H9C2 cells were obtained from KeyGEN BioTECH (KG444, Nanjing, China), and cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM, 0030034DJ, GIBCO, New York, USA) supplemented with 10% fetal bovine serum (FBS, 16140071, GIBCO, New York, USA) and 1% penicillin/streptomycin (15140122, GIBCO, New York, USA). The cells were incubated in a tri-gas incubator with 5% CO₂ at 37°C.

Cells were inoculated at 3,000–4,000/well in 96-well plates. After treatment, 10% cell counting kit-8 (CCK-8, HY-K0301, MCE, New Jersey, United States) reagent was added and incubated for 1–3 h. The absorbance was measured using a Microplate Reader (Thermo Fisher Scientific, Massachusetts, United States) at 450 nm.

H9C2 cells were treated with no reagents (C), erastin (5 μM, GC16630, California, GLPBIO, United States) for 24 h (E), and propofol (50 μM, D126608, Sigma, Missouri, United States) for 1 h before erastin (E + P). Each experiment was repeated three times (Figure 1A).

H9C2 cells were inoculated in 12-well plates and treated as described above. Next, 0.4% trypan blue solution (T8070, Solarbio, Beijing, China) was added to the cells for 3 min. Cell viability was calculated by a cell counter (Countstar^® BioTech, ALIT Life Science, Shanghai, China).

H9C2 cells were inoculated into 12-well plates overnight and treated as described above, then 10 µM dihydroethidium solution (DHE, S0063, Beyotime, Shanghai, China) was added to each well and the plates were incubated in the dark for 30 min at 37°C. The cells were observed under a fluorescence microscope (Nikon, Tokyo, Japan) and analyzed with Image J software (National Institutes of Health, Maryland, United States).

H9C2 cells from each group were collected and used to generate a 10% homogenate, which was placed at −20°C and repeatedly thawed on ice 3 times. The supernatant was centrifuged at 2,500 r/min for 10 min at 4°C. The MDA levels and SOD activity were determined according to the kit instruction (A003-1-2 and A001-3-2, Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The iron levels in H9C2 cells were detected using a kit (DIFE-250, BioAssay Systems, California, USA) and in the myocardium were determined according to the kit instructions (A039-2-1, Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The protein concentration was measured with a BCA kit (P0012S, Beyotime, Shanghai, China).

After incubation in 12-well plates for 24 h, the H9C2 cell medium was replaced with a serum-free medium (Opti-MEM I, Thermo Fisher Scientific, Massachusetts, United States). The siRNA (Gene Pharma, Shanghai, China) was diluted in serum-free medium and mixed with diluted Lipofectamine 2000 (Lipo 2000, Thermo Fisher Scientific, Massachusetts, USA) for 20 min at room temperature. Then, the mixture was added into 12-well plates and incubated for 6–8 h, replacing the medium with a complete medium once. After being incubated for 24 h, the cells were treated for follow-up experiments as the design above. Proteins were extracted from H9C2 cells and handled as western blot analysis. The siRNA sequence of AKT was as follows: F: 5′-CCA​AGC​ACC​GUG​UGA​CCA​UTT-3′, R: 5′-AUG​GUC​ACA​CGG​UGC​UUG​GTT-3′.

Adult male rats (Wistar, weight 180–240 g), purchased from the Experimental Animal Center of Shandong University, were housed in a constant temperature (25 ± 2°C) room, fed with standard water and laboratory diet.

Rats were acclimatized to the laboratory environment for 3 days, anesthetized by 10% chloral hydrate, and sequentially injected with heparin (250 U/kg, H8060, Solarbio, Beijing, China). The hearts were rapidly resected and dipped into 40 ml ice-cold KH solution (pH 7.4, containing in mM: NaCl 115, MgSO₄ 1.2, KCl 4.7, KH₂PO₄ 1.2, CaCl₂ 1.8, glucose 11, and NaHCO₃ 25). Finally, the hearts were cannulated using a retrofitted Langendorff apparatus and perfused with KH at a velocity of 10 ml/min, which was gassed with 5% CO₂ and 95% O₂ at 37°C. Myocardial ischemia was caused by stopping perfusion for 30 min, and reperfusion resulted from recanalization for another 60 min.

All rats were randomly divided into five groups. After stabilizing for 15 min, rat hearts were treated with KH for 120 min (C), with (I/R + P) or without (I/R) propofol for 20 min before stopping perfusion for 30 min and reperfusion for 60 min, with (I/R + P + MK) or without propofol (I/R + MK) after MK2206 (15 nM, GC16304, New York, GLPBIO, USA) for 10 min before stopping perfusion (N = 6, Figure 1B).

After recanalization, the rat heart left ventricle anterior wall was cut. MDA, iron levels (A039-2-1, A039-2-1, Nanjing Jiancheng Bioengineering Institute, Nanjing, China), and the GSH/GSSG ratio (S0053, Beyotime, Shanghai, China) were assessed using their respective assay kits. The concentration of protein was detected by a BCA kit and the absorbance was determined with a microplate reader.

Hearts were fixed overnight in 4% paraformaldehyde, dehydrated with alcohol, and made transparent with xylene. They were then fixed in paraffin and finally cut into 3–4-µm-thick slices, which were placed in xylene and alcohol and then stained with hematoxylin-eosin (HE) (C0105S, Beyotime, Shanghai, China). Finally, the sections were sealed with neutral resin and imaged under a microscope (Nikon, Tokyo, Japan).

After reperfusion as described above, the left ventricle anterior wall was cut into a size of 2 mm × 5 mm × 10 mm and fixed overnight in electron microscope fixative (G1102, Servicebio, Wuhan, China). The sections were then embedded in phosphate buffer and baked in the oven. Sequentially, they were cut to a size 50–60 nm and stained with 2% uranyl acetate lead. Images were acquired under a transmission electron microscope (HITACHI, Tokyo, Japan).

Proteins were extracted from H9C2 cells and myocardium lysates of the left ventricle anterior wall. Protein was separated by 12% SDS-PAGE, transferred to polyvinylidene fluoride (PVDF) membranes, blocked with 5% non-fatty milk for 60 min, and incubated with primary antibodies overnight at 4°C. Then, the membranes were incubated with secondary antibodies for 60 min and detected with enhanced chemiluminescence (ECL) detection system. Images were analyzed by ImageJ software.

After dewaxing and rehydrating according to a previous description, paraffin sections were repaired in citrate solution (P0083, Beyotime, Shanghai, China) for 30 min, blocked with 5% bovine serum albumin (BSA) for 60 min, and incubated with primary antibodies overnight at 4°C. Subsequently, they were incubated with secondary antibodies for 60 min and DAPI (C1002, Beyotime, Shanghai, China) for 15 min, followed by visualization using an immunofluorescence microscope and analyzed with ImageJ software.

The following antibodies were used: anti-AKT (ab179463, Abcam, Cambridgeshire, UK), anti-AKT (phospho Ser473, 4060, CST, Boston, United States), anti-P53 (2,524, CST, Boston, United States), anti-P53 (phospho S392, ab33889, Abcam, Cambridgeshire, UK), anti-Ferritin (ab75973, Abcam, Cambridgeshire, UK), anti-GPX4 (ab125066, Abcam, Cambridgeshire, UK), anti-XCT (ab175186, Abcam, Cambridgeshire), anti-α-tubulin (ab7291,Abcam, Cambridgeshire, UK). HRP-conjugated secondary antibodies (ZB-2301 or ZB-2305, ZSGB-BIO, Beijing, China) and fluorochrome conjugated secondary antibody (ab150064, Abcam, Cambridgeshire).

Data are expressed as mean ± standard deviation (SD) and were analyzed by one-way ANOVA with Tukey’s post hoc test or the t-test. GraphPad Prism version 9.0 (San Diego, California, United States) was used for analysis, and a p value < 0.05 was considered to indicate a significant difference.

First, we examined the effect of erastin on H9C2 cells and found a linear inhibition of cell viability with increasing erastin concentrations using the CCK-8 assay. We found that 2.5 µM erastin markedly decreased cell viability; hence it was selected for follow-up experiments (Figure 2A). The cell viability was lower in Group E than in Group C, as revealed by the optical microscope and the trypan blue assay, which was increased by propofol pretreatment (Figures 2B,D,E). Similarly, the ROS, MDA, and iron levels were higher in Group E than in Group C as revealed by DHE staining, MDA, and iron test. All of these parameters were also reduced by propofol pretreatment (Figures 2C,F–H). The change of SOD activity was similar to the trypan blue results (Figure 2I). In addition, western bolt analysis showed a significant decrease in the expression of anti-ferroptosis enzymes (FTH1 and GPX4) in Group E compared with Group C, which was again reduced by propofol pretreatment (Figures 2J–L). Taken together, these results demonstrated that erastin induced ferroptosis in H9C2 cells and propofol pretreatment alleviates this form of death, suggesting an important protective role of propofol in the H9C2 cells ferroptosis.

Given the central role of AKT in myocardial protection, we investigated whether AKT was involved in the anti-ferroptosis effect of propofol. We first knocked out the AKT gene of H9C2 cells with AKT siRNA. Western bolt analysis showed that AKT siRNA significantly reduced AKT and P-AKT expressions levels (Figures 3A–C). In addition, it showed that AKT siRNA significantly reduced the expression of anti-ferroptosis enzymes (FTH1, XCT, and GPX4) in H9C2 cells, which was further reduced by erastin, while propofol slightly inhibited the effect of erastin (Figures 3D–G). Similarly, the cell viability of H9C2 cells in the CCK8 experiment was similar to the above enzymes (Figure 3H). These results indicated that propofol might inhibit ferroptosis in H9C2 cells through the AKT signaling pathway.

To understand the effect of propofol on myocardial tissue ferroptosis, we established an isolated myocardial I/R model with the Langendorff system. Similarly, MDA and iron levels were higher in Group I/R than in Group C, as revealed by the MDA and iron test; they were also reduced by propofol pretreatment (Figures 4A,B). In addition, western bolt analysis revealed a significant decrease in the expression of anti-ferroptosis enzymes (FTH1, XCT, and GPX4) in the I/R compared with Group C, which was also reduced by propofol pretreatment (Figures 4C–F). Taken together, these results demonstrated that I/R induced myocardial tissue ferroptosis and propofol pretreatment alleviates this phenomenon.

Next, we investigated whether AKT was involved in the anti-ferroptosis effect of propofol on the myocardium. We pretreated myocardial tissue with AKT inhibitor (MK2206). I/R showed a lower GSH/GSSG ratio than C, which was reversed with propofol. However, MK2206 inhibited all the effects of propofol (Figure 4G). MK2206 significantly increased myocardium iron levels in Group I/R + MK compared with Group I/R and inhibited the effect of propofol (Figure 4H). Similarly, HE staining showed an abundance of severely wavy and injured myofibers in Group I/R compared with Group C, which was prevented by propofol (Figure 4I). TEM revealed that enlarging or distorting myocardial mitochondrial ridges in Group I/R compared with Group C, which was significantly diminished in Group I/R + P. MK2206 attenuated all these effects (Figure 4J). Changes in p53 and p-p53 protein were common with increased myocardium iron (Figure 4K–M). In addition, western blot showed that the changes in anti-ferroptosis enzymes (FTH1, XCT, and GPX4) were similar to those in the GSH/GSSG ratio (Figures 5A–D). These results suggest that propofol pretreatment inhibits ferroptosis in myocardial tissue through the AKT/p53 signaling pathway.

Western blot analysis showed that AKT phosphorylation, which was inhibited by I/R or MK2206, was activated by propofol (Figures 5E,F). Immunofluorescence levels showing P-AKT phosphorylation were identical to the western blot results shown above (Figures 5G,H). There was no difference in immunofluorescence levels of AKT among the groups (Figures 5I,J).