Cell Culture and Grouping

In this study, H9c2 cells ( provided by the Chinese Academy of Sciences Shanghai Cell Bank) were used as experimental cells in vitro. A complete culture medium was prepared using DMEM medium (C11995500BT, Thermo Fisher Scientific, USA), 10% fetal bovine serum (Thermo Fisher Scientific, USA), and 1% bispecific antibody (P1400, Solarbio, Beijing, China), and culture cells in a constant temperature incubator at 37℃ and 5% CO2. When the cells reached approximately 80% confluence, they were digested with 0.25% trypsin solution (C0207; Beyotime, Shanghai, China) and passaged at a 1:3 ratio. H9c2 cells were divided into four groups: the LPS group, stimulated with 10 μg/mL LPS (L2360, Sigma, Germany) for 24 h to induce the LPS-induced cardiac injury model [27]; the LPS + LV-sh-NC group and the LPS + LV-sh-FBXO32 group, which underwent respective cell transfections followed by treatment with 10 μg/mL LPS for 24 h; and the Control group, which received an equal volume of culture medium.

Ribonucleic Acid Sequencing (RNA-seq)

Cell samples from the Control and LPS groups (with three biological replicates per group) were collected, and total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). RNA purity and quantity were evaluated using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA). Libraries were constructed using the TruSeq Stranded mRNA LT Sample Prep Kit (Illumina, San Diego, CA, USA). After quality control using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA), the libraries were sequenced on an Illumina HiSeq XTen platform by Shanghai OE Biotech. Co., Ltd. (Shanghai, China), and raw RNA-Seq data were exported in fastq format. Quality control and adapter removal were performed using Trimmomatic software, and differential gene expression analysis was conducted with the DESeq (2012) R package [28], applying screening criteria of log2 fold change (log2FC) > 1 or < −1 and FDR < 0.05.

Cell Transfection

shRNA lentiviruses targeting Fbxo32 were designed and synthesized by GeneChem Co., Ltd. (Shanghai, China), and small interfering RNAs targeting Anxa1 were designed and synthesized by GenePharma (Shanghai, China). According to the manufacturer’s instructions, HitransG A infection enhancer was employed to transfect sh-NC (control siRNA) and sh-FBXO32 (5’-GCAAAGTCACAGCTCACATCC-3’). Stable cell lines were established by continuous screening with 2 μg/mL puromycin for three generations, after which they were used for subsequent experiments. Alternatively, in sh-NC and sh-FBXO32 cell lines, si-NC and si-ANXA1 (5’-GCAGGAAUAUGUUCAAGCUTT-3’) were transfected with GP-transfect-Mate reagent. Following transfection, cells were cultured for another 48 h before being used in subsequent experiments.

Cell Viability Assay

Cell viability was assessed using the CCK-8 (Biosharp, Hefei, China). Specifically, H9c2 cells were seeded in 96-well plates at a density of 5 × 103 cells/well. After experimental treatments, 10 μL of CCK-8 solution was added to each well, and plates were incubated at 37 °C for 2 h. Absorbance was measured at 450 nm using a microplate reader, and cell viability (%) was calculated [29].

Annexin V-FITC/PI Double Staining

H9c2 cells were seeded into 6-well plates. After treatment, the cells (including those in the culture supernatant) from different experimental groups were collected, centrifuged, washed, and resuspended in flow cytometry tubes. According to the instructions of the Annexin V-FITC/PI Apoptosis Kit (MULTI SCIENCES, Hangzhou, China), 5 μL of Annexin V-FITC and 10 μL of PI were added to each tube. Subsequently, the samples were incubated in the dark at room temperature for 5 min and then analyzed using a flow cytometer (NovoCyte, ACEA Biosciences, USA).

Intracellular ROS Detection

Intracellular ROS levels were detected using a ROS detection kit (S0033S; Beyotime, Shanghai, China). H9c2 cells were seeded in 6-well plates and treated according to the experimental groups. After treatment, cells were collected and incubated with 1 mL of 10 μmol/L DCFH-DA dilution at 37 °C in the dark for 20 min. The cells were subsequently washed thrice and analyzed using a flow cytometer (NovoCyte, ACEA Biosciences, USA).

Mitochondrial Membrane Potential Detection

The mitochondrial membrane potential was measured using a JC-1 kit (C2006, Beyotime, Shanghai, China) according to the manufacturer’s instructions. H9c2 cells were seeded in 6-well plates and treated according to the experimental groups. After treatment, the cells were collected, resuspended in 500 μL JC-1 working solution, and incubated at 37 °C for 20 min. The cells were washed twice and analyzed using a flow cytometer (NovoCyte, ACEA Biosciences, USA). According to the instructions, the maximum excitation wavelength of JC-1 monomer was 514 nm, and the maximum emission wavelength was 529 nm; The maximum excitation wavelength of JC-1 polymer (J-aggregates) was 585 nm, and the maximum emission wavelength was 590 nm. Therefore, FITC and PE channels were selected to detect the corresponding fluorescence signals.

CoIP-MS

Cell lysates from the LPS group were subjected to co-immunoprecipitation (Co-IP). The Co-IP products underwent western blotting and silver staining analyses. Subsequently, liquid chromatography-tandem mass spectrometry (LC–MS/MS) was performed by Guangzhou RiboBio Co., Ltd. (Guangzhou, China). To identify FBXO32 interacting proteins, the resulting mass spectrometry data were analyzed using PEAKS software, searching against the UniProt rat proteome database (UniProt-proteome_UP000002494_20220418.fasta).

Co-IP

Co-IP was performed using an immunoprecipitation kit (P2179S; Beyotime, Shanghai, China) according to the manufacturer’s instructions. Briefly, cells were lysed in a buffer containing the protease inhibitor cocktail provided with the kit. An appropriate amount of supernatant was retrieved as an input control, whereas the remaining supernatant was reserved for subsequent Co-IP. Next, an antibody working solution was prepared, consisting of FBXO32 antibody (GTX47819, Gene Tex), ANXA1 antibody (21,990–1-AP, Proteintech), and Normal Rabbit IgG, all diluted to a ratio of 1:200). This solution was incubated with Protein A + G magnetic beads at room temperature for 1 h. Protein samples were then incubated with antibody-bound beads in a rotary mixer at 4 °C overnight. The beads were subsequently washed with lysis buffer, and the supernatant was discarded. After adding sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) sample loading buffer (1X), the samples were heated at 95 °C for 5 min. After magnetic separation, supernatants were subjected to western blot analysis.

Ubiquitination Level Analysis

Cells were pretreated with 20 μM MG132 (S2619, Selleck) for 6 h before collection. Next, the ubiquitination enzyme inhibitor N-ethylmaleimide (S3692, Selleck) was added to the protein lysis buffer at a final concentration of 10 μM. The product from cell lysis was then immunoprecipitated with protein A + G beads bound to ANXA1 antibody. Protein ubiquitination levels were detected by western blotting using an anti-ubiquitin (Ub) antibody (sc-8017, Santa Cruz).

Experimental Animals and Grouping

SPF-grade male SD rats aged 7–8 weeks and weighing 180–210 g were provided by the Animal Experiment Center of Lanzhou University and housed in the SPF-grade animal laboratory of Lanzhou University. Rats had ad libitum access to food and water in a controlled environment (temperature: 20–26 °C, relative humidity: 40–70%, 12-h light/dark cycle) and were acclimated for one week before experiments. All animal experiments complied with the “Guide for the Care and Use of Laboratory Animals” published by the National Institutes of Health (NIH publication No. 85–23, revised 1996) and were approved by the Animal Ethics Committee of the First Hospital of Lanzhou University (Approval No. LDYYLL2024-405). The rats were divided into four groups using a random number table: Control, LPS, LPS + AAV9-sh-NC, and LPS + AAV9-sh-FBXO32, with each group containing six rats. In the LPS group, cardiac injury was induced by an intraperitoneal injection of 10 mg/kg LPS (diluted to 1 mL with saline) [30]. Alternatively, the LPS + AAV9-sh-NC and LPS + AAV9-sh-FBXO32 groups received in situ myocardial injections of AAV9-NC or AAV9-shRNA-FBXO32 before modeling. The Control group received an equal volume of saline via intraperitoneal injection at the same time points.

Myocardial Injection of Adeno-Associated Virus

AAV9 targeting Fbxo32 coupled with a cardiac troponin T (cTNT) promoter was designed and synthesized by Genechem Co., Ltd. (Shanghai, China). Myocardial injection of AAV9-NC or AAV9-shRNA-FBXO32 (5’-GCAAAGTCACAGCTCACATCC-3´) was subsequently performed. Rats were anesthetized with an intraperitoneal injection of 40 mg/kg sodium pentobarbital. Once anesthetized, the rats were placed in a supine position on a surgical table, intubated, and connected to a ventilator (HX-100E, TECHMAN, Chengdu, China) for assisted ventilation. The chest was opened to expose the heart, and a total of 25 μL of AAV9 viral solution (titer: 1.92E + 13 vg/mL) was injected into five different sites using a microsyringe. The chest was then closed, and the rats were allowed to recover naturally before being transferred to their cages. Four weeks later, the knockdown efficiency of Fbxo32 was verified using quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) and western blotting. Upon successful knockdown, LPS was administered intraperitoneally to induce the model.

Hematoxylin and Eosin (HE) Staining

The myocardial tissues from each group were fixed in 4% paraformaldehyde for 48 h, dehydrated using a graded series of ethanol, embedded in paraffin, and sectioned. The sections were deparaffinized, rehydrated, stained with HE, dehydrated, and mounted. The morphology of the myocardial tissue was examined under a microscope.

Terminal Deoxynucleotidyl Transferase-Mediated dUTP Nick-End Labeling (TUNEL) Staining

Paraffin-embedded myocardial tissue sections were subjected to TUNEL staining according to the instructions of the TUNEL Apoptosis Detection Kit (C1091, Beyotime, Shanghai, China). The sections were observed and photographed under an Olympus microscope. Five random fields were selected from each section to calculate the percentage of TUNEL-positive cells. The rate of myocardial apoptosis was calculated by dividing the number of TUNEL-positive myocardial cells by the total number of cells, multiplied by 100.

Transmission Electron Microscopy (TEM) Observation of Myocardial Tissue Ultrastructure

Myocardial tissues were pre-fixed in 3% glutaraldehyde solution, post-fixed in 1% osmium tetroxide, washed, dehydrated using a graded ethanol series, and embedded in epoxy resin. Ultrathin sections were prepared, mounted on copper grids, stained sequentially with uranyl acetate and lead citrate, and observed under a transmission electron microscope (JEM-1400FLASH, JEOL, Japan) to assess ultrastructural changes in myocardial cells.

Hemodynamic Monitoring of the Heart

Thirty minutes before the observation time point, the SD rats were weighed and anesthetized via an intraperitoneal injection of 40 mg/kg sodium pentobarbital. Once anesthetized, the rats were placed in a supine position on a surgical table. They were then intubated and connected to a ventilator (HX-100E, TECHMAN, Chengdu, China) to maintain assisted ventilation throughout the procedure. A PE50 polyethylene tube was connected to an integrated signal acquisition and processing system (BL-420I, TECHMAN, Chengdu, China) using a PT-102N pressure transducer. Next, the right common carotid artery was isolated, and the catheter was retrogradely inserted into the left ventricle through the common carotid artery and fixed. Once the waveform stabilized, the following cardiac parameters were recorded: LVSP, LVEDP, maximal rate of increase in left ventricular pressure during systole (+ dp/dtmax), and maximal rate of decrease in left ventricular pressure during diastole (-dp/dtmax).

Enzyme-Linked Immunosorbent Assay (ELISA)

According to the instructions provided with the ELISA kits, the levels of inflammatory cytokines IL-6 (ERC003.48, NeoBioscience, Shenzhen, China) and TNF-α (ERC102a.48, NeoBioscience, Shenzhen, China), as well as myocardial injury markers CK-MB (MB-6930B, Jiangsu Meibiao Biotechnology Co., Ltd, Jiangsu, China) and cTnT (MB-7278B, Jiangsu Meibiao Biotechnology Co., Ltd, Jiangsu, China), and the cardiac function marker NT-proBNP (MB-1870B, Jiangsu Meibiao Biotechnology Co., Ltd, Jiangsu, China) were measured in rat plasma. The optical density (OD) values at 450 nm were measured using a microplate reader (Multiskan Spectrum 1500, Thermo Scientific), and the concentrations of each sample were calculated based on a standard curve.

qRT-PCR

Total RNA was extracted from myocardial tissue or H9c2 cells using TRIzol reagent (15,596,026, Invitrogen). Subsequently, cDNA was synthesized using a reverse transcription kit (R223; Vazyme, Nanjing, China) and amplified using the ChamQ Universal SYBR qRT-PCR Master Mix (Q711, Vazyme, Nanjing, China) on a QuantStudio 5 Real-Time PCR System (Applied Biosystems). With Gapdh as the internal control gene, the relative expressions of target genes were then calculated using the 2−ΔΔCT method. The primer sequences for PCR in this study were designed and synthesized by Sangon Biotech (Shanghai, China) as follows: Fbxo32 forward: 5’-ACTCATACGGGAACTTCTCCAGACC-3’; Fbxo32 reverse: 5’-GCTGCTGTTGCCAGTGTAGAGTG-3’; Anxa1 forward: 5’-GGAAGCCCCTGGATGAAACCTTG-3’; Anxa1 reverse: 5’-CCTTCATGGCAGCACGGAGTTC-3’; Gapdh forward: 5’-AGTTCAACGGCACAGTCAAGGC-3’; and Gapdh reverse: 5’-CGACATACTCAGCACCAGCATCAC-3’.’

Western Blotting

Proteins were extracted from myocardial tissue or H9c2 cells using western blotting and IP cell lysis buffer (P0013, Beyotime, Shanghai, China) and quantified using a BCA protein assay kit (P0012S, Beyotime, Shanghai, China). Proteins (30 μg) were separated using 12% SDS-PAGE (P1200, Solarbio, Beijing, China) and transferred to polyvinylidene fluoride (PVDF) membranes. The PVDF membranes were subsequently blocked with QuickBlock Western blocking solution and shaken at room temperature for 10 min. The membranes were then incubated overnight at 4 °C with different primary antibodies, including FBXO32 (GTX47819, Gene Tex, 1:500), ANXA1 (21,990–1-AP, Proteintech, 1:5000), Bcl-2 (Ab32124, Abcam, 1:1000), Bax (Ab32503, Abcam, 1:1000), Cleaved Caspase-3 (AF7022, Affinity, 1:1000), Caspase-3 (Ab32351, Abcam, 1:1000), AKT (YT0177, Immunoway, 1:500), p-AKT (Ab81283, Abcam, 1:1000), PI3K (Ab151549, Abcam, 1:1000), p-PI3K (bs-6417R, BIOSS, 1:1000), Ub (sc-8017, Santa Cruz, 1:500), and GAPDH (YN5585, Immunoway, 1:5000). The membranes were then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (RS0002; Immunoway) at room temperature for 2 h. Finally, a hypersensitive chemiluminescent substrate (BL520B; Biosharp, Hefei, China) was evenly added to the protein side of the PVDF membranes. Membranes were visualized using a chemiluminescence imaging system (MiniChemi 610, SINSAGE, Beijing, China). The grayscale values of the protein bands were measured using ImageJ 1.52a software. The ratio of the grayscale values of the target protein to that of the internal control protein, GAPDH, was calculated to represent the relative expression of the target protein.

Statistical Methods

GraphPad Prism 9.0 software (GraphPad Software, San Diego, CA, USA) was employed for graphing and statistical analysis. The results are presented as the mean ± standard deviation. The t-test was used for comparisons between two independent samples. One-way analysis of variance was adopted for comparisons among multiple groups, and Tukey’s method was used for pairwise comparisons. A two-tailed P-value < 0.05 was considered statistically significant.