Neonatal rat cardiomyocytes were cultured as reported previously (23). The whole hearts of 1–3-day-old neonatal Wistar rats were purchased from Slac Laboratory Animals, Shanghai, China, and cut into ~1–2 mm sections and digested in a digestion solution (0.025% collagen type II, 0.06% trypsin, and 20 µg/ml DNase) at 37°C three times, for 15 min each time. The homogenate was loaded onto a 45.5% percoll gradient over another 58.5% percoll gradient, followed by centrifugation at 15°C with 3,800 × g for 30 min. The dissociated cardiomyocytes were collected and seeded at 1×10⁵ cells per cm², followed by culture in Dulbecco's modified Eagle medium (Thermo Fisher Scientific, Inc., Waltham, MA, USA) with 10% fetal bovine serum (Thermo Fisher Scientific, Inc.) at 37°C with 5% CO₂ and 95% O₂. In all experiments, different concentrations of dexmedetomidine (10, 100 and 1,000 nM; Sigma; Merck Millipore, Darmstadt, Germany) were used for cardiomyocyte treatment at 37°C for 24 h. All experiments were approved by the Animal Care and Use Committee of Shanghai General Hospital (Shanghai, China) and performed in accordance with the relevant guidelines and regulations of the Bio-X Institutes of Shanghai Jiao Tong University (Shanghai, China).

Cells were cultured in multi-glass slides. Following dexmedetomidine treatment, cells were washed in sterilized PBS three times (5 min each) and fixed in cold acetone for 20 min, followed by washing with TBS three times. Following blocking with BSA (Sigma; Merck Millipore) for 1 h at room temperature, the cells were analyzed using a TdT-FragEL DNA Fragmentation Detection kit (Sigma; Merck Millipore) to quantify apoptosis. Counterstaining with fluorescence mounting medium containing DAPI (blue; Thermo Fisher Scientific, Inc., Waltham, MA, USA) was performed to visualize normal nuclei. Sections were observed using a fluorescence microscope (Olympus FluoView™ FV1000; Olympus Corporation, Tokyo, Japan). Measurements of the apoptotic nuclei percentage were obtained and analyzed using ImageJ (version 1.47; National Institutes of Health) from five randomly selected visual fields, and the average TUNEL-positive percentage (%) was calculated.

Cardiomyocytes were cultured in a multi 6-well dish. Cells were pretreated with dexmedetomidine at 37°C for 24 h, and then incubated with H₂O₂ (200 µM) at 37°C for ~6 h. Cell were washed with PBS three times and then images were captured using fluorescence microscopy as aforementioned (Olympus FluoView™ FV1000; Olympus Corporation, Tokyo, Japan). To determine cell survival, the area of live cells from five randomly selected visual fields using ImageJ (version 1.47; National Institutes of Health, Bethesda, MD, USA) was analyzed. The percentage of live cells area within a visual field area was defined as the cell survival (% field).

FACS analysis was performed to detect the apoptosis of cardiomyocytes induced by H₂O₂, as reported previously (24). Briefly, the neonatal rat cardiomyocytes were cultured in 6-well dishes. For cellular apoptosis, H₂O₂ (200 µM) was added to cells and incubated at 37°C for 4 h, following exposure of the cells to dexmedetomidine pretreatment for 24 h. The cardiomyocytes (10×10⁵ cells/500 µl) were labeled fluorescently for the detection of apoptotic and necrotic cells by adding 50 µl binding buffer and 5 µl Annexin V-FITC (BD Pharmingen, San Diego, CA, USA) and 2 µl of propidium iodide (Cedarlane Laboratories, Hornby, ON, Canada). The samples were incubated at room temperature for 15 min following gentle mixing. A minimum of 20,000 cells within a gated region were analyzed using FACS (Coulter Epics Altra flow cytometer; Beckman Coulter, Fullerton, CA, USA).

The primary neonatal rat cardiomyocytes were cultured in collagen-coated 96-well dishes. MitoSOX (cat. no. M36008; Invitrogen; Thermo Fisher Scientific, Inc.) or TMRE (cat. no. ab113852; Abcam; Cambridge, UK) were used following dexmedetomidine stimulation for 24 h. For the MitoSOX assay, on the day of measurement, the cells were washed twice in sterilized PBS followed by stimulation with H₂O₂ at 37°C for 15 min. The cells were then incubated with 5 µM MitoSOX-containing HBSS medium at 37°C for 10 min and were measured at Ex/Em: 510/580 nm. On the day of TMRE measurement, the cells were incubated with 1 µM TMRE at 37°C for 15 min, and then washed with PBS with 0.2% BSA three times to remove excess TMRE, followed by measurement at Ex/Em: 510/580 nm. For the carbonilcyanide p-triflouromethoxyphenylhydrazone (FCCP) inhibition assay, prior to TMRE incubation, the cells were stimulated by 0.5 or 1 µM FCCP (Agilent Technologies, Inc., Santa Clara, CA, USA) at 37°C for 30 min.

Neonatal rat cardiomyocyte OCR was measured using a Seahorse XF 24 extracellular analyzer (Agilent Technologies, Inc.). The cells were seeded at a density of 0.5×10⁵ cells/well. Cellular mitochondrial respiratory complexes were inhibited by injecting 1 µm oligomycin (inhibitor of complex V), 0.5 µm FCCP, and a combination of 0.5 µm rotenone and 0.5 µm antimycin A (R/A; inhibitors of complex I and complex III). Basic parameters were calculated as follows: Cellular respiration, basic OCR prior to oligomycin injection; mitochondria respiration, final rate measurement prior to oligomycin injection-minimum rate measurement following R/A injection; ATP production, final rate measurement prior to oligomycin injection-minimum rate measurement following oligomycin injection; maximal respiration, maximum rate measurement following FCCP injection-minimum rate measurement following R/A injection; proton leak, minimum rate measurement following oligomycin injection-minimum rate measurement following R/A injection; coupling efficiency, ATP production rate/cellular respiration rate. For measurement of the cellular response to ROS, H₂O₂ was injected three times following basic measurements.

The cultured cardiomyocytes were scraped and collected, and the centrifuged (1,500 × g at 4°C for 5 min) cardiomyocytes were dissolved in lysis buffer as reported previously (23). Protein concentration was quantified using protein assay buffer (cat. no. 500–0006; Bio-Rad Laboratories, Inc., Hercules, CA, USA). Briefly, original protein solution and standard curve protein was diluted (500-fold) with protein assay buffer in a 96 microplate and analyzed using an iMark microplate reader (absorbance 595 nm; Bio-Rad Laboratories, Inc., Hercules, CA, USA). Total protein (10 µg) was loaded and separated using SDS-PAGE (8–20% gel) for 90 min, and then transferred onto PVDF membranes for another 90 min. Following blocking in 3% skim milk for 1 h, the membranes were incubated in primary antibodies at 4°C overnight. The unbound antibodies were then washed off in TBST 3–5 times the subsequent day, followed by incubations with horseradish peroxidase-conjugated sheep anti-rabbit immunoglobulin G (1:2,000; cat. no. HAF016; Bio-Techne Ltd., Oxford, UK) at room temperature for 1 h. A FluorChem E (Cell Biosciences, Inc., Shanghai, China) imaging system was used to visualize the signals. The primary antibodies used were as follows: α2a, α2b and α2c adrenergic receptor (1:1,000; cat. nos. ab85570, ab151727 and ab151618, respectively; Abcam, Cambridge, UK), B-cell lymphoma 2 (1:1,000; BCL2; cat. no. ab59348; Abcam), Bcl-2-associated X protein (1:2,000; BAX; cat. no. 2772; Cell Signaling Technology, Inc., Danvers, MA, USA), total OXPHOS rodent WB antibody cocktail (1:10,000; cat. no. ab110413; Abcam) and GAPDH (1:2,000; cat. no. 2118; Cell Signaling Technology, Inc.).

Total RNA was extracted from cultured cardiomyocytes using TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.), and cDNA (50 ng/µl) was synthesized using oligo (dT) primers with the Transcriptor First Strand cDNA Synthesis kit (cat. no. 04896866001; Roche Diagnostics, Shanghai, China). Total DNA was extracted from the cultured cardiomyocytes using a QIAamp DNA Mini kit (Qiagen, Inc., Shanghai, China). Following analysis using NanoDrop™ 2000/2000c (Thermo Fisher Scientific, Inc.), DNA was diluted with RNase-free distilled H₂O (Takara Biotechnology Co., Ltd., Dalian, China) to a total volume of 20 ng/µl. The mitochondrial DNA was then measured by detecting the cytochrome B (CYTB) gene. The real-time PCR amplifications were quantified using SYBR-Green (cat. no. 04887352001; Roche Diagnostics) and the reaction system was composed of 10 µl SYBR premix Ex Taq II, 0.2 µl primers, 8.8 µl H₂O, 1 µl DNA. There thermocycling conditions were as follows: 5 sec at 95°C followed by 30 sec at 60°C for 42 cycles using a thermal cycler dice real-time system (Takara Biotechnology Co., Ltd.) (25). The results were normalized by the gene expression of 18s rRNA. The primers used in the present study are presented in Table I.

All experiments were repeated two or three times. All results are reported as the mean ± standard error of the mean. The normality of distribution was analyzed using the D'Agostino-Pearson omnibus normality test using GraphPad Prism software (version 6.0; GraphPad Software, Inc., La Jolla, CA, USA). Comparisons between two groups were analyzed using Student's t-test. Multiple comparisons between groups were performed using one-way analysis of variance with Tukey's multiple comparisons test (GraphPad Prism version 6.0). P<0.05 was considered to indicate a statistically significant difference.

Prior to experiments, it was detected that α2 adrenergic receptors were expressed in cardiomyocytes using western blot analysis (Fig. 1A), which is consistent with a previous report (14). To determine whether dexmedetomidine treatment is beneficial to cardiomyocytes, the present study investigated the survival and apoptosis of dexmedetomidine-pretreated cardiomyocytes following H₂O₂ stimulation. First, the cardiomyocytes were exposed to various doses of dexmedetomidine (26) and it was demonstrated that dexmedetomidine pretreatment attenuated the loss of cardiomyocytes induced by H₂O₂ (Fig. 1B). As the beneficial effect of dexmedetomidine against ROS may suppressed apoptosis, apoptosis was detected using TUNEL staining and FACS, and it was identified that dexmedetomidine pretreatment suppressed H₂O₂-induced cardiomyocyte apoptosis, as presented in Figs. 1C, 2A and B. These results suggested that dexmedetomidine pretreatment created protective cellular conditions, which resisted ROS-induced apoptosis and increased cellular survival.

To verify the protective role of dexmedetomidine pretreatment, the present study investigated several mitochondria-associated parameters, as mitochondria are the primary site for ROS generation and the main target of ROS (27,28). As mitochondrial DNA and biogenesis are crucial in the maintenance of cellular and mitochondrial function under oxidative stress (29), the results demonstrated that dexmedetomidine did not affect mitochondrial DNA synthesis (Fig. 3A), but significantly decreased (P<0.05) the expression of genes involved in mitochondrial biogenesis (Fig. 3B), in addition to respiratory complex I, II and IV-related proteins, in a dose-dependent manner (Fig. 3C). However, marginal changes were observed in integrated mitochondrial respiratory complexes (Fig. 3D). These results led to the investigation of whether suppressed mitochondria were accompanied by any adverse functional phenotypes. Therefore, an extracellular flux analyzer was used to detect the respiratory functions of dexmedetomidine-treated cardiomyocytes (Fig. 4). Dexmedetomidine pretreatment did not affect extracellular acidification rate, nor cellular, mitochondria or ATP-linked respiration (Fig. 4B and C), indicating that the decreased mitochondria biogenesis and respiratory complexes induced by dexmedetomidine were not accompanied by any pathological alterations. However, dexmedetomidine decreased FCCP-induced cellular maximal respiration (Fig. 4D), indicating a potential role for dexmedetomidine in resistance to FCCP. It was hypothesized that mitochondria respiratory complexes declined but with no respiratory impairment due to elevated respiratory efficiency. As expected, dexmedetomidine decreased proton leakage, which reduced the uncoupling proton influx and increased coupling efficiency (Fig. 4E).

The Δψ_m protects cells against ROS and apoptosis (30), therefore, the present study examined whether Δψ_m was affected by dexmedetomidine. It was identified that dexmedetomidine upregulated Δψ_m in a dose-dependent manner (Fig. 5A). As evidence indicates that BCL2 family members protect against Δψ_m depolarization and apoptosis (31–33), the present study examined BCL2 family proteins and demonstrated that dexmedetomidine treatment significantly induced the protein expression of BCL2 without any change in BAX expression (Fig. 5B). Subsequently, whether the improvement in Δψ_m by dexmedetomidine contributed to its anti-apoptotic role was investigated. Cardiomyocytes were exposed to 0.5 and 1 µM FCCP, a mitochondrial uncoupler reported to impair Δψ_m (34). The results confirmed that FCCP induced Δψ_m loss; however, dexmedetomidine pretreatment inhibited FCCP-induced mitochondria Δψ_m loss (Fig. 5C). These results indicated that dexmedetomidine reinforced Δψ_m, which suppressed its collapse induced by FCCP. To account for the crucial role of BCL2 in regulating Δψ_m, the present study examined tested the upregulated BCL2 induced by dexmedetomidine is involved in resisting apoptosis. Cardiomyocytes were exposed to a BCL2 inhibitor (ABT-199), and it was demonstrated that dexmedeto-midine did not suppress FCCP-induced loss of Δψ_m following BCL2 inhibition (Fig. 5D), indicating that BCL2 was involved in the protective effect of dexmedetomidine against Δψ_m loss.

As mitochondria are a main target for ROS damage, it was hypothesized that dexmedetomidine attenuates the mitochondrial response to ROS, thus reducing ROS-induced damage. Additional ROS has been reported to induce ROS release, which promotes mitochondrial and cell damage (35). In the present study, ROS levels were measured under normal conditions and following H₂O₂ incubation. It was demonstrated that dexmedetomidine marginally reduced cardiomyocyte ROS levels under normal conditions and significantly reduced ROS levels following H₂O₂ incubation (Fig. 6A and B). As different ROS levels were inhibited by dexmedetomidine [H₂O₂(−) in Fig. 6A] the ROS response rate to ROS stimulation was calculated [H₂O₂(+)/H₂O₂(−)], and a reduced ROS response towards to H₂O₂ was observed (Fig. 6B and C). Mitochondrial ROS generation is accompanied by the improved function of respiratory complexes (27,28), which leads to high cellular OCR, and ROS has been reported to induce cellular OCR (36). Therefore, the present study subsequently measured the OCR response toward H₂O₂ in cardiomyocytes incubated with dexmedetomidine. The dexmedetomidine-treated cardiomyocytes significantly inhibited the cellular increase of OCR following H₂O₂ incubation (Fig. 7A and B).

In summary, all results indicate that dexmedetomidine preconditioning protects against ROS-induced apoptosis of cardiomyocytes by reducing sensitivity of the mitochondrial response towards ROS through decreasing mitochondrial respiratory complexes, reinforcing Δψ_m, causing resistance to Δψ_m loss (Fig. 7C).