OGT-mediated O-GlcNAc of FOXO1 promotes the progression of neonatal heart failure via regulating pyroptosis

Abstract

Neonatal heart failure (HF) is a progressive disease caused by cardiovascular and non-cardiovascular abnormalities. O-linked beta-N-acetylglucosamine (O-GlcNAc), a dynamic post-translational modification, is rapidly cycled on and off proteins by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), respectively. This study aimed to investigate the role of O-GlcNAc in neonatal HF and the underlying mechanism. Twenty-eight neonatal HF patients and 22 healthy control volunteers were included. In vivo and in vitro HF models were established. Western blotting and RT-qPCR were used to detect the expression of O-GlcNAc, OGT, OGA, and pyroptosis-related indicators. CCK-8 was used to detect the cell viability. Lactate dehydrogenase and propidium iodide staining commercial kits were used to assess the cytotoxicity and apoptosis. The concentrations of interleukin (IL)-1β and IL-18 were analyzed by ELISA. Co-immunoprecipitation was performed to verify the interaction between OGT and forkhead box O1 (FOXO1). Results showed that OGT-mediated O-GlcNAc was elevated in HF. Besides, OGT deficiency suppressed pyroptosis in Angiotensin (Ang)II-treated H9c2 cells. Mechanically, OGT regulated the O-GlcNAc of FOXO1 at S41 site in H9c2 cells. Subsequent rescue experiments indicated that FOXO1 overexpression promoted pyroptosis in AngII-treated H9c2 cells. Final animal studies illustrated that OGT inhibition alleviated myocardial tissue necrosis, myocardial fibrosis, and pathological cardiac dysfunction. In conclusion, OGT-mediated O-GlcNAc of FOXO1 promoted the progression of neonatal HF via regulating pyroptosis, which might provide a new insight for neonatal HF treatment.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-00850-5.

Introduction

Neonatal heart failure (HF) is characterized by a condition in which the heart’s ability to pump blood is compromised, resulting in insufficient blood flow to satisfy the metabolic demands of various organs and tissues¹. The causes of neonatal HF are multifaceted and may include dysfunction in multiple organ systems, such as pulmonary hypertension, myocarditis, severe anemia, and congenital renal dysplasia. Clinically, neonatal HF presents with symptoms such as sympathetic activation, cardiac dysfunction, congestion in pulmonary circulation, and venous congestion in systemic circulation. Currently, the primary approach to treating neonatal HF involves addressing the underlying causes and triggers, enhancing hemodynamic stability, and supporting the compromised heart. Despite these efforts, neonatal HF is associated with a poor prognosis and elevated rates of cardiovascular morbidity and mortality². Consequently, investigating the fundamental mechanisms underlying neonatal HF is essential for the development of effective treatment options.

Protein glycosylation represents a significant post-translational modification (PTM) of proteins, involving the enzymatic attachment of sugar molecules to proteins. This process initiates in the endoplasmic reticulum and concludes in the Golgi apparatus³. Through glycosylation, proteins are transformed into glycoproteins, which play a crucial role in regulating protein functions and facilitating proper protein folding. O-glycosylation, or O-linked N-acetylglucosamine (O-GlcNAc) glycosylation, refers to a single O-GlcNAc connected to the oxygen atom of the serine or threonine hydroxyl group of a protein by O-glucoside bonds⁴. This modification is integral to various complex cellular processes, including signal transduction, gene transcription, protein translation, cellular responses, and protein degradation. A number of established factors would affect the O-GlcNAc state, e.g. glutamine-fructose-6-phosphate amidotransferase (GFAT), which is involved in the upstream metabolic pathways related to O-GlcNAcylation⁵. The addition and removal of O-GlcNAc modification groups are mediated by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), respectively. OGT facilitates the attachment of GlcNAc to the hydroxyl group of target protein serine or threonine, while OGA is responsible for the removal of GlcNAc from the protein’s hydroxyl group⁶. O-GlcNAc plays a significant role in the physiology and pathology of different cardiovascular diseases, including HF⁷.

Programmed cell death refers to an active process of cell death in order to maintain the stability of the internal environment after receiving certain signals or being stimulated by certain factors⁸. Pyroptosis is a newly discovered programmed death mode of inflammatory cells, characterized by activation of the NOD-like receptor protein 3 (NLRP3) inflammasome⁹. It mainly mediates the activation of various Caspases including Caspase-1 through inflammatosomes, resulting in the shear and polymerization of various gasdermin (GSDM) family members including GSDMD, resulting in cell perforation, and then cell death. GSDMD-N acts as the ultimate executor of pyroptosis⁹. Compared with apoptosis, pyroptosis occurs more quickly and is accompanied by the release of a large number of pro-inflammatory factors, including interleukin (IL)-1β and IL-18. Role of pyroptosis in cardiovascular diseases and its therapeutic implications has been clarified before¹⁰. Whereas, the process of pyroptosis in neonatal HF has not been fully explored.

In this study, we collected clinical samples and established the in vivo and in vitro HF models to explore the role of O-GlcNAc and pyroptosis in neonatal HF, in order to provide a reference for the exploration of the mechanism of neonatal HF.

Methods and materials

Patients

A total of 28 neonatal HF patients (1–28 d) and 22 age- and sex-matched healthy control volunteers were included in the study from Renmin Hospital of Wuhan University. Peripheral blood samples was collected from all participants. The analysis of peripheral blood specimens adhered to the guidelines set forth in the Declaration of Helsinki and received approval from Renmin Hospital of Wuhan University (approval number: WDRY2025-K075). All participants or their families provided informed consent by signing the necessary documentation.

Cell culture, treatment, and transfection

Rat cardiac myocytes (H9c2; #JSY-CC1776; JSCall Biotechnology Co., LTD, Shanghai, China) were cultured in commercial complete medium (#JSY-1382; JSCall) in a 37 °C incubator with 5% CO₂. H9c2 cells were treated with Angiotensin (Ang) II (1 µM; #HY-13948; Merck) for 48 h.

To inhibit the expression of OGT and overexpress that of FOXO1, short hairpin (sh)OGT and pcDNA3.1-FOXO1 and their negative controls (shNC and pcDNA3.1-vector) were obtained from Ribio Biotechnology Co. Ltd., (Guangzhou, China). Cell transfection were performed using the commercial transfection reagent (#C931109; Macklin Inc., Shanghai, China) when the H9c2 cell confluence reached 80%. Finally, the mRNA expression of OGT and FOXO1 were measured by reverse transcription-quantitative polymerase chain reaction (RT-qPCR).

Protein extraction and Western blotting

Proteins from serum, tissues, and H9c2 cells were extracted using the protein extraction reagent (#BB-31013; Bestbio Co., Ltd, Nanjing, China). The concentration of the extracted proteins was determined using the bicinchoninic acid (BCA) quantitative kit (#BB-3401; Bestbio). An equal quantity of total protein (30 µg) was subjected to separation via 10% SDS-PAGE gels, followed by transfer to polyvinylidene fluoride (PVDF; #BB-3511; Bestbio) membranes. The membranes were then blocked with 5% skim milk for 1 h, after which they were incubated with the primary antibodies O-GlcNAc (#PTM-951RM; 1/1000; PTM Biotechnology Co., LTD, Hangzhou, China), OGT (rabbit anti-human/rat; #ab177941; 1/2000; Abcam Cambridge, MA, USA), OGA (#ab124807; 1/5000; Abcam), β-actin (rabbit anti-human/rat; #ab8227; 1/5000; Abcam), NLRP3 (#ab263899; 1/1000; Abcam), ASC (ab309497; 1/1000; Abcam), Caspase 1 (#ab286125; 2 µg/mL; Abcam), GSDMD-N (#PA5-116815; 1/1000; Thermo Fisher), and FOXO1 (#MA5-14846; 1/1000; Abcam) at 4 °C overnight, and subsequently with secondary antibody (rabbit anti-goat IgG; #31402; 1/50,000; Thermo Fisher) at room temperature for 1 h. Finally, the membranes were treated with chemiluminescence developing agents (#BB-3501; Bestbio). The band intensities were quantified using ImageJ software.

RT-qPCR

Total RNA was isolated from serum, tissues, and H9c2 cells using Trizol reagent (Takara, Osaka, Japan). The cDNA was synthesized using the SuperScript™ IV reverse transcriptase (#18090200; Thermo Fisher) and subjected to qRT-PCR assay with PowerUp™ SYBR™ Green (#A25741; Thermo Fisher). The primers used in this study were obtained from Sangon Biotechnology Co., LTD (Shanghai, China) and were shown below: OGT (rat), forward, 5′-GACGCAACCAAACTTTGCAGT-3′ and reverse, 5′-TCAAGGGTGACAGCCTTTTCA-3′; OGT (human), forward, 5′-TCCTGATTTGTACTGTGTTCGC-3′ and reverse, 5′-AAGCTACTGCAAAGTTCGGTT-3′; FOXO1, forward, 5′-CCCAGGCCGGAGTTTAACC-3′ and reverse, 5′-GTTGCTCATAAAGTCGGTGCT-3′; glyceraldehyde-3-phosphate dehydrogenase (GAPDH; rat), forward, 5′-AATGGATTTGGACGCATTGGT-3′ and reverse, 5′-TTTGCACTGGTACGTGTTGAT-3′; GAPDH (human), forward, 5′-TGTGGGCATCAATGGATTTGG-3′ and reverse, 5′-ACACCATGTATTCCGGGTCAAT-3′.

Cell viability

H9c2 cells (1 × 10³ cells per well) were seeded into 96-well plates in 100 µL of complete medium and incubated at 37 °C for 24 h. Then, 10 µL of CCK-8 reagent (#HY-K0301; Merck) was added to each well, and the cells were further incubated at 37 °C for 2 h. Subsequently, the optical density value at the absorbance of 450 nm was determined using a microplate reader.

Lactic dehydrogenase (LDH) release

H9c2 cells (1 × 10⁵ cells/mL) were seeded on 96-well plates, followed by centrifugation to collect the supernatants. LDH assay kit (#ab65393) was acquired from Abcam and utilized to assess the release of LDH in the supernatant of the cell culture medium, following the guidelines provided by the manufacturer. Finally, the colorimetric change of cells at the absorbance of 492 nm was determined using a microplate reader.

Propidium iodide (PI) staining

PI staining was performed to analyze the apoptosis in H9c2 cells using a commercial PI kit (#HY-D0815; Merck). First, the 40 µg/mL of PI working solution was prepared. H9c2 cells were then cultured on sterile cover slides. Next, add 100 µL dye working solution to the cells, gently shake the dye to completely cover the cells, and incubate for 15 min at 37 °C. Finally, fluorescence microscopy was used to observe. The percentage of positive cells was quantified, and the mean fluorescence intensity was evaluated using Image Pro advanced software.

Co-immunoprecipitation (Co-IP)

The Co-IP procedure was conducted utilizing the Pierce Crosslink Magnetic IP/Co-IP kit (#88805; Thermo Fisher) in accordance with the manufacturer’s guidelines. In summary, H9c2 cells were harvested and resuspended in cold lysis buffer. After centrifugation at 13,000 g for 15 min at 4 °C, the resulting lysates were incubated overnight at 4 °C with 2 µg of antibodies against OGT (1/5000; #ab96718; Abcam) and FOXO1 (1/500; #MA5-14846; Abcam). Subsequently, the lysates were treated with protein A/G-agarose beads at 4 °C for 1.5 h. The proteins that bound to the beads were then eluted and analyzed through Western blotting.

IP

The assessment of O-GlcNAc level of FOXO1 in H9c2 cells was conducted using an IP assay in conjunction with Western blotting. In summary, lysates from H9c2 cells were prepared and subjected to immunoprecipitation utilizing an anti-FOXO1 antibody along with protein A/G agarose (Novex, Oslo, Norway), after which Western blotting was performed to detect O-GlcNAc.

Prediction of O-GlcNAc sites of FOXO1

The Department of Health Technology research database (DTU; https://services.healthtech.dtu.dk/services/YinOYang-1.2/) was used to predict the O-GlcNAc sites of FOXO1.

Site mutation

To identify FOXO1 was O-glycosylated at which site, we commissioned Genescript Co. Ltd., (Nanjing, China) to mutate serine (S)41, S246, S284, and S415 to alanine (S41A, S246A, S284A, and S415A). These mutated plasmids were then transfected into H9c2 cells.

Protein stability assessment

Protein stability assessment was performed to verify the protein stability of FOXO1 after OGT inhibition in H9c2 cells treated. H9c2 cells were treated with cycloheximide (CHX, 100 µg/mL; #HY-12320; Merck), the protein level of FOXO1 at different time points (0, 8, 16, and 24 h) was detected.

Luciferase reporter assay

The cells were transfected with FOXO1-Luciferase reporter plasmid with 6 × FOXO1-binding site (S41) using the transfection reagent. A Renilla-Luc reporter plasmid was used for normalization. A luminometer was used to measure the luminescence (Pharmingen Moonlight 3010, BD Biosciences).

Animal study

Twenty-four male neonatal Sprague-Dawley (SD) rats (1–10 d; 8–15 g) were obtained from Sja Laboratory Animal Co., LTD. (Changsha, China) and housed in cages with a temperature of 24 °C and a 12-h light/dark cycle, and given ad libitum access to food and water. The animal experiments were approved by the Animal Care and Use Ethics Committee of Renmin Hospital of Wuhan University (No.WDRM20250106B). After one week of adaptive feeding, all rats were divided into sham, HF, HF + Lv-shNC, and HF + Lv-shOGT groups, with 6 rats in each group. The lentiviruses expressing either OGT shRNA or control shRNA were constructed from GenePharma Co., Ltd (Shanghai, China). In order to establish a rat model of HF, rats in the model groups were implanted with mini-osmotic pumps (DURECT Corp., Cupertino, CA) as previously described¹¹. Pumps were filled with Ang II dissolved in saline to deliver at rates of 0.1 µl/h/side at a total dose of 1 ng/kg per min, for consecutive 14 days. Sham group rats were implanted with pumps that delivered saline. For delivery of lentiviruses to the myocardium, approximately 5 µL/heart (1 × 10⁹ TU/ml) of shOGT or shNC lentivirus was into the left ventricle cavity through the heart apex. An equivalent volume of normal saline was injected as a control. Afterwards, all rats were euthanized via pentobarbital overdose (i.p.: 100 mg/kg) injection, after which blood and heart samples were collected individually.

Assessment of cardiac functions

The cardiac functions of rats were assessed using the Mylab X5 Vet Ultrasound imaging system (Esaote Ltd., Hong Kong, China). Measurements taken via echocardiography in the left ventricular long axis included left ventricular (LV) end-systolic diameter (LVESD), LV end-diastolic diameter (LVEDD), LV ejection fraction (LVEF), and LV fractional shortening (LVFS).

Histological detection

The degree of myocardial injury and fibrosis were evaluated by hematoxylin & eosin (H&E) and Masson, respectively. The heart isolated from rats were fixed using 4% paraformaldehyde solution for 24 h, and embedded in paraffin. Then, the embedded tissues were sliced into 4 μm sections followed by staining with H&E (#C0105S; Beyotime) and Masson (#C0189S; Beyotime). All operations were conducted according to the manufacturer’s instructions. Finally, the sections were observed by a microscope (Olympus, Tokyo, Japan).

Enzyme-linked immunosorbent assay (ELISA)

Commercial ELISA kits were obtained from Saipei Biotech Co., Ltd (Wuhan, China) to analyze the concentrations of interleukin (IL)-1β (#SP12225) and IL-18 (#SP12234) in H9c2 cells and the brain natriuretic peptide (BNP; #SP30135) in serum of rats. All protocols were carried out following the manufacturer’s provided instructions. Subsequently, the microplate reader from Thermo Fisher Scientific (Waltham, MA, USA) was employed to measure the OD value of each well. The acquired outcomes were then adjusted based on the total protein concentration in each sample to enable comparison between samples.

Statistical analysis

The SPSS 21.0 software was used to analyze data. Data are expressed as mean ± standard deviation (SD). Student’s t-test was used for comparison between two groups and one-way ANOVA was used for comparison between multiple groups. Statistical analyses were performed using GraphPad Prism software (v8.0.1, GraphPad Software Inc., San Diego, CA, USA). p < 0.05 indicates that the difference is statistically significant.

Results

OGT-mediated O-GlcNAc was elevated in HF

O-GlcNAc represents a unique form of O-glycosylation occurring within the nucleocytoplasmic compartment¹². The cycling of O-GlcNAc is regulated by two specific enzymes: OGT, which facilitates the addition of O-GlcNAc, and OGA, which is responsible for its removal⁶. Prolonged activation of cardiac O-GlcNAc is frequently linked to changes in cellular metabolism, which can adversely affect cardiovascular function and increase the risk of cardiovascular diseases, including hypertension, cardiac remodeling, heart failure, and arrhythmias⁷. While the involvement of O-GlcNAc in HF has been extensively researched, its significance in neonatal HF remains underexplored. This study aims to investigate the role of O-GlcNAc in patients with neonatal HF. Western blotting analysis revealed elevated levels of O-GlcNAc and OGT proteins in the neonatal HF group compared with the HC group, whereas the OGA level did not exhibit any notable differences between the two groups (Fig. 1A). In addition, RT-qPCR results showed that compared with the HC group, the mRNA expression level of OGT was higher in the neonatal HF group (Fig. 1B), while the expression of OGA was not significantly different between the two groups (Fig. 1C). For further verification, the in vivo and in vitro HF models were established. RT-qPCR and Western blotting results displayed increased OGT mRNA and protein levels in HF rat heart tissues and AngII-treated H9c2 cells (Fig. 1D–G). These findings suggested that OGT-mediated O-GlcNAc was elevated in HF.

Fig. 1.

OGT-mediated O-GlcNAc was elevated in HF. (A) Western blotting was performed to detect the serum protein levels of O-GlcNAc, OGT, and OGA in the HC (N = 22) and neonatal HF (N = 28) groups; RT-qPCR was used to assess the serum mRNA expression of (B) OGT and (C) OGA in the HC (N = 22) and neonatal HF (N = 28) groups; (D) RT-qPCR and (E) Western blotting were used to analyze the expression of OGT in the heart tissues of sham and HF group rats (N = 6); The mRNA and protein levels of OGT in the control and Ang II group H9c2 cells were analyzed via (F) RT-qPCR and (G), Western blotting (N = 3). O-GlcNAc, O-linked N-acetylglucosamine; OGT, O-GlcNAc transferase; OGA, O-GlcNAcase; HC, healthy control; HF, heart failure; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; HF, heart failure; Ang II, Angiotensin II.

OGT deficiency suppressed pyroptosis in AngII-treated H9c2 cells

To further explore the role of OGT in HF, we introduced shNC and shOGT into H9c2 cells through transfection. The findings revealed that the expression of OGT was decreased in shOGT group in contrast to the shNC group (Fig. 2A). When the cell membrane ruptures, lactate dehydrogenase (LDH) is released from the cell into the medium. Thus, cytotoxicity can be quantitatively analyzed by detecting LDH release¹³. In comparison to the control group, AngII-treated H9c2 cells showed downregulated cell viability and upregulated LDH release. In addition, compared with the shNC group, OGT knockdown increased the cell viability and decreased the LDH release in H9c2 cells (Fig. 2B,C), suggesting that loss of OGT inhibited cytotoxicity in H9c2 cells. Moreover, PI staining results showed that AngII treatment increased the percentage of PI positive cells compared with the control group. Compared with the shNC group, OGT inhibition downregulated the percentage of PI positive cells in H9c2 cells (Fig. 2D,E). Pyroptosis, a type of programmed cell death closely related to inflammation, has been shown to be involved in the progression of HF¹⁴. In the classical process of pyroptosis, Caspase-1 is activated through inflammasomes, particularly the NLRP3 inflammasome. This activation leads to the inflammatory cleavage of GSDMD, resulting in the formation of water channels within the cytoplasmic membrane. Consequently, this process causes localized cell swelling, triggers cell lysis, and facilitates the release of cellular contents along with inflammatory mediators such as IL-1β and IL-18¹⁵. However, the regulation of pyroptosis in HF by OGT has not been explored. In this study, we found that AngII-treated H9c2 cells upregulated the IL-1β and IL-18 concentrations and the pyroptosis-related (NLRP3, ASC, Caspase 1, and GSDMD-N) protein levels compared with the control group. Additionally, OGT knockdown decreased the concentrations of IL-1β and IL-18 and the protein levels of NLRP3, ASC, Caspase 1, and GSDMD-N in H9c2 cells in contrast to the shNC group (Fig. 2F–H). These outcomes demonstrated that OGT deficiency suppressed pyroptosis in AngII-treated H9c2 cells.

Fig. 2.

OGT deficiency suppressed pyroptosis in AngII-treated H9c2 cells. (A) Measurement of relative expression of OGT in the shNC and shOGT groups (N = 3); (B) CCK-8 was used to detect the cell viability in each group (N = 3); (C) LDH release in each group was measured by a commercial kit (N = 3); (D) PI staining was performed to analyze the apoptosis in each group (N = 3); (E) Percentage of PI positive cells in each group (N = 3); The concentrations of (F) IL-1β and (G) IL-18 in each group were analyzed by ELISA (N = 3); (H) Western blotting was performed to measure the protein levels of NLRP3, ASC, Caspase 1, and GSDMD-N in each group (N = 3). OGT, O-GlcNAc transferase; shRNA, shot hairpin RNA; CCK-8, cell counting-kit 8; LDH, lactic dehydrogenase; PI, propidium iodide; IL, interleukin; ELISA, enzyme-linked immunosorbent assay; NLRP3, NOD-, LRR- and pyrin domain-containing protein 3; ASC, apoptosis-associated speck-like protein containing a caspase recruitment domain; GSDMD-N, N-terminus of gasdermin D.

OGT regulated the O-GlcNAc of FOXO1 at S41 site in H9c2 cells

Forkhead box O1 (FOXO1) is a transcription factor involved in the regulation of a variety of physiological processes, including glucose metabolism, lipogenesis, apoptosis, and autophagy. FOXO1 activity is modulated by changes in protein expression and PTMs in response to different physiological or pathogenic conditions¹⁶. In some studies, O-GlcNAc has been found to regulate FOXO1^17,18. In the present research, we found that OGT inhibition decreased the protein levels of FOXO1 and FOXO1-O-Linked N-Acetylglucosamine (RL2) in H9c2 cells (Fig. 3A). Then, Co-IP was performed to detect the interaction between OGT and FOXO1. Results indicated that OGT could interact with FOXO1 in H9c2 cells (Fig. 3B). Next, we used the DTU research database to screen O-GlcNAc sites of FOXO1, and found four possible O-GlcNAc sites of FOXO1 (S41, S246, S284, and S415) (Fig. 3C,D). Therefore, we introduced alanine mutations at these four sites to explore the binding sites of OGT and FOXO1. Western blotting and IP results indicated that site mutation of S41A showed decreased protein levels of FOXO1 and FOXO1-RL2 compared with the WT group in H9c2 cells. Whereas, site mutation of S246A, S284A, and S415A showed no significant differences in FOXO1 and FOXO1-RL2 protein levels compared with the WT group in H9c2 cells (Fig. 3E). Furthermore, OGT deficiency decreased the luciferase activity of FOXO1 in H9c2 cells (Fig. 3F). In addition, protein stability assay results illustrated that OGT inhibition accelerated the degradation of FOXO1 in H9c2 cells (Fig. 3G). These results suggested that OGT regulated the O-GlcNAc of FOXO1 at S41 site in H9c2 cells.

Fig. 3.

OGT regulated the O-GlcNAc of FOXO1 at S41 site in H9c2 cells. (A) The protein levels of FOXO1 and FOXO1-RL2 were detected by Western blotting after OGT inhibition (N = 3); (B) Co-IP was used to detect the interaction between OGT and FOXO1 in H9c2 cells (N = 3); (C) The DTU research database was used to screen O-GlcNAc sites of FOXO1; (D) Four possible O-GlcNAc sites of FOXO1; (E) The protein levels of FOXO1 and FOXO1-RL2 in each group were assessed using Western blotting (N = 3); (F) Luciferase activity in the shNC and shOGT was detected by luciferase reporter assay (N = 3); (G) The H9c2 cells in each group were treated with CHX, then the protein level of FOXO1 was assayed by Western blotting at different time points (0, 8, 16, and 24 h) (N = 3). FOXO1, Forkhead box O1; RL2, O-Linked N-Acetylglucosamine; OGT, O-GlcNAc transferase; Co-IP, co-immunoprecipitation; DTU, Department of Health Technology research database; O-GlcNAc, O-linked N-acetylglucosamine; shRNA, shot hairpin RNA; CHX, cycloheximide.

FOXO1 overexpression promoted pyroptosis in AngII-treated H9c2 cells

To further explore the role of FOXO1 in AngII-treated H9c2 cells, FOXO1 overexpression and empty vectors were introduced into H9c2 cells. Results demonstrated that FOXO1 overexpression increased the mRNA level of FOXO1 in H9c2 cells compared with the vector group (Fig. 4A). Besides, in comparison to the vector group, FOXO1 overexpression decreased the cell viability and increased the LDH release, the percentage of PI positive cells, the concentrations of IL-1β and IL-18, and the protein levels of NLRP3, ASC, Caspase 1, and GSDMD-N in AngII-treated H9c2 cells (Fig. 4B–H). These findings suggested that FOXO1 overexpression promoted pyroptosis in AngII-treated H9c2 cells.

Fig. 4.

FOXO1 overexpression promoted pyroptosis in AngII-treated H9c2 cells. (A) Relative expression of FOXO1 was detected by RT-qPCR after FOXO1 overexpression (N = 3); (B) The cell viability in each group was assessed by CCK-8 assay (N = 3); (C) Measurment of LDH release in each group (N = 3); (D) The apoptosis in each group was analyzed by PI staining (N = 3); (E) Percentage of PI positive cells in each group (N = 3); ELISA was performed to assess the concentrations of (F) IL-1β and (G) IL-18 in each group (N = 3); H, The protein levels of NLRP3, ASC, Caspase 1, and GSDMD-N in each group were analyzed via Western blotting (N = 3). FOXO1, Forkhead box O1; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; CCK-8, cell counting-kit 8; LDH, lactic dehydrogenase; PI, propidium iodide; IL, interleukin; ELISA, enzyme-linked immunosorbent assay; NLRP3, NOD-, LRR- and pyrin domain-containing protein 3; ASC, apoptosis-associated speck-like protein containing a caspase recruitment domain; GSDMD-N, N-terminus of gasdermin D.

OGT Inhibition alleviated myocardial tissue necrosis, myocardial fibrosis, and pathological cardiac dysfunction

Finally, we established a HF rat model to further analyze the effects of OGT in vivo. The heart tissues of rats were isolated for H&E and Masson staining. H&E staining revealed that the myocardial fibers of rats in the sham operation group were organized in a distinct and orderly fashion, exhibiting no significant deformation. In contrast, the myocardial tissues of the HF model group displayed fragmented and necrotic fibers, accompanied by noticeable infiltration of inflammatory cells in the affected areas. Besides, the inhibition of OGT significantly reduced the damage observed in the HF rats. In addition, Masson staining revealed that, in contrast to the sham rats, the myocardial tissue of HF rats exhibited a disorganized arrangement. A significant presence of blue fibrotic tissues was observed within the myocardial interstitium, indicating a marked increase in both the area of collagen fibers and the extent of myocardial fibrosis. Notably, in comparison to the shNC group, OGT deficiency led to a reduction in the collagen fiber area, while the myocardial cells maintained structural integrity and were organized in a neat arrangement (Fig. 5A). BNP is a cardiac neuroendocrine hormone mainly secreted by ventricular myocytes during the increase of ventricular pressure load and ventricular volume expansion, and is one of the most widely used biomarkers in the diagnosis and treatment of HF¹⁹. In this study, HF group showed higher serum BNP concentration compared with the sham group. Besides, compared with the Lv-shNC group, OGT inhibition decreased the serum BNP level (Fig. 5B). Then, the cardiac function of each group rats was evaluated. Results indicated that HF group rats showed the decreased percentages of LVEF and LVFS and the increased LVEDD and LVESD compared with the sham group rats. Besides, in contrast to the Lv-shNC group, loss of OGT upregulated the percentage of LVEF and LVFS and downregulated the LVEDD and LVESD (Fig. 5C–F). These outcomes suggested that loss of OGT reversed HF-induced myocardial tissue necrosis, myocardial fibrosis, and pathological cardiac dysfunction.

Fig. 5.

OGT inhibition alleviated myocardial tissue necrosis, myocardial fibrosis, and pathological cardiac dysfunction. (A) H&E and Masson staining were used to detect the pathological changes of heart tissues in each group rats (N = 6); (B) Serum BNP concentration in each group was analyzed by a commercial ELISA kit (N = 6); (C) LVEF, (D) LVFS, (E) LVEDD, and (F) LVESD of each group rats were measured (N = 6). H&E, hematoxylin and eosin; BNP, brain natriuretic peptide; ELISA, enzyme-linked immunosorbent assay; LVEF, left ventricular ejection fraction; LVFS, left ventricular fractional shortening; LVEDD, left ventricular end-diastolic diameter; LVESD, left ventricular end-stage systole diameter.

Discussion

O-GlcNAc glycosylation, similar to phosphorylation, is a prevalent post-translational modification. Both modifications specifically target serine or threonine residues within proteins²⁰. However, a significant distinction lies in the regulatory mechanisms: while there exists a multitude of protein kinases and phosphatases that oversee phosphorylation processes in cells, O-GlcNAc glycosylation is regulated by a singular pair of enzymes, OGT and OGA²¹. The physiological role of O-GlcNAc glycosylation is crucial for maintaining metabolic and immune homeostasis. Current understanding suggests that persistently elevated levels of O-GlcNAc glycosylation can be detrimental to human health²². In this study, we gathered clinical samples and developed both in vivo and in vitro models of HF to investigate the implications of O-GlcNAc in neonatal HF. Our findings revealed that OGT-mediated O-GlcNAc levels were significantly increased in HF. Similarly, attenuation of O-GlcNAc is beneficial against pressure overload-induced HF²². Besides, a review indicates that chronic abnormal elevations in proteinO-GlcNAc glycosylation are associated with various cardiovascular conditions, such as atherosclerosis, myocardial hypertrophy, heart failure, hypertension, and vascular dysfunction²³. In addition, the changes of cardiomyocyte O-GlcNAc level regulates store-operated calcium entry in neonatal cardiomyocytes²⁴. Interestingly, a previous study finds that the increase of O-GlcNAc protects neonatal cardiomyocytes from ischemia-reperfusion injury²⁵.

Pyroptosis is a highly inflammatory cell death that uses the pores created in the cell membrane to disrupt cellular homeostasis and cause cell death. Various studies have shown that pyroptosis is related to different stages of HF²⁶. In this study, we discovered that OGT silencing suppressed the pyroptosis of AngII-treated H9c2 cells by inhibiting the formation of NLRP3 inflammasome, the activation of Caspase-1, and the release of inflammatory cytokines (IL-1β and IL-18), suggesting that OGT promoted the progression of HF via regulating pyroptosis. Similar with our results, OGT is involved in the progression of various inflammatory diseases by regulating pyroptosis. Yang et al.²⁷ discover that inhibition of OGT improved lipopolysaccharide-induced pyroptosis of human gingival fibroblasts. Besides, OGT-induced O-GlcNAcylation promotes osteoarthritis progression by promoting chondrocyte pyroptosis²⁸. In addition, the NLRP3/Caspase-1/GSDMD pyrogenic pathway can be used as a therapeutic target for a variety of heart diseases, including chronic HF and heart injury^29,]30.

FOXO genes belong to a transcription factor family³¹. In mammals, FOXO1 is involved in many physiological and biochemical processes, including angiogenesis, gluconeogenesis, apoptosis and cell cycle, and plays an important role in development, metabolism and tumor inhibition³¹. FOXO protein is regulated by various protein PTMs such as phosphorylation and O-glycosylation, and helps organisms respond to various additional stimuli such as nutritional conditions and peroxides by activating or inhibiting the expression of related genes. In this study, we found that OGT regulated the O-GlcNAc of FOXO1 at S41 site in H9c2 cells. Similarly, Kuo et al. indicate that treatments that increase protein O-GlcNAc glycosylation induce O-GlcNAc modification of FOXO1 in human embryonic kidney (HEK)-293T cells³². Besides, O-GlcNAc regulates hepatic FOXO1 activation in response to glucose in diabetes¹⁷. Moreover, FOXO1 overexpression promoted pyroptosis in AngII-treated H9c2 cells in this study, implying that OGT-mediated O-GlcNAc of FOXO1 promoted the progression of HF via regulating pyroptosis.

In final animal studies, we found that OGT inhibition alleviated myocardial tissue necrosis, myocardial fibrosis, and pathological cardiac dysfunction. Similarly, previous studies have found that OGT regulates cardiac function and structural phenotype in pathologic hypertrophy and diabetic heart^33,]34. In summary, this research demonstrated that OGT-mediated O-GlcNAc of FOXO1 promoted the progression of neonatal HF via regulating pyroptosis. At present, there are some targeted inhibitors for pyroptosis to improve cardiovascular diseases, including HF³⁵. Our study suggested that developing OGT inhibitors may be a more effective HF treatment strategy. In this study, there are still some limitations. For example, the in vitro model was still established with reference to ordinary HF. In the future, in vitro models more consistent with neonatal HF are worthy of further exploration.

Electronic supplementary material

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Author contributions

All authors participated in the design, interpretation of the studies and analysis of the data and review of the manuscript. J W drafted the work and revised it critically for important intellectual content; Z X, X L and B H were responsible for the acquisition, analysis and interpretation of data for the work; Q Z and Q D made substantial contributions to the conception or design of the work. All authors read and approved the final manuscript.

Funding

The work was supported by The National Natural Science Fund under Grant Number 82170312.

Data availability

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.

Ethical approval and consent to participate

This study was approved by the Ethics Committee of Renmin Hospital of Wuhan University. This study was performed in line with the principles of the Declaration of Helsinki. Written informed consent was obtained from the parents. All animal experiments should comply with the ARRIVE guidelines. All methods were carried out in accordance with relevant guidelines and regulations.