E3 Ubiquitin Ligase FBXO32 Promotes Sepsis-Induced Cardiomyopathy by Regulating ANXA1/PI3K/AKT Signaling

doi:10.21203/rs.3.rs-4874434/v1

Download PDF

Research Article

E3 Ubiquitin Ligase FBXO32 Promotes Sepsis-Induced Cardiomyopathy by Regulating ANXA1/PI3K/AKT Signaling

https://doi.org/10.21203/rs.3.rs-4874434/v1

This work is licensed under a CC BY 4.0 License

You are reading this latest preprint version

Sepsis-induced cardiomyopathy (SIC) is a severe complication of sepsis. Therefore, understanding SIC pathogenesis and developing new therapeutic targets are of great significance. This study investigated the role of F-box-only protein 32 (FBXO32) in SIC pathogenesis. SIC models were established in rats and H9c2 cells using lipopolysaccharide. The effects of FBXO32 on myocardial apoptosis and mitochondrial structure and function were determined using electron microscopy, reactive oxygen species detection, and JC-1 staining. The molecular mechanism was elucidated using western blotting and co-immunoprecipitation. The results showed elevated FBXO32 expression in both in vivo and in vitro SIC models. FBXO32 knockdown alleviated apoptosis and mitochondrial and cardiac dysfunction. Mechanistic analysis revealed that FBXO32 promoted ubiquitination and degradation of annexin A1 (ANXA1), inhibiting the phosphatidylinositol 3-kinase (PI3K) and protein kinase B (AKT) pathways. Rescue experiments demonstrated that ANXA1 knockdown reversed the effects of FBXO32 knockdown. This study suggests that FBXO32 exacerbates SIC progression by mediating ANXA1 ubiquitination and inhibiting the PI3K/AKT signaling pathway.

Sepsis-induced cardiomyopathy

Ubiquitination

FBXO32

Annexin A1

Cell apoptosis

Sepsis-induced cardiomyopathy (SIC) is one of the most severe complications of sepsis and is a leading cause of mortality among affected patients. Current treatment strategies for SIC include infection control, fluid management, pharmacological treatments with vasoactive drugs and inotropes, and non-pharmacological interventions such as circulatory support [1]. Despite advances in SIC treatment, the high costs and substantial consumption of medical resources have seriously compromised patient safety and quality of life [2]. Therefore, developing new therapeutic targets to overcome the bottlenecks in SIC treatment is crucial.

Studies have confirmed that apoptosis plays a critical role in sepsis-induced myocardial damage, and inhibiting apoptosis can partially reverse the myocardial dysfunction caused by sepsis [3, 4]. Apoptosis can be regulated through the ubiquitin-proteasome system (UPS), which is vital in cardiovascular diseases such as heart failure and ischemia-reperfusion injury [5, 6]. As key components of the UPS, E3 ubiquitin ligases exert their biological functions by specifically recognizing target proteins and regulating the ubiquitination of substrate proteins.

F-box only protein 32 (FBXO32), an E3 ubiquitin ligase and member of the F-box protein family, is highly expressed in myocardial tissue and plays a significant role in cardiac-related diseases such as myocardial hypertrophy, ischemia, and heart failure by regulating the degradation of myocardial substrate proteins [7, 8]. Research [7] has shown that FBXO32 expression is significantly elevated in myocardial tissue during acute myocardial infarction in mice. Additionally, FBXO32 has been found to play an essential role in mediating excessive inflammation, including neutrophil infiltration, inflammasome formation, and the production of pro-inflammatory cytokines through the activation of nuclear factor-κB (NF-κB), thereby promoting cardiac rupture post-myocardial infarction. Al-Yacoub et al. [9] have found that by binding to its target protein and activating transcription factor 2 (ATF2), FBXO32 can mediate its ubiquitination. Moreover, the upregulation of ATF2 due to FBXO32 mutations is associated with the activation of the endoplasmic reticulum stress apoptosis pathway, further contributing to the progression of dilated cardiomyopathy.

Xie et al. [10] established a myocardial ischemia-reperfusion injury model using primary cultured neonatal rat cardiomyocytes. Using this model, they found that FBXO32 promotes the ubiquitination and degradation of the substrate protein mitogen-activated protein kinase phosphatase-1 (MKP-1), activating the c-Jun N-terminal kinase (JNK) signaling pathway and exacerbating cardiomyocyte apoptosis. This finding revealed that FBXO32 is a critical mediator in controlling cardiomyocyte apoptosis after ischemia-reperfusion injury. However, the effect of FBXO32 on myocardial apoptosis in SIC remains unclear.

In this study, we investigated the role of FBXO32 in myocardial apoptosis in lipopolysaccharide (LPS)-induced SIC, providing a theoretical basis for identifying effective preventive and therapeutic targets for SIC.

FBXO32 is Upregulated in the Myocardial Cells of LPS-Induced SIC Rats

To identify potential ubiquitination molecules associated with the pathogenesis of SIC, RNA-seq was conducted on H9c2 cells treated with LPS for 24 h, followed by a comparison with untreated H9c2 cells. The data showed that FBXO32 was upregulated in the LPS group (Figs. 1a-c). To validate the RNA-seq results, qRT-PCR analysis was conducted, revealing that the mRNA expression of FBXO32 was upregulated in LPS-treated H9c2 cells (Fig. 1d), consistent with the sequencing results. Additionally, the expression of FBXO32 was elevated in LPS-treated cells based on LPS intervention (Fig. 1e). Similarly, the mRNA and protein expression levels of FBXO32 were enhanced in the myocardial tissues of LPS-treated rats (Figs. 1f, g).

Knockdown of FBXO32 Alleviates LPS-Induced Mitochondrial Dysfunction and Apoptosis in H9c2 Cells

To explore whether FBXO32 knockdown affects LPS-induced apoptosis in H9c2 cells, lentivirus-mediated shRNA was used to knockdown FBXO32 in vitro, and successful transfection was confirmed by qRT-PCR and western blotting (Figs. 2a, b). The CCK-8 assay showed that cell viability in the LPS group was significantly lower than that in the control group. However, there was no significant difference in cell viability between the LPS and LPS + LV-sh-NC groups. Conversely, cell viability was significantly higher in the LPS + LV-sh-FBXO32 group than in the LPS + LV-sh-NC group (Fig. 2c). Further quantification of the effect of FBXO32 knockdown on LPS-induced apoptosis in cardiomyocytes using Annexin V-FITC/PI flow cytometry revealed a significant increase in apoptosis in the LPS group compared with that in the control group. There was no significant difference in apoptosis between the LPS and LPS + LV-sh-NC groups. In contrast, apoptosis was significantly decreased in the LPS + LV-sh-FBXO32 group compared with that in the LPS + LV-sh-NC group (Fig. 2d). Additionally, western blotting revealed that cleaved caspase-3 expression increased and the Bax/Bcl-2 ratio increased in the LPS group compared with those in the control group. No significant difference was identified between the LPS and LPS + LV-sh-NC groups. However, the expression of cleaved caspase-3 and the Bax/Bcl-2 ratio decreased in the LPS + LV-sh-FBXO32 group compared with those in the LPS + LV-sh-NC group (Fig. 2e).

To assess the effect of FBXO32 on mitochondrial function, the DCFH-DA probe was used to measure ROS production in H9c2 cells in vitro. Flow cytometry results showed a significant enhancement in ROS fluorescence intensity in the LPS group compared with that in the control group. In contrast, ROS intensity was significantly lower in the LPS + LV-sh-FBXO32 group than in the LPS + LV-sh-NC group, indicating that FBXO32 knockdown mitigated intracellular ROS production (Fig. 2f).

The effect of FBXO32 on the mitochondrial membrane potential in LPS-treated H9c2 cells was subsequently assessed using JC-1 staining. Flow cytometry results suggested that for cells in the LPS group, JC-1 failed to accumulate in the mitochondrial matrix, manifesting as a decrease in the red/green fluorescence ratio compared with that in the control group, indicating a significant reduction in the mitochondrial membrane potential. Moreover, the red/green fluorescence ratio increased substantially in the LPS + LV-sh-FBXO32 group compared with that in the LPS + LV-sh-NC group, suggesting that FBXO32 knockdown improved the mitochondrial membrane potential. This indicates protection against LPS-induced mitochondrial membrane potential loss in H9c2 cells (Fig. 2g).

Knockdown of FBXO32 Alleviates LPS-Induced Mitochondrial Dysfunction and Apoptosis in Rat Myocardial Cells

To determine whether FBXO32 knockdown affects LPS-induced apoptosis in rat myocardial cells, in vivo experiments were conducted using a myocardial in situ injection of AAV9 carrying FBXO32-shRNA to knock down FBXO32 expression. Measurements at four weeks revealed that FBXO32 mRNA and protein expression levels were significantly reduced (Figs. 3a, b). TUNEL assays were also employed to detect the impact of FBXO32 knockdown on myocardial cell apoptosis. The results suggested that the apoptosis rate of myocardial cells in the LPS group was significantly higher than that in the control group. Conversely, the LPS + AAV9-sh-FBXO32 group exhibited a considerably lower rate of apoptosis than the LPS + AAV9-sh-NC group (Fig. 3c). In addition, western blotting was performed to measure the expression of apoptosis-related proteins in the myocardial tissues of the different groups of mice. The results indicated that cleaved caspase-3 levels and the Bax/Bcl-2 ratio were higher in the LPS group than in the control group. Moreover, these values were lower in the LPS + AAV9-sh-FBXO32 group than in the LPS + AAV9-sh-NC group (Fig. 3d). These consistent results from in vitro and in vivo experiments indicate that FBXO32 knockdown reduces myocardial cell apoptosis.

To investigate the effect of FBXO32 on mitochondrial function in the rat myocardium, TEM was performed to observe mitochondrial morphological changes. In the control group, the mitochondria appeared to be oval-shaped and neatly arranged and had clear and straight cristae. Conversely, in the LPS and LPS + AAV9-sh-NC groups, many mitochondria were swollen, exhibiting reduced or broken cristae, with a sparse matrix, and some even appeared vacuolated with a decreased electron density. In the LPS + AAV9-sh-FBXO32 group, the mitochondria appeared oval-shaped and neatly arranged, exhibiting clear and straight cristae, uniform electron density, and fewer swollen mitochondria, with a mildly dissolved and sparse matrix (Fig. 3e). These observations suggested that FBXO32 knockdown significantly improved mitochondrial morphology.

Knockdown of FBXO32 Mitigates LPS-Induced Cardiac Injury and Dysfunction

To evaluate whether FBXO32 knockdown affects LPS-induced cardiac dysfunction, right common carotid artery catheterization was performed to measure hemodynamic parameters. The LPS group showed lower left ventricular ± dp/dt max and LVSP and higher LVEDP than the control group. In contrast, the LPS + AAV9-sh-FBXO32 group exhibited higher ± dp/dt max and LVSP and lower LVEDP than the LPS + AAV9-sh-NC group (Fig. 4a), indicating improved cardiac function due to FBXO32 knockdown. Further assessment of myocardial injury markers in serum demonstrated that the levels of inflammatory markers IL-6 and TNF-α, myocardial injury markers CK-MB and cTnT, and the cardiac function marker NT-proBNP, were substantially higher in the LPS group than in the control group. In contrast, the LPS + AAV9-sh-FBXO32 group exhibited significantly lower levels of these markers than the LPS + sh-NC group (Fig. 4b).

Histological analysis using HE staining confirmed the protective effects of FBXO32 knockdown on myocardial injury. In the LPS and LPS + AAV9-sh-NC groups, the rat myocardial fibers exhibited blurred boundaries, disorganized arrangement, vacuolar degeneration, cytoplasmic swelling, and infiltration of inflammatory cells within the tissue. In contrast, in the LPS + AAV9-sh-FBXO32 group, the myocardial fibers appeared smoother with a more orderly arrangement, reduced vacuolar degeneration, and significantly less infiltration of inflammatory cells (Fig. 4c). These data demonstrate that FBXO32 knockdown reverses LPS-induced cardiac injury and dysfunction in vivo.

Knockdown of FBXO32 Promotes Activation of the PI3K/AKT Signaling Pathway

To understand the impact of altered FBXO32 expression on the PI3K/AKT signaling pathway, western blotting was conducted to detect changes in the phosphorylation levels of PI3K and AKT following FBXO32 knockdown. In vitro results showed lower p-PI3K and p-AKT protein levels in LPS-treated H9c2 cells than in the control group. Conversely, the LPS + LV-sh-FBXO32 group exhibited higher p-PI3K and p-AKT protein levels than the LPS + LV-sh-NC group (Fig. 5a). FBXO32 knockdown reversed the decrease in p-PI3K and p-AKT levels in the myocardial tissues of LPS-treated rats in vivo (Fig. 5b). Collectively, these results indicated that FBXO32 knockdown promoted the activation of the PI3K/AKT signaling pathway.

FBXO32 Promotes Ubiquitination and Degradation of ANXA1

To further explore how FBXO32 influences apoptosis in SIC, CoIP-MS was employed to detect interactions between FBXO32 and other proteins in LPS-treated H9c2 cells for 24 h, identifying ANXA1 as one of the proteins involved (Figs. 6a, b). Subsequent validation confirmed the elevated ANXA1 protein expression in both cell and animal models exposed to LPS (Figs. 6c, d). Given FBXO32’s role as an F-box protein, which is part of the ubiquitin E3 ligase complex, we hypothesized that FBXO32 regulates ANXA1 through ubiquitination. To verify this hypothesis, we examined how variations in FBXO32 mRNA expression affected ANXA1 mRNA levels under LPS-induced conditions. The qRT-PCR results indicated that FBXO32 knockdown did not significantly affect ANXA1 mRNA levels compared with those in the control group (Fig. 6e). However, further examination using western blotting revealed a notable increase in ANXA1 protein levels following FBXO32 knockdown compared with those in the control group (Fig. 6f), suggesting that FBXO32 might regulate ANXA1 protein expression through post-translational modifications.

Subsequently, FBXO32’s negative regulation of ANXA1 was investigated by examining the degradation pathways of ANXA1. Under LPS treatment, the proteasome inhibitor, MG132, effectively blocked ANXA1 protein degradation, whereas the lysosomal inhibitor, NH4Cl, exerted no such effect. This finding indicates that LPS triggers ANXA1 degradation predominantly through the proteasome pathway rather than the lysosome pathway (Figs. 6g, h). Moreover, FBXO32 knockdown resulted in a more pronounced increase in ANXA1 protein expression after MG132 treatment than no FBXO32 knockdown, confirming that FBXO32 modulates ANXA1 protein expression through proteasome-dependent regulation (Fig. 6i). Subsequent Co-IP experiments verified the direct binding between FBXO32 and ANXA1 proteins (Fig. 6j). Next, a gradient of CHX was administered to inhibit protein synthesis and evaluate the effect of FBXO32 on ANXA1 protein degradation. FBXO32 knockdown significantly prolonged the half-life of ANXA1, suggesting that FBXO32 knockdown attenuated its role in mediating ANXA1 degradation (Fig. 6k). Finally, Co-IP analysis was used to examine the impact of FBXO32 on ANXA1 ubiquitination, revealing a decrease following FBXO32 knockdown (Fig. 6l).

FBXO32 Regulates LPS-Induced Apoptosis and Mitochondrial Dysfunction Through ANXA1

To investigate whether ANXA1 contributes to the pro-apoptotic effects of FBXO32 in LPS-induced myocardial injury, rescue experiments were conducted by knocking down ANXA1 in H9c2 cells with stable FBXO32 shRNA using siRNA. qRT-PCR and western blotting results confirmed a significant reduction in ANXA1 expression with ANXA1 siRNA (Figs. 7a, b).

The CCK-8 assay revealed higher cell viability in the LPS + sh-FBXO32 + si-NC group than in the LPS + sh-NC + si-NC group. Moreover, cell viability was lower in the LPS + sh-FBXO32 + si-ANXA1 group than in the LPS + sh-FBXO32 + si-NC group (Fig. 7c). In addition, annexin V-FITC/PI flow cytometry, western blotting, and DCFH-DA staining revealed lower apoptosis rates, expression of apoptosis-related proteins, and intracellular ROS in the LPS + sh-FBXO32 + si-NC group than in the LPS + sh-NC + si-NC group. Conversely, these levels were significantly higher in the LPS + sh-FBXO32 + si-ANXA1 group than in the LPS + sh-FBXO32 + si-NC group (Figs. 7d-f).

JC-1 staining indicated a higher mitochondrial membrane potential in the LPS + sh-FBXO32 + si-NC group than in the LPS + sh-NC + si-NC group. Conversely, it was lower in the LPS + sh-FBXO32 + si-ANXA1 group than in the LPS + sh-FBXO32 + si-NC group (Fig. 7g). Furthermore, western blotting revealed higher levels of p-PI3K and p-AKT in the LPS + sh-FBXO32 + si-NC group than in the control group, whereas these levels were lower in the LPS + sh-FBXO32 + si-ANXA1 group than in the LPS + sh-FBXO32 + si-NC group (Fig. 7h).

Collectively, these findings suggest that ANXA1 knockdown effectively reversed the effects of FBXO32 knockdown on cell viability, apoptosis, ROS levels, and mitochondrial function. Additionally, it significantly mitigated the effects of FBXO32 on the PI3K/AKT signaling pathway.

In this study, FBXO32 expression was significantly elevated in both in vivo and in vitro rat models of SIC. FBXO32 knockdown mitigated apoptosis, mitochondrial dysfunction, and cardiac dysfunction. Further mechanistic analyses revealed that FBXO32 promotes the ubiquitination and degradation of ANXA1 by binding to it, thereby inhibiting the activation of the PI3K/AKT pathway.

Apoptosis is a tightly regulated form of cell death in which mitochondria play a critical role in regulation and execution. Mitochondria are abundant in cardiomyocytes and are essential for maintaining cardiac function and cell survival. Mitochondrial dysfunction is a key factor that can promote apoptosis [11, 12] and significantly impacts the onset, progression, and prognosis of myocardial dysfunction in septic patients [1]. Suliman et al. [13] speculated that bacterial toxins (such as LPS) might lead to excessive production of cytokines and ROS, causing mitochondrial DNA oxidation and subsequent mitochondrial damage. In their study on the cytotoxicity and molecular mechanisms of LPS in human alveolar epithelial A549 cells, Chuang et al. [14] found that LPS increased ROS levels and reduced the mitochondrial membrane potential, thereby driving the intrinsic apoptotic pathway in the mitochondria. Xie et al. [10] demonstrated that FBXO32 promotes apoptosis in myocardial ischemia-reperfusion injury. In this study, FBXO32 expression was significantly elevated in the SIC models. FBXO32 knockdown increased the mitochondrial membrane potential, inhibited intracellular ROS production, improved abnormal mitochondrial morphology, and alleviated apoptosis.

LPS stimulation has been confirmed to be a major mechanism underlying cardiac dysfunction through the production of pro-inflammatory cytokines [15]. Excessively activated inflammatory responses can induce cardiomyocyte apoptosis [16, 17]. As an inflammatory trigger, TNF-α is involved in the production of IL-1β and the subsequent induction of secondary inflammatory factors such as IL-6, ultimately leading to an inflammatory cascade reaction [18]. In this study, LPS induction resulted in elevated levels of the inflammatory cytokines TNF-α and IL-6 in rat plasma. Furthermore, these levels decreased following FBXO32 knockdown. HE staining demonstrated that FBXO32 knockdown alleviated LPS-induced inflammatory cell infiltration in the rat myocardium.

The PI3K/AKT signaling pathway is crucial for regulating cardiomyocyte apoptosis and inflammatory responses. Xing et al. [19] discovered that activation of the β3-adrenergic receptor-mediated PI3K/AKT signaling pathway alleviated apoptosis and cardiac dysfunction in SIC rats. Yang et al. [20] demonstrated that aloesin lowered mortality in mice with CLP-induced sepsis by activating the PI3K signaling pathway. A study on the cardioprotective effects of dexmedetomidine during sepsis [21] indicated that dexmedetomidine prevented sepsis-induced cardiac dysfunction in rats by regulating cardiomyocyte autophagy via the PI3K/AKT pathway. In this study, we evaluated the effect of FBXO32 on the activation of the PI3K/AKT pathway. The results indicated that in both the in vivo and in vitro SIC models, p-PI3K and p-AKT protein levels decreased, whereas FBXO32 knockdown reversed this reduction, suggesting that FBXO32 may influence SIC-induced cardiomyocyte apoptosis and inflammatory responses via the PI3K/AKT pathway.

ANXA1 is an endogenous anti-inflammatory protein involved in various cellular processes including apoptosis, cell proliferation, inflammation, and carcinogenesis [22]. Qin et al. [23] found that in LPS-induced sepsis models, annexin-A1 short peptide (ANXA1sp) alleviated myocardial damage and reduced apoptosis by upregulating silent information regulator 3 (SIRT3). Alternatively, Zhang et al. [24] induced sepsis-related cardiac dysfunction in rat and H9c2 cell models using cecal ligation and puncture (CLP) and 10 µg/mL LPS stimulation. They observed that the ANXA1 mimetic peptide Ac2-26 mitigated sepsis-induced cardiomyocyte apoptosis in vivo and in vitro, potentially by modulating the lipoxin A4 (LXA4)/PI3K/AKT signaling pathway. In this study, Co-IP was performed to screen FBXO32 interacting proteins, revealing that ANXA1 not only served as a binding molecule for FBXO32 but also negatively regulated its expression. Additionally, ubiquitination experiments confirmed that FBXO32 knockdown reduced the ubiquitination level of ANXA1, thereby verifying the impact of FBXO32 on the ubiquitination of ANXA1.

Subsequent rescue experiments in FBXO32-knockdown H9c2 cells, involving additional ANXA1 knockdown, significantly reversed the increased cell viability and mitochondrial membrane potential, as well as the reduced the apoptosis rate, apoptosis-related proteins, and intracellular ROS levels associated with FBXO32 knockdown. Moreover, the activation of the PI3K/AKT signaling pathway was also weakened. Collectively, these findings suggest that ANXA1 is a target of FBXO32, which may promote SIC-induced myocardial apoptosis by enhancing the ubiquitination and degradation of ANXA1.

In conclusion, FBXO32 is upregulated in rat SIC models and facilitates myocardial apoptosis in this context. The molecular mechanism involves FBXO32 interacting with ANXA1, which increases ANXA1 ubiquitination and promotes its degradation, thereby inhibiting the PI3K/AKT signaling pathway. This suggests that FBXO32 is an important regulator of SIC cell apoptosis, positioning it as a promising therapeutic target for SIC.

Limitations of the study

The aim of this study was to evaluate the causal relationship and specific mechanisms underlying abnormal FBXO32 expression and apoptosis in SIC cells. However, this study had certain limitations. First, although LPS-induced SIC models have been widely used, they do not fully replicate the pathophysiological state of patients with SIC. Therefore, further human studies are warranted to assess the potential of FBXO32 as a therapeutic target for SIC. Secondly, although this study used Co-IP to confirm the interaction between FBXO32 and ANXA1, the specific binding sites between these two proteins require further investigation.

Methods

Cell Culture and Grouping

The rat cardiomyocyte cell line (H9c2) used in this study was purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences. Cells were cultured in high-glucose DMEM (C11995500BT, Thermo Fisher Scientific, USA) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, USA) and 1% penicillin/streptomycin mix (P1400, Solarbio, Beijing, China) in a 5% CO₂ incubator at 37°C (HeraCell VIOS 160i, Thermo Fisher Scientific, USA). 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 hours h to induce the SIC model [25]; 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.

RNA 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 [26], applying screening criteria of log2 fold change (log2FC) > 1 or < -1 and FDR < 0.05.

Cell Transfection

Short hairpin RNA (shRNA) lentiviruses targeting FBXO32 were designed and synthesized by GeneChem Co., Ltd. (Shanghai, China), and small interfering RNAs (siRNAs) 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 Cell Counting Kit-8 (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 [27].

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 Reactive Oxygen Species (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 minutes. 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).

Co-Immunoprecipitation and Mass Spectrometry (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 (21990-1-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, SIC was induced by an intraperitoneal injection of 10 mg/kg LPS (diluted to 1 mL with saline) [28]. 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

Adeno-associated virus serotype 9 (AAV9) targeting FBXO32 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: left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (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 (15596026, 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% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (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 (21990-1-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. Data with a normal distribution were expressed as the mean ± standard deviation (_x ± s). 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.

Competing Interests:

The authors have no relevant financial or non-financial interests to disclose.

Ethics approval:

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).

Funding:

This work was supported by the Natural Science Foundation of Gansu Province(24JRRA616); Wu Jieping Medical Foundation (320.6750.2024-2-3); the Health Industry Research Program Funding Project of Gansu Province (GSWSKY2021-073); and the Lanzhou Science and Technology Plan Project (2022-5-74).

Author Contribution

Conceptualization: Chen De, Liu Jian, and Liang Xuan; Methodology: Chen De, Liu Jian, and Liang Xuan; Formal analysis and investigation: Chen De, Liu Jian, and Liang Xuan; Writing - original draft preparation: Chen De; Writing - review and editing: Liu Jian, Zhang Lei, Zuo Kun, and Guo Hong; Funding acquisition: Chen De, Liu Jian, Liang Xuan, Zhang Lei, and Du Song; Resources: Zhang Jingjing, Gao Lina, Yan Dong, and Du Song; Supervision: Liu Jian, Zhang Lei, Zuo Kun, and Guo Hong. All authors read and approved the final manuscript.

Acknowledgement

We would like to express our deepest gratitude to the Gansu Key Laboratory of Cardiovascular Diseases for the provision of essential equipment that significantly contributed to this research. This project was funded by the Natural Science Foundation of Gansu Province(24JRRA616); Wu Jieping Medical Foundation (320.6750.2024-2-3); the Health Industry Research Program Funding Project of Gansu Province (GSWSKY2021-073); and the Lanzhou Science and Technology Plan Project (2022-5-74).

Data Availability

The raw sequence data reported in this paper have been deposited in the Genome Sequence Archive in National Genomics Data Center , China National Center for Bioinformation / Beijing Institute of Genomics, Chinese Academy of Sciences (GSA: CRA018060) that are publicly accessible at https://ngdc.cncb.ac.cn/gsa.The original contributions presented in the study are included in the article/supplementary material; further inquiries can be directed to the corresponding author.

Ravikumar, Nidhruv, Mohammed A. Sayed, Chanaradh J. Poonsuph, Rijuvani Sehgal, Manasi M. Shirke, and Amer Harky. 2021. Septic Cardiomyopathy: From Basics to Management Choices. Current Problems in Cardiology 46: 100767. https://doi.org/10.1016/j.cpcardiol.2020.100767.
Evans, Laura, Andrew Rhodes, Waleed Alhazzani, Massimo Antonelli, Craig M. Coopersmith, Craig French, Flávia R. Machado, Lauralyn Mcintyre, Marlies Ostermann, Hallie C. Prescott, Christa Schorr, Steven Simpson, W. Joost Wiersinga, Fayez Alshamsi, Derek C. Angus, Yaseen Arabi, Luciano Azevedo, Richard Beale, Gregory Beilman, Emilie Belley-Cote, Lisa Burry, Maurizio Cecconi, John Centofanti, Angel Coz Yataco, Jan De Waele, R. Phillip Dellinger, Kent Doi, Bin Du, Elisa Estenssoro, Ricard Ferrer, Charles Gomersall, Carol Hodgson, Morten Hylander Møller, Theodore Iwashyna, Shevin Jacob, Ruth Kleinpell, Michael Klompas, Younsuck Koh, Anand Kumar, Arthur Kwizera, Suzana Lobo, Henry Masur, Steven McGloughlin, Sangeeta Mehta, Yatin Mehta, Mervyn Mer, Mark Nunnally, Simon Oczkowski, Tiffany Osborn, Elizabeth Papathanassoglou, Anders Perner, Michael Puskarich, Jason Roberts, William Schweickert, Maureen Seckel, Jonathan Sevransky, Charles L. Sprung, Tobias Welte, Janice Zimmerman, and Mitchell Levy. 2021. Surviving Sepsis Campaign: International Guidelines for Management of Sepsis and Septic Shock 2021. Intensive Care Medicine 47: 1181–1247. https://doi.org/10.1007/s00134-021-06506-y.
Li, Ning, Heng Zhou, Haiming Wu, Qingqing Wu, Mingxia Duan, Wei Deng, and Qizhu Tang. 2019. STING-IRF3 Contributes to Lipopolysaccharide-Induced Cardiac Dysfunction, Inflammation, Apoptosis and Pyroptosis by Activating NLRP3. Redox Biology 24: 101215. https://doi.org/10.1016/j.redox.2019.101215.
Wang, Yiyang, Yuan Wang, Duomeng Yang, Xiaohui Yu, Hongmei Li, Xiuxiu Lv, Daxiang Lu, and Huadong Wang. 2015. β₁-Adrenoceptor Stimulation Promotes LPS-Induced Cardiomyocyte Apoptosis through Activating PKA and Enhancing CaMKII and IκBα Phosphorylation. Critical Care 19: 76. https://doi.org/10.1186/s13054-015-0820-1.
Orlowski, Robert Z. 1999. The Role of the Ubiquitin-Proteasome Pathway in Apoptosis. Cell Death & Differentiation 6: 303–313. https://doi.org/10.1038/sj.cdd.4400505.
Li, Hui-Hua., Monte S. Willis, Pamela Lockyer, Nathaniel Miller, Holly McDonough, David J. Glass, and Cam Patterson. 2022. Atrogin-1 Inhibits Akt-Dependent Cardiac Hypertrophy in Mice via Ubiquitin-Dependent coactivation of Forkhead Proteins. Journal of Clinical Investigation 132: e157373. https://doi.org/10.1172%2FJCI157373
Usui, Soichiro, Akio Chikata, Osamu Takatori, Shin-Ichiro Takashima, Oto Inoue, Takeshi Kato, Hisayoshi Murai, Hiroshi Furusho, Ayano Nomura, Daniela Zablocki, Shuichi Kaneko, Junichi Sadoshima, and Masayuki Takamura. 2019. Endogenous Muscle Atrophy F–Box Is Involved in the Development of Cardiac Rupture After Myocardial Infarction. Journal of Molecular & Cellular Cardiology 126: 1–12. https://doi.org/10.1016/j.yjmcc.2018.11.002.
Yu, Ying, James C. Fuscoe, Chen Zhao, Chao Guo, Meiwen Jia, Tao Qing, Desmond I. Bannon, Lee Lancashire, Wenjun Bao, Tingting Du, Heng Luo, Zhenqiang Su, Wendell D. Jones, Carrie L. Moland, William S. Branham, Feng Qian, Baitang Ning, Yan Li, Huixiao Hong, Lei Guo, Nan Mei, Tieliu Shi, Kevin Y. Wang, Russell D. Wolfinger, Yuri Nikolsky, Stephen J. Walker, Penelope Duerksen-Hughes, Christopher E. Mason, Weida Tong, Jean Thierry-Mieg, Danielle Thierry-Mieg, Leming Shi, and Charles Wang. 2014. A Rat RNA-Seq Transcriptomic BodyMap Across 11 Organs and 4 Developmental Stages. Nature Communications 5: 3230. https://doi.org/10.1038/ncomms4230.
Al-Yacoub, Nadya, Dilek Colak, Salma A. Mahmoud, Maya Hammonds, Kunhi Muhammed, Olfat Al-Harazi, Abdullah M. Assiri, Jehad Al-Buraiki, Waleed Al-Habeeb, and Coralie Poizat. 2021. Mutation in FBXO32 Causes Dilated Cardiomyopathy Through Up-Regulation of ER-Stress Mediated Apoptosis. Communications Biology 4: 884. https://doi.org/10.1038/s42003-021-02391-9.
Xie, Ping, Shubin Guo, Yongna Fan, Hua Zhang, Dongfeng Gu, and Huihua Li. 2009. Atrogin-1/MAFbx Enhances Simulated Ischemia/Reperfusion-Induced Apoptosis in Cardiomyocytes Through Degradation of MAPK phosphatase-1 and Sustained JNK Activation. Journal of Biological Chemistry 284: 5488–5496. https://doi.org/10.1074/jbc.M806487200.
Abate, Marianna, Agostino Festa, Michela Falco, Angela Lombardi, Amalia Luce, Anna Grimaldi, Silvia Zappavigna, Pasquale Sperlongano, Carlo Irace, Michele Caraglia, and Gabriella Misso. 2020. Mitochondria as Playmakers of Apoptosis, Autophagy and Senescence. Seminars in Cell & Developmental Biology 98: 139–153. https://doi.org/10.1016/j.semcdb.2019.05.022.
Tian, Lei, Nan Chu, H. Yang, Jun Yan, Bencheng Lin, Wei Zhang, Kang Li, Wenqing Lai, Liping Bian, Huanliang Liu, Zhuge Xi, and Xiaohua Liu. 2021. Acute Ozone Exposure Can Cause Cardiotoxicity: Mitochondria Play an Important Role in Mediating Myocardial Apoptosis. Chemosphere 268: 128838. https://doi.org/10.1016/j.chemosphere.2020.128838.
Suliman, Hagir B., Martha S. Carraway, and Claude A. Piantadosi. 2003. Postlipopolysaccharide Oxidative Damage of Mitochondrial DNA. American Journal of Respiratory & Critical Care Medicine 167: 570–579. https://doi.org/10.1164/rccm.200206-518OC.
Chuang, Chi-Yuan, Ta-Liang Chen, Yih-Giun Cherng, Yu-Tyng Tai, Tyng-Guey Chen, and Ruei-Ming Chen. 2011. Lipopolysaccharide Induces Apoptotic Insults to Human Alveolar Epithelial A549 Cells Through Reactive Oxygen Species-Mediated Activation of an Intrinsic Mitochondrion-Dependent Pathway. Archives of Toxicology 85: 209–218. https://doi.org/10.1007/s00204-010-0585-x.
Yu, Xiaohui, Baoyin Jia, Faqiang Wang, Xiuxiu Lv, Xuemei Peng, Yiyang Wang, Hongmei Li, Yanping Wang, Daxiang Lu, and Huadong Wang. 2014. α₁ Adrenoceptor Activation by Norepinephrine Inhibits LPS-Induced Cardiomyocyte TNF-α Production via Modulating ERK1/2 and NF-κB Pathway. Journal of Cellular & Molecular Medicine 18: 263–273. https://doi.org/10.1111/jcmm.12184.
Zhu, Zhao, Guoxiu Zhang, Dahuan Li, Xiaojun Yin, and Tianzhong Wang. 2022. Silencing of Specificity Protein 1 Protects H9c2 Cells Against Lipopolysaccharide-Induced Injury via Binding to the Promoter of Chemokine CXC Receptor 4 and Suppressing NF-κB Signaling. Bioengineered 13: 3395–3409. https://doi.org/10.1080/21655979.2022.2026548.
Huang, P., Yuhong Guo, Xiao Hu, Xiaolei Fang, Xiaolong Xu, and Qingquan Liu. 2024. Mechanism of Shenfu Injection in Suppressing Inflammation and Preventing Sepsis-Induced Apoptosis in Murine Cardiomyocytes Based on Network Pharmacology and Experimental Validation. Journal of Ethnopharmacology 322: 117599. https://doi.org/10.1016/j.jep.2023.117599.
Wang, Ziwen, Lin Bu, Peng Yang, Shoujie Feng, and Feng Xu. 2019. Alleviation of SepsisInduced Cardiac Dysfunction by Overexpression of Sestrin2 Is Associated with Inhibition of pS6K and Activation of the pAMPK Pathway. Molecular Medicine Reports 20: 2511–2518. https://doi.org/10.3892/mmr.2019.10520.
Xing, Yun, Tian Tian, Xue Zhang, Duomeng Yang, Chanjuan Zhang, Miao Wang, Yiyang Wang, Tao Luo, Zhi Wang, Huadong Wang, and Hongmei Li. 2024. Endogenous β 3 -ADRENERGIC Receptor Activation Alleviates Sepsis-Induced Cardiomyocyte Apoptosis via PI3K/AKT Signaling Pathway. Shock 61: 915–923. https://doi.org/10.1097/SHK.0000000000002354.
Yang, Sumin, Wonhwa Lee, Bong-Seon Lee, Changhun Lee, Eui K. Park, Sae-Kwang Ku, and Jong-Sup Bae. 2019. Aloin Reduces HMGB1-Mediated Septic Responses and Improves Survival in Septic Mice by Activation of the SIRT1 and PI3K/Nrf2/HO-1 Signaling Axis. American Journal of Chinese Medicine 47: 613–633. https://doi.org/10.1142/S0192415X19500320.
Yu, Tianyi, Dan Liu, Min Gao, Peilang Yang, Meng Zhang, Fei Song, Xiong Zhang, and Yan Liu. 2019. Dexmedetomidine Prevents Septic Myocardial Dysfunction in Rats via Activation of α7nAChR and PI3K/Akt- Mediated Autophagy. Biomedicine & Pharmacotherapy 120: 109231. https://doi.org/10.1016/j.biopha.2019.109231.
Foo, Sok L., Gracemary Yap, Jianzhou Cui, and Lina H. K. Lim. 2019. Annexin-A1 – A Blessing or a Curse in Cancer?. Trends in Molecular Medicine 25: 315–327. https://doi.org/10.1016/j.molmed.2019.02.004.
Qin, Song, Yingcong Ren, Banghai Feng, Xiaoqin Wang, Junya Liu, Jie Zheng, Kang Li, Hong Mei, Qiuyu Dai, Hong Yu, and Xiaoyun Fu. 2024. Annexin-A1 Short Peptide Alleviates Septic Myocardial Injury by Upregulating SIRT3 and Inhibiting Myocardial Cell Apoptosis. Histology & Histopathology 39: 947–957. https://doi.org/10.14670/HH-18-691.
Zhang, L., Yan-Lei Zheng, Rong-Hua Hu, L. Zhu, Chen-Chen Hu, Fei Cheng, Shi Li, and Jian-Guo Li. 2018. Annexin A1 Mimetic Peptide AC2-26 Inhibits Sepsis-Induced Cardiomyocyte Apoptosis Through LXA4/PI3K/AKT Signaling Pathway. Current Medical Science 38: 997–1004. https://doi.org/10.1007/s11596-018-1975-1.
Zheng, Yanlei, Shi Li, Ronghua Hu, Fei Cheng, and L. Zhang. 2020. GFI-1 Protects Against Lipopolysaccharide-Induced Inflammatory Responses and Apoptosis by Inhibition of the NF-κB/TNF-α Pathway in H9c2 Cells. Inflammation 43: 74–84. https://doi.org/10.1007/s10753-019-01095-x.
Anders, Simon, and Wolfgang Huber. 2013. Differential Expression of RNA-Seq Data at the Gene Level-The DeSeq Package. European Molecular Biology Laboratory.
Niu, Xiaowei, Jingjing Zhang, Shuwen Hu, Wenhui Dang, Kaiwen Wang, and Ming Bai. 2024. lncRNA Oip5-as1 Inhibits Excessive Mitochondrial Fission in Myocardial Ischemia/Reperfusion Injury by Modulating DRP1 Phosphorylation. Cellular & Molecular Biology Letters 29: 72. https://doi.org/10.1186/s11658-024-00588-4.
Steven, Sebastian, Michael Hausding, Swenja Kröller-Schön, Michael Mader, Yuliya Mikhed, Paul Stamm, Elena Zinßius, Amanda Pfeffer, Philipp Welschof, Saule Agdauletova, Stephan Sudowe, Huige Li, Matthias Oelze, Eberhard Schulz, Thomas Klein, Thomas Münzel, and Andreas Daiber. 2015. Gliptin and GLP-1 Analog Treatment Improves Survival and Vascular Inflammation/Dysfunction in Animals with Lipopolysaccharide-Induced Endotoxemia. Basic Research in Cardiology 110: 6. https://doi.org/10.1007/s00395-015-0465-x.

No competing interests reported.

Download PDF

Reviews received at journal
06 Sep, 2024
Reviews received at journal
24 Aug, 2024
Reviewers agreed at journal
19 Aug, 2024
Reviewers agreed at journal
15 Aug, 2024
Reviewers agreed at journal
12 Aug, 2024
Reviewers invited by journal
12 Aug, 2024
Editor assigned by journal
07 Aug, 2024
Submission checks completed at journal
07 Aug, 2024
First submitted to journal
07 Aug, 2024

You are reading this latest preprint version

E3 Ubiquitin Ligase FBXO32 Promotes Sepsis-Induced Cardiomyopathy by Regulating ANXA1/PI3K/AKT Signaling

Status:

Version 1

Abstract

Figures

Introduction

Results

FBXO32 is Upregulated in the Myocardial Cells of LPS-Induced SIC Rats

Knockdown of FBXO32 Alleviates LPS-Induced Mitochondrial Dysfunction and Apoptosis in H9c2 Cells

Knockdown of FBXO32 Alleviates LPS-Induced Mitochondrial Dysfunction and Apoptosis in Rat Myocardial Cells

Knockdown of FBXO32 Mitigates LPS-Induced Cardiac Injury and Dysfunction

Knockdown of FBXO32 Promotes Activation of the PI3K/AKT Signaling Pathway

FBXO32 Promotes Ubiquitination and Degradation of ANXA1

FBXO32 Regulates LPS-Induced Apoptosis and Mitochondrial Dysfunction Through ANXA1

Discussion

Limitations of the study

Methods

Cell Culture and Grouping

RNA Sequencing (RNA-seq)

Cell Transfection

Cell Viability Assay

Annexin V-FITC/PI Double Staining

Intracellular Reactive Oxygen Species (ROS) Detection

Mitochondrial Membrane Potential Detection

Co-Immunoprecipitation and Mass Spectrometry (CoIP-MS)

Co-IP

Ubiquitination Level Analysis

Experimental Animals and Grouping

Myocardial Injection of Adeno-Associated Virus

Hematoxylin and Eosin (HE) Staining

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

Transmission Electron Microscopy (TEM) Observation of Myocardial Tissue Ultrastructure

Hemodynamic Monitoring of the Heart

Enzyme-Linked Immunosorbent Assay (ELISA)

qRT-PCR

Western Blotting

Statistical Methods

Declarations

Competing Interests:

Funding:

Author Contribution

Acknowledgement

Data Availability

References

Additional Declarations

Status:

Version 1