5′-ACG UGA CAC GUU CGG AGA ATT-3′. The siRNA diluted solution was mixed with lipofectamine RNAiMAX to form an siRNA/lipofectamine RNAiMAX complex, which was added to the wells containing the cells and medium (no antibiotics). The cell culture plate was gently shaken and then incubated for 48 h at 37 °C in a CO₂ incubator.

In the present study, siRNA-NC or siRNA-BNIP3 was transfected into cells, and after transfection, cells were exposed to hypoxic conditions for 3 h and reoxygenation for 3 h (SpostC: cells were exposed to 2.4% sevoflurane for 15 min at the beginning of reoxygenation after 3 h of hypoxia, and then incubated in a 5% CO₂ cell incubator for 165 min). The treatments included hypoxia/reoxygenation + siRNA-NC (H/R+NC); hypoxia/reoxygenation + siRNA-BNIP3 (H/R+siBNIP3); hypoxia/reoxygenation + sevoflurane + siRNA-negative control (H/R+SPostC+NC); and hypoxia/reoxygenation + sevoflurane + siRNA-BNIP3 (H/R+SPostC+siBNIP3) (Fig. 2).

Determination of cell viability

Cell viability was determined by the CCK-8 method. A total of 100 μl of 10% CCK-8 solution was added to each well of a 96-well plate, which was then incubated at 37 °C. After 2 h, the optical density values were measured at a wavelength of 450 nm using a plate reader.

Determination of lactate dehydrogenase (LDH) content

According to the instructions of the LDH cytotoxicity test kit, cell supernatants of each well of the 96-well plate were collected and transferred to a new 96-well plate in each experimental group, and 60 µl of LDH test solution was added to each well. The plate was wrapped with foil and placed on a shaker to incubate for 30 min at room temperature. The absorbance values were then measured at 490 nm. The absorbance value was proportional to the LDH content.

Apoptosis detection

Apoptosis was detected using an Annexin V-FITC/PI Assay Kit (Sigma, St. Louis, MO, USA). The cells were washed with precooled PBS and then resuspended in 100 μl of Annexin V binding solution to prepare single-cell suspensions. Then, 10 μl of Annexin V-FITC and 10 μl of PI staining solution were added and mixed gently, and the plate was placed at 4 °C for 15 min in the dark. An additional 500 μl of Annexin V binding solution was added. Flow cytometry detection was performed within 30 min.

TEM analysis

A total of one ml of 2.5% glutaraldehyde was added to each group of H9C2 cells. Cells were fixed at 4 °C for 2 h, rinsed three times with precooled PBS and dehydrated with acetone. Next, 1% osmic acid was added at room temperature for 2 h. The cells were embedded in epoxy resin and dried, and ultrathin sectioning was performed after trimming. The cells were stained with uranyl acetate and lead citrate for 15 min and 5 min, respectively. The ultrastructural changes and autophagosomes in each group of H9C2 cells were observed under a transmission electron microscope. For autophagosome quantification, five micrographs, primary magnification × 20,000, were blindly taken from each group, and the total amount of autophagosomes was counted. Mitochondria were scored using a semi-quantitative analysis of FlaMeng as described previously, the higher the score, the more severe the mitochondrial damage (Jiang et al., 2016).

Western blot analysis

H9C2 cells were lysed in ice–cold radioimmunoprecipitation assay lysis buffer, at 4 °C for 60 min and then the homogenate was incubated and centrifuged. The supernatant was collected, and the protein concentration was determined using the bicinchoninic acid protein assay kit according to the manufacturer’s protocol (Beyotime, Haimen, China). The supernatant was mixed with ×5 loading buffer and heated for 5 min at 100 °C, and then 30 micrograms of sample was subjected to electrophoresis using an SDS-PAGE gel system, transferred to a membrane and blocked at 37 °C for 2 h. Diluted primary antibodies to HIF-1α (1:200, abcam, ab1), BNIP3 (1:500, abcam, ab10433), and Beclin-1 (1:500, abcam, ab62557) were added, and the membrane was incubated overnight (4 °C). The membrane was washed with TBST solution and incubated with HRP-conjugated secondary antibody (1:10,000, sanggon, D110024) for 1 h at room temperature. Enhanced Chemiluminescence (ECL) was used for visualization and imaging. Gray value analysis of the target protein bands was performed using the Quantity One image analysis system.

Statistical analysis

All experiments were independently repeated in the laboratory at least three times. SPSS 19.0 statistical software was used for the statistical analysis. Data are expressed as the mean ± standard deviation. Statistical significance was determined using a Student’s t-test for the comparison between two groups. Other comparisons were performed using a one-way ANOVA analysis with LSD post-hoc test for data with homogeneous variances, or Tamhane post-hoc test for data with non-homogeneous variances. A value of P < 0.05 was considered statistically significant. GraphPad Prism 5.0 was used to prepare graphs.

SpostC alleviated cell injury induced by hypoxia and reoxygenation

Cell viability, LDH activity, and apoptosis reflect the extent of cell damage. In this study, the cell viability of the H/R group was significantly lower than that of the control group (P < 0.05), while the cell viability of the SpostC group was increased compared to the H/R group (P < 0.05) (Fig. 3A). The LDH assay showed that compared to the control group, the LDH activity of the H/R group was increased (P < 0.05), while the LDH activity of the SpostC group was decreased compared to the H/R group (P < 0.05) (Fig. 3B). In addition, compared to the control group, the apoptotic rate of the H/R group was significantly increased (P < 0.05), while the apoptosis rate of the SpostC group was significantly lower than that of the H/R group (P < 0.05) (Fig. 3C).

SpostC promoted mitochondrial autophagy via HIF-1α

The morphological changes of mitochondria and autophagosomes were observed by transmission electron microscopy. The mitochondria in the H/R group were significantly swollen and contained a large number of autophagosomes compared to the control group; however, in the SpostC group, the mitochondrial morphology was normal, and the number of autophagosomes was significantly reduced (Figs. 4F and 4G). BCL2/ adenovirus E1B 19-kDa-interacting protein 3 is a key protein that regulates mitochondrial autophagy and a downstream target gene of HIF-1α. The results of this study showed that the expression levels of the HIF-1α and BNIP3 proteins in the SpostC group were significantly higher than those in the H/R group (Figs. 5B and 5C). However, the expression levels of the HIF-1α and BNIP3 proteins in the 2ME2 and MSP groups were significantly decreased after administration of 2ME2, an HIF-1α inhibitor (Figs. 5B and 5C), which was accompanied by mitochondrial swelling and accumulation of autophagosomes (Fig. 4), suggesting that SpostC promotes mitochondrial autophagy via HIF-1α.

BNIP3 participated in mitochondrial autophagy promoted by SpostC

To confirm the role of BNIP3 in myocardial protection mediated by SpostC, the BNIP3 gene was silenced, which showed that cell viability was decreased (Fig. 6A), LDH activity was increased (Fig. 6B), the apoptotic rate was increased (Fig. 6C), mitochondrial damage was significant, and autophagosomes accumulated (Figs. 7E and 7F), BNIP3 expression was decreased (Fig. 8C) in the H/R+SPostC+siBNIP3 group and H/R+ siBNIP3 group compared to the H/R+SPostC+NC group. These results suggest that BNIP3 participates in mitochondrial autophagy promoted by SpostC through HIF-1α.