Male Sprague–Dawley rats aged 8–10 weeks and weighing 220 g were obtained from the Nanjing Biomedical Research Institute of Nanjing University. All animal experiments complied with the Animal Research: Reporting in vivo Experiments (ARRIVE) guidelines (Supplementary Table 1. The ARRIVE guidelines 2.0 author checklist). The protocol was approved by the Ethics Committee of the Affiliated Hospital of the Nanjing University of Chinese Medicine. Following acclimatization for 1 week, the rats were divided into five groups of six rats each before the experiment. The establishment of the myocardial I/R model was based on previous studies (14). Sodium pentobarbital (45 mg/kg, i.p.) was used to anesthetize the rats, and the left coronary artery (LCA) was exposed using left thoracotomy at the fifth intercostal space. Following the LCA ligation with 7-0 silk sutures, a smooth catheter was applied to the artery to achieve ischemia for 30 min. The rats were then sacrificed 120 min after reperfusion. Rats in the sham group (without the LCA I/R) underwent surgery and were treated with saline. The miR-135b-3p group rats were injected with miR-135b-3p overexpression virus or knockdown lentivirus (1 × 10⁸ U/ml, 0.2 ml), respectively, for five consecutive days before surgery. The detailed animal grouping information in this study is listed in Supplementary Figure 1.

The rat myocardial cell line H9C2 was purchased from the Cell Resource Center of the Shanghai Academy of Sciences. H9C2 cells were cultured in DMEM supplemented with 10% fetal bovine serum and 100 units/ml of penicillin-streptomycin (MP Biomedicals). The cells were maintained in a humidified incubator containing 5% CO₂ at 37°C. When the cells reached 80% confluence, the DMEM was replaced with serum-free and sugar-free medium. The cells were then placed in a 37°C hypoxia incubator containing 95% N₂ and 5% CO₂ for 6 h (17, 18). After hypoxia, the medium was replaced with a fresh medium and refilled with air containing 5% CO₂ to establish I/R injury in cells.

H9C2 cells cultured in 100-mm plastic dishes were allowed to adhere to the plate at 37°C in 5% CO₂ for 6 h. Subsequently, cells were treated with 50 μM ferroptosis activator Erastin (MCE, China) or 1 μM ferroptosis inhibitor ferrostatin-1 (Fer-1, MCE, China) and incubated for 24 h. The cells were seeded in six-well plates at 1.0 × 10⁵/ml. When the confluency of cells reached 60%, transfection was performed. The GPX4 overexpression plasmid, miR-135b-3p mimics, and inhibitor were purchased from Synthgene (Nanjing, China), and the transfection was performed using Lipofectamine 2000 (Thermo-Scientific, USA) according to the manufacturer's instructions.

Rat blood was collected after reperfusion. After centrifugation at 3,000 rpm for 10 min at 4°C, 100 μl of serum was obtained. The activities of specific marker enzymes, including creatine phosphokinase (CK), lactic dehydrogenase (LDH), and cardiac troponin T (cTnT), were assessed according to the manufacturer's instructions (R&D Systems).

Intracellular ferrous iron (Fe²⁺) levels in rat myocardial tissues or H9C2 cells were measured using an iron assay kit (Abcam, USA) according to the manufacturer's instructions. Briefly, samples were collected and washed in cold PBS and then homogenized in 5X volumes of iron assay buffer on ice. The supernatant was collected, an iron reducer was added to each sample before mixing, and the samples were incubated at 25°C for 30 min. Thereafter, an iron probe was added to each sample before mixing and incubating at 25°C for 60 min. The output was measured immediately using a colorimetric microplate reader (optical density [OD], 593 nm).

Cells were seeded in 96-well plates at a density of 2 × 10³ cells/well (200 μl/well) and cultured for 24 h. Subsequently, 5 mg/ml of the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) reagent (Sigma, USA) was added (20 μl/well) and incubated at 37°C for another 4 h. Thereafter, the medium was removed and replaced with 150 μl/well DMSO (Sigma, USA), followed by vigorous shaking at room temperature for 10 min to solubilize the dark blue formazan crystals formed. Cell viability was evaluated by measuring the optical absorbance at 490 nm (OD490) using an Elx800 enzyme immunoassay analyzer (Bio-TEK, USA). The cell viability index was calculated as the experimental OD value/control OD value.

Total RNA was isolated from the myocardial tissues or cultured cells using TRIzol® reagent (Thermo-Scientific, USA) and RNA was reverse transcribed to cDNA from 1 μg of total RNA using a PrimeScript RT reagent kit with gDNA Eraser (Takara, Japan) according to the manufacturer's protocol. The reaction conditions were as follows: 42°C for 2 min, 37°C for 15 min, and 85°C for 5 s. RT-PCR was performed using the SYBR green PCR kit on an Applied Biosystems 7300 sequence detection system (Applied Biosystems, USA). U6 levels were used to normalize the relative abundance of miR-135b-3p, and Gapdh was used to normalize the expression of Gpx4, ferritin heavy chain 1 (Fth1), Ascl4, nicotinamide adenine dinucleotide phosphate oxidase 1 (Nox1), and cyclooxygenase 2 (Cox2). RT-qPCR reactions were performed in a 96-well plate at 95°C for 10 min, followed by 40 cycles of 95°C for 15 s, and 60°C for 60 s, according to the manufacturer's specifications. The primers used in this study are listed in Supplementary Table 2.

Total protein from H9C2 cells or myocardial tissues was extracted using RIPA lysis buffer. Proteins in the samples (20 μg) were separated via 10% SDS-PAGE and then transferred to a polyvinylidene difluoride membrane (Millipore, USA). Membranes were then incubated with 5% non-fat milk containing 0.1% PBST for 2 h at room temperature to block nonspecific binding and incubated with primary antibodies against GPX4 (1:1,000, Abcam), FTH1 (1:1,000, Abcam), ACSL4 (1:5,000, Abcam), NOX1 (1:5,000, Abcam), COX1 (1:500, Abcam), and GAPDH (1:1,000, Abcam) at 4°C overnight, followed by incubation with goat anti-rabbit HRP-conjugated secondary antibody (1:5,000, Abcam) at room temperature for 2 h. Protein bands were visualized using an enhanced chemiluminescence kit (Synthgene, China), and GAPDH was used as an internal control for the relative protein expression. Protein bands were quantified using the ImageJ software.

The entire 3′-UTR of Gpx4 containing the predicted binding sites for miR-135b-3p was amplified and inserted into a luciferase reporter plasmid (Synthgene, China). To assess the binding specificity, the sequences that interacted with miR-135b-3p were mutated, and the mutant Gpx4 3′-UTR was inserted into an equivalent luciferase reporter plasmid. For the luciferase reporter assay, cells were plated in 24-well plates, and each well was transfected with 1 μg of luciferase reporter plasmid, 1 μg of β-galactosidase plasmid (internal control), and 100 pmol of miR-135b-3p mimic or control mimic using Lipofectamine 2000 (Thermo Fisher Scientific, USA). After 48 h, luciferase signals were measured using a luciferase assay kit according to the manufacturer's protocol (Promega Corporation, USA) (19, 20).

After excising the myocardial tissue from rats, the tissues were fixed with 4% paraformaldehyde for 24 h and paraffin embedded. Sections (4 μm) were cut and stained with HE (21).

At the end of 120 min of reperfusion, the hearts were collected and frozen at −20°C. Frozen hearts were cut into 1-mm sections that were incubated in 1% TTC solution at 37°C for 10 min and then fixed in 4% paraformaldehyde for 24 h. The sections were photographed using a digital camera. The infarcted areas were not stained by TTC (22, 23). The infarcted (unstained) and non-infarcted (stained) areas were measured in each section using ImageJ by a blinded investigator.

Cells were treated as indicated, trypsinized, and resuspended in a medium supplemented with 10% FBS. A 10 μM solution of C11-BODIPY (Thermo Fisher, USA) was added, and samples were incubated for 30 min at 37°C with 5% CO₂ and protection from light. The cells were washed twice with PBS to remove excess C11-BODIPY. The fluorescence of C11-BODIPY was measured using a fluorescence microscope (Nikon, Japan) (24).

Cardiac function and structure were assessed using the MyLab™Eight Platform (Esaote, Italy). Briefly, rats were anesthetized with isoflurane (5%) using ventilation equipment, fur was carefully removed from the left chest, and two-dimensional echocardiographic measurements were obtained. Left ventricular internal diastolic and systolic diameter (LVIDd and LVIDs, respectively) and the left ventricular ejection fraction and fractional shortening (LVEF and LVFS, respectively) were measured using M-mode tracing.

All data are presented as the mean ± standard deviation (SD) of three independent experiments. One-way ANOVA and Duncan's multiple range tests were used to evaluate the mean differences between groups and within groups. Statistical significance was set at p < 0.05.

Initially, we established a myocardial I/R injury model in rats and collected myocardial tissue and serum samples. We performed HE staining and ELISA to detect tissue structure and cardiac injury marker expression, respectively. The serum CK, LDH, and cTnT levels were significantly increased in the I/R group compared to those in the sham group (Figure 1A). As shown in Figure 1B, the myocardial cells were well-ordered with regular structure and myocardial fibers were intact in the sham group, but myocardial cells were disordered and a few myocardial fibers were broken in the I/R group. Results of the analysis of the cardiac injury biomarker expression and HE staining revealed significant damage in the hearts of the rats in the I/R group. To verify the involvement of ferroptosis in the process of cardiac injury, we used the iron assay kit to measure ferrous iron levels in myocardial tissues, and the results showed that in the I/R group, the normalized Fe²⁺ levels were elevated (Figure 1C). The ferroptosis-related gene expression results are shown in Figures 1D–F. The protein and mRNA expression of GPX4, FTH1, ACSL4, NOX1, and COX2 was detected through Western blotting and RT-qPCR, respectively. ACSL4, NOX1, and COX2 mRNA and protein levels were significantly higher in the I/R group than in the sham group. However, we found that GPX4 and FTH1 were significantly reduced at the protein level in the I/R group but not at the mRNA level, suggesting the presence of factors inhibiting the translation of these two genes.

To visualize the cardiac damage in the I/R group, we performed transthoracic echocardiography and M-mode tracings in rats in the I/R group. The results showed that LVIDs were significantly higher while LVFS and LVEF% were significantly lower in the I/R group than those in the sham group. In addition, the mean LVIDd values were higher in the I/R group than the sham group, and there were no statistically significant differences between the two groups (Figures 1G–J). The above results showed that the heart function of rats was disrupted after I/R injury.

To explore the miRNA-mediated regulation of Gpx4 expression, we used TargetScan, miRWalk, and miRada to predict and screen the miRNAs targeting Gpx4 (Supplementary Dataset 1), and the results were presented in the form of a Venn diagram (Figure 2A), which was drawn using the tool available from http://bioinformatics.psb.ugent.be/webtools/Venn/. The expression of miRNAs found by database analysis, in the sham and I/R groups, was examined using RT-qPCR. We found that miR-135b-3p expression was significantly increased in myocardial I/R (Figure 2B). The prediction results of the TargetScan Release 7.0 database showed that the 3′-UTR of Gpx4 mRNA possesses putative binding sites for miR-135b-3p (Figure 2C). To further confirm the direct binding of miR-135b-3p to the Gpx4 mRNA 3′UTR, a luciferase activity assay was performed. H9C2 cells were co-transfected with Gpx4-3′UTR-WT, Gpx4-3′UTR-MUT, miR-135b-3p, or negative control mimics. miR-135b-3p significantly inhibited the luciferase activity of Gpx4-3′UTR-WT, whereas that of Gpx4-3′UTR-MUT was not decreased (Figure 2D), suggesting that miR-135b-3p was able to suppress the translation by binding to the 3′UTR of Gpx4 mRNA. These results demonstrate that the miR-135b-3p expression increases in myocardial I/R tissue and directly regulates the expression of Gpx4 in H9C2 cells.

Erastin is an oncogenic RAS-selective lethal small molecule that triggers a unique iron-dependent form of non-apoptotic cell death in various cell types. Fer-1 inhibits the accumulation of cytoplasmic and lipid ROS induced by erastin (25). To further explore the roles of ferroptosis in myocardial I/R, we cultured rat myocardial H9C2 cells under H/R conditions to induce I/R damage. Cell viability was reduced in the H/R group compared to the control group, and treatment with erastin further reduced cell viability, whereas treatment with Fer-1 restored the viability of H9C2 cells subjected to H/R (Figure 3A). These results confirmed that H/R treatment could promote ferroptosis in cells. An iron assay kit was used to measure ferrous iron levels in H9C2 cells. The results showed that the normalized Fe²⁺ level in the H/R group was significantly higher than that in the control group. The normalized Fe²⁺ level in H/R cells was increased after treatment with erastin but decreased after Fer-1 treatment (Figure 3B). Subsequently, we examined the expression of ferroptosis-associated proteins by Western blotting. GPX4 and FTH1 expression decreased and ACSL4, NOX1, and COX2 expression increased in H/R cells compared to the control cells. When H/R cells were treated with erastin, GPX4 and FTH1 expression was inhibited, while ACSL4, NOX1, and COX2 expression increased. As shown in Figures 3C,D, the ferroptosis-related gene expression in Fer-1-treated cells was completely contrary to that in the erastin-treated cells. Immunofluorescence results showed that ROS levels were upregulated in H/R cells and the erastin-treated H/R cells, whereas Fer-1 treatment downregulated the ROS levels in H/R cells (Figure 3E). These results suggest that ferroptosis is involved in myocardial H/R injury. Furthermore, we used RT-qPCR to detect changes in miR-135b-3p expression in different groups and found that miR-135b-3p expression was positively correlated with the severity of ferroptosis (Figure 3F). These results suggest that miR-135b-3p may be involved in myocardial cell ferroptosis.

Previous studies have demonstrated that miR-135b-3p can target GPX4 and is aberrantly expressed in ferroptosis; therefore, we determined whether miR-135b-3p promotes ferroptosis by regulating GPX4 expression. To explore the miR-135b-3p effect in H/R-induced H9C2 cells, we first transfected miR-135b-3p mimics, negative control mimics, miR-135b-3p inhibitor, and negative control inhibitor in H/R-induced H2C9 cells. miR-135b-3p expression detected by RT-qPCR was used to confirm the transfection efficiency (Figure 4A). Western blotting results showed a significant negative correlation between miR-135b-3p and GPX4 (Figures 4B,C). miR-135b-3p increased the normalized Fe²⁺ levels (Figure 4D). The cell viability assay showed that miR-135b-3p mimics significantly decreased the cell viability compared to the negative control mimics group, whereas the viability of cells transfected with miR-135b-3p inhibitor was increased compared to the negative control group (Figure 4E). Based on these results, we concluded that miR-135b-3p regulated the expression of GPX4 and ferroptosis in H/R-induced H9C2 cells.

To further explore the influence of miR-135b-3p on ferroptosis through GPX4, we used the GPX4 plasmid to increase the expression of GPX4, which was confirmed by Western blotting. The Western blot results also showed that the increased expression of GPX4 and miR-135b-3p leads to increased expression of FTH1 and decreased expression of ACSL4, NOX1, and COX2 in H/R myocardial cells, compared to the cells transfected only with miR-135b-3p mimics (Figures 5A,B). Results from the assays for normalized Fe²⁺ levels, cell viability, and ROS levels confirmed that restoration of GPX4 expression could reduce cell ferroptosis and increase cell viability (Figures 5C–E). These data suggest that miR-135b-3p promotes cell ferroptosis by inhibiting GPX4 expression in H/R myocardial cells.

To confirm that miR-135b-3p promotes cell ferroptosis by suppressing GPX4 expression in vivo, we constructed an I/R rat model by injecting the control virus, miR-135b-3p overexpression virus, or knockdown virus into the tail vein. After reperfusion, the myocardial tissue and serum were collected. ELISA results showed a positive correlation between miR-135b-3p and CK, LDH, and cTnT levels, indicating that miR-135b-3p could aggravate myocardial I/R injury (Figure 6A). HE staining showed that overexpression of miR-135b-3p could lead to myocardial cell disorder and a rise in cell death, and knockdown of miR-135b-3p could restore these effects (Figure 6B). Western blot results showed that GPX4 expression was reduced in the I/R group, but inhibiting miR-135b-3p expression restored the GPX4 expression (Figures 6C,D).

To visually assess the infarct volume, we performed TTC staining, which showed that the hearts of rats in the I/R group showed distinct areas of infarction compared to the sham group and that the infarction became more severe after miR-135b-3p overexpression (Figures 6E,F). In addition, we performed transthoracic echocardiography and M-mode tracing in rats from five groups. The results showed that LVIDd and LVIDs values were significantly higher, whereas LVFS% and LVEF% were significantly lower, in the I/R group than in the sham group, as demonstrated in Figure 1. In addition, we found that miR-135b-3p expression had a significant effect on these four indicators: miR-135b-3p overexpression led to an increase in LVIDd and LVIDs and a decrease in LVFS% and LVEF% in rats (Figures 6G–J).

Taken together, the in vivo experiments confirmed that miR-135b-3p promotes cellular ferroptosis by downregulating GPX4 expression, thereby exacerbating myocardial I/R injury.