The myocardial I/R model was established as previously described (32). A total of 18 rats were used in the study, with 6 rats in each group. Rats were randomly assigned to three groups: The sham group underwent thoracotomy without left anterior descending (LAD) ligation; the I/R group received 4 weeks of daily normal saline gavage before I/R surgery; and the I/R + Sal group received 4 weeks of daily Sal (50 mg/kg/day) gavage before I/R surgery. This dosage was selected based on a previous study demonstrating its robust cardioprotective efficacy against myocardial I/R injury without inducing systemic toxicity (33). Rats were monitored daily for general health and behavior throughout the experimental period, including activity, food and water intake, body weight, posture and signs of pain or distress. Animals were euthanized if they exhibited severe or persistent signs of distress, including marked reductions in activity or food/water intake, severe respiratory distress, inability to ambulate or maintain normal posture or other signs of severe pain that could not be alleviated. No animals met the predefined humane endpoint criteria during the present study. The total experimental period was 4 weeks of daily gavage followed by myocardial I/R surgery and 2 h of reperfusion. Briefly, rats were anesthetized with isoflurane (induction at 3%, maintenance at 1.5%) and mechanically ventilated, followed by hair removal and a left thoracotomy at the fourth intercostal space to expose the heart. The LAD coronary artery was ligated with a 7-0 silk suture for 30 min of ischemia, after which the ligature was released to allow 2 h of reperfusion. The chest was closed in layers with a 4-0 silk suture after evacuating residual air from the pleural cavity. At the end of the experiment, echocardiographic assessment was performed, followed by blood collection from all rats while they remained under anesthesia. According to the American Veterinary Medical Association Guidelines for the Euthanasia of Animals (34), rats were euthanized by a deep anesthetic overdose using 5% isoflurane, and death was confirmed by cessation of respiration and heartbeat. No rats died during surgery or the experimental period; no animals were found dead and no animals were excluded from the final analysis. All 18 rats were euthanized at the end of the experiment.

Under isoflurane anesthesia, left ventricular function was assessed using M-mode echocardiography [V6 lead, 23 MHz linear transducer; VINNO Technology (Suzhou) Co., Ltd.] to measure left ventricular fractional shortening (LVFS) and left ventricular ejection fraction (LVEF).

After echocardiographic measurement and while under isoflurane anesthesia, ~2 ml blood was collected from the abdominal aorta. The rats were then immediately euthanized. Blood samples were stored at 4°C overnight and then centrifuged at 12,000 × g for 15 min at 4°C to obtain serum. LDH and CK-MB levels were determined using corresponding assay kits (LDH, cat. no. A020-2-2; CK-MB, cat. no. H197-1-1; Nanjing Jiancheng Bioengineering Institute) as per the manufacturer's protocol.

Following euthanasia, the hearts were immediately excised, washed with PBS and frozen at -20°C for 30 min. Each heart was sliced into 2 mm sections, followed by incubation in 2% TTC solution (cat. no. C0652; Beyotime Biotechnology) at 37°C in the dark for 30 min. Digital images of the heart slices were obtained and analyzed using ImageJ software (version 1.8.0; National Institutes of Health). To quantify myocardial infarct size, the infarcted area and the total ventricular slice area were measured manually in a blinded manner. The infarct size was calculated as the percentage of the infarcted area relative to the total ventricular slice area (used as the denominator).

Myocardial tissue samples used for H&E staining were collected separately from those used for TTC staining and were not subjected to the freezing procedure. Following the respective treatments, the tissues were fixed in 4% paraformaldehyde at room temperature for 24 h. The fixed tissues were then dehydrated, embedded in paraffin and sectioned at a thickness of 5 μm. According to the manufacturer's instructions (cat. no. G1076; Wuhan Servicebio Technology Co., Ltd.), sections were stained with hematoxylin at room temperature for 10 min, followed by eosin staining at room temperature for 2 min. Stained sections were observed and imaged at ×400 magnification using an optical microscope (LX73; Olympus Corporation).

H9c2 cells were purchased from the Cell Bank/Stem Cell Bank of the Chinese Academy of Sciences. The cells were cultured in Dulbecco's Modified Eagle Medium (DMEM; Beijing Solarbio Science & Technology Co., Ltd.; cat. no. 12100) supplemented with 10% fetal bovine serum (FBS; cat. no. ZQ0500; Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd.) and 1% penicillin-streptomycin and maintained at 37°C in a humidified atmosphere containing 5% CO₂.

In accordance with a previous study, an H9c2 cell A/R injury model was established to simulate in vivo I/R injury (35). Briefly, cells were incubated in an anoxic buffer containing 1.0 mM CaCl₂, 20.0 mM HEPES, 10.0 mM KCl, 1.2 mM MgSO₄, 98.5 mM NaCl, 0.9 mM NaH₂PO₄, 6.0 mM NaHCO₃ and 40.0 mM sodium lactate (pH 6.8) for 4 h at 37°C under anoxia conditions (95% N₂ and 5% CO₂). Subsequently, the medium was replaced with a reoxygenation solution composed of 1.0 mM CaCl₂, 5.5 mM glucose, 20.0 mM HEPES, 5.0 mM KCl, 1.2 mM MgSO₄, 129.5 mM NaCl, 0.9 mM NaH₂PO₄ and 20.0 mM NaHCO₃ (pH 7.4), and cells were further incubated for 4 h at 37°C in a sealed chamber equilibrated with 95% O₂ and 5% CO₂.

To investigate the role of USP11 in myocardial I/R injury, genetic manipulations were performed in H9c2 cells to knock down or overexpress USP11. For USP11 knockdown, small interfering (si) RNA (si-USP11) and its negative control (si-NC) (Shanghai Focus Bioscience Co., Ltd.; Table SI) were transfected into H9c2 cells at a final concentration of 50 nM using jetOPTIMUS transfection reagent (cat. no. 101000006; Polyplus-transfection SA) according to the manufacturer's instructions. The cells were incubated at 37°C in a humidified atmosphere containing 5% CO₂, and subsequent experiments were performed after 48 h of transfection. For USP11 overexpression, the USP11 empty vector (USP11-EV; pCMV-3xFLAG-Neo) and USP11 overexpression plasmid [USP11-OE; pCMV-Usp11(rat)-3xFLAG-Neo] were constructed using the same pCMV-3xFLAG-Neo backbone and obtained from Wuhan MiaoLing Biotech Science Co., Ltd. The plasmids were transfected into H9c2 cells using 4-6 μg of plasmid DNA per transfection with HighGene plus transfection reagent (cat. no. RM09014P; ABclonal Biotech Co., Ltd.) following the manufacturer's protocol. The cells were incubated at 37°C in a humidified atmosphere containing 5% CO₂ for 6 h during transfection, after which the culture medium was replaced. Subsequent experiments were performed 48 h after transfection.

All treatments were performed at 37°C. The experimental design consisted of the following groups: i) Control group of H9c2 cells maintained under standard culture conditions; ii) A/R group of H9c2 cells subjected to A/R injury; iii) Sal concentration-dependent group of H9c2 cells pretreated with varying concentrations of Sal (1.25, 2.5, 5, 10, 20 and 40 μM) for 48 h; iv) Sal concentration-dependent + A/R group of H9c2 cells pretreated with Sal at concentrations of 1.25, 2.5, 5, 10, 20 and 40 μM for 48 h, followed by A/R injury; v) 10 μM Sal + A/R group of H9c2 cells pretreated with 10 μM Sal for 48 h prior to A/R injury; vi) Fer-1 + A/R group of H9c2 cells pretreated with 5 μM Fer-1 for 2 h prior to A/R injury; vii) erastin group of H9c2 cells pretreated with 10 μM erastin for 24 h prior to protein extraction (this group served strictly as a positive methodological control to validate the efficacy of the ferroptosis detection assays, rather than to model A/R injury); viii) 10 μM Sal + si-USP11 + A/R group of H9c2 cells transfected with si-USP11 and pretreated with 10 μM Sal for 48 h before A/R injury; and ix) USP11-OE + A/R group of H9c2 cells transfected with USP11-OE for 48 h prior to A/R injury.

Cell viability was assessed using the Cell Counting Kit-8 (CCK-8; cat. no. GK10001; GLPBIO Technology LLC) according to the manufacturer's instructions. H9c2 cells were seeded into 96-well plates and subjected to the indicated treatments. Subsequently, serum-free DMEM containing 10% CCK-8 solution was added. The cells were then incubated at 37°C for 30 min and the optical density was measured at a wavelength of 450 nm.

Lipid peroxidation levels in H9c2 cells were evaluated by measuring 4-hydroxynonenal (4-HNE) and malondialdehyde (MDA) contents. MDA levels were quantified using an MDA assay kit (cat. no. S0131; Beyotime Biotechnology) and 4-HNE levels were determined using a 4-HNE assay kit (cat. no. JL46304; Jianglai Biotechnology Co., Ltd.), according to the manufacturers' instructions.

To further assess lipid peroxidation, a boron-dipyrromethene (BODIPY) 581/591 C11 lipid peroxidation assay kit (cat. no. S0043; Beyotime Biotechnology) was used according to the manufacturer's instructions. The BODIPY 581/591 C11 probe was diluted 1:1,000 to a final concentration of 2 μM and incubated with H9c2 cells at 37°C in the dark for 20 min. After three washes with PBS, fluorescence images were captured using an optical microscope (LX73; Olympus Corporation) at ×200 magnification. Red fluorescence indicates the non-oxidized form of the BODIPY 581/591 C11 probe, whereas green fluorescence indicates the oxidized form, reflecting increased lipid peroxidation.

After the designated treatments, cell lysates were collected from each group. SOD activity (cat. no. S0101S; Beyotime Biotechnology) and the levels of reduced GSH and oxidized GSSG (cat. no. S0053; Beyotime Biotechnology) were measured according to the manufacturer's instructions. The GSH/GSSG ratio was subsequently calculated.

Intracellular ROS levels were measured using a ROS assay kit (cat. no. S0033S; Beyotime Biotechnology). The 2',7'-dichlorodihydrofluorescein diacetate probe was diluted to a final concentration of 10 μM and incubated with H9c2 cells at 37°C in the dark for 20 min. Cells were then washed three times with PBS and fluorescence images were captured using fluorescence microscopy (LX73; Olympus Corporation) at ×100 magnification.

Ferrous ion levels in H9c2 cells were assessed using a ferrous ion detection kit (cat. no. F374; Dojindo Molecular Technologies, Inc.). According to the manufacturer's instructions, the FerroOrange working solution was prepared and diluted with Hank's balanced salt solution to a final concentration of 1 μM. Cells were incubated with the 1 μM working solution at 37°C in the dark for 30 min. Fluorescence images were subsequently acquired using an optical microscope (magnification, ×200; LX73; Olympus Corporation).

Cells were lysed in RIPA buffer (cat. no. R0010; Beijing Solarbio Science & Technology Co., Ltd.) supplemented with protease and phosphatase inhibitors. Lysates were centrifuged at 12,000 × g for 15 min at 4°C and total protein concentrations were determined using a BCA protein assay kit. To ensure equal total protein input across all experimental groups, equal amounts of total protein from each sample were adjusted to the same final volume and used for each IP reaction. Each reaction contained 600 μl of lysate and was incubated with 3 μl of anti-PRDX2 antibody at 4°C with gentle rotation overnight. Protein A+G agarose beads (50 μl suspension; cat. no. P2055; Beyotime Biotechnology) were then added and the mixture was incubated for an additional 8 h at 4°C with gentle rotation. The agarose beads were collected by centrifugation at 3,000 × g for 30 sec at room temperature, washed three times with PBST (0.1% Tween-20) and boiled directly in SDS-PAGE loading buffer. The immunoprecipitants were subsequently separated by 10% SDS-PAGE and analyzed using western blotting.

Following the respective treatments, cells were lysed in RIPA buffer containing 1% PMSF (cat. no. R0010; Beijing Solarbio Science & Technology Co., Ltd.). The lysates were collected and protein concentrations were determined using a BCA Protein Assay Kit (cat. no. GK10009; GLPBIO Technology LLC). Equal amounts of protein (30 μg) were separated by 10% SDS-PAGE and transferred onto PVDF membranes. The membranes were blocked with 5% non-fat milk for 2 h at room temperature, followed by incubation with primary antibodies at 4°C overnight. The primary antibodies used were PTGS2, GPX4, USP11, PRDX2, ubiquitin and β-actin (all at 1:1,000). After incubation with HRP-conjugated secondary antibodies (1:5,000) for 2 h at room temperature, protein bands were visualized using an Ultra-High Sensitivity ECL Kit (cat. no. GK10008; GLPBIO Technology LLC) and imaged with a FluorChem FC3 system (ProteinSimple). All immunoblot experiments were performed in at least three independent biological replicates. Target protein expression was normalized to the corresponding β-actin internal loading control. Band intensities were semi-quantified using ImageJ software (version 1.8.0; National Institutes of Health).

Cell lysates were prepared as aforementioned and the supernatants were collected. Each lysate was divided into two equal portions and incubated with Sal (10 μM) or DMSO (0.1%) at room temperature for 30 min. The samples were then aliquoted into six equal fractions and heated at 50, 54, 58, 62, 66 or 70°C for 3 min. Following heat treatment, samples were centrifuged at 12,000 × g for 15 min at 4°C, and the resulting supernatants were collected, mixed with loading buffer, boiled and subsequently subjected to western blot analysis. As a control experiment to assess the thermal stability of USP11 at 50°C, cell lysates were divided into equal portions and treated with Sal (10 μM) or DMSO (0.1%) at room temperature for 30 min, followed by heating at 34, 38, 42, 46 or 50°C for 3 min. Samples were then processed for western blot analysis, and USP11 band intensities were quantified. Notably, a conventional loading control (such as β-actin) was not employed in this assay, as the majority of cellular proteins undergo unpredictable thermal denaturation across the applied temperature gradient. Instead, the reliability of the assay was ensured by strictly equalizing the starting protein concentration and dividing the lysates into equal aliquots prior to heat treatment, in accordance with established pioneering CETSA protocols (36-38). USP11 does not undergo significant thermal denaturation at 50°C; therefore, for quantitative analysis the band intensity at 50°C was defined as the intact baseline reference (100%) and the subsequent temperature points were normalized to this value to determine the thermal denaturation curves.

H9c2 cells were transfected with si-USP11 and/or treated with Sal (10 μM), as indicated. Subsequently, cells were treated with CHX (10 μg/ml; cat. no. HY-12320; MedChemExpress) to inhibit de novo protein synthesis. Cells were collected at 0, 6, 12 and 24 h after CHX treatment, and total protein was extracted and analyzed by western blotting. The protein levels of USP11 and PRDX2 were determined, with β-actin used as the loading control. PRDX2 stability was assessed by comparing its relative protein abundance at each time point with that at 0 h.

H9c2 cells were treated with Sal (10 μM) and/or transfected with si-USP11 according to the experimental design. MG132 (10 μM; cat. no. HY-13259; MedChemExpress) was added 4 h before protein collection to inhibit proteasomal degradation. Cell lysates were subjected to immunoprecipitation with an anti-PRDX2 antibody following the procedure described for the Co-IP assay. The resulting immunoprecipitants were subsequently analyzed by western blotting with an anti-ubiquitin antibody to assess PRDX2 ubiquitination.