Mechanistic role of RND3-regulated IL33/ST2 signaling on cardiomyocyte senescence

doi:10.21203/rs.3.rs-3208029/v1

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Mechanistic role of RND3-regulated IL33/ST2 signaling on cardiomyocyte senescence

https://doi.org/10.21203/rs.3.rs-3208029/v1

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BACKGROUND: Hyperinflammatory responses are pivotal in the pathophysiology of cardiomyocyte senescence, with IL33 serving as a crucial pro-inflammatory mediator. Our previous findings highlighted RND3's suppressive effect on IL33 expression. This study delves into the influence of RND3 on IL33/ST2 signaling activation and cardiomyocyte senescence.

METHODS: AC16 cardiomyocytes were subjected to treatments involving recombinant IL33, NF-κB inhibitor PDTC, or ST2 antibody Astegolimab. SA-β-gal and γH2AX staining were utilized to monitor alterations in cell senescence and DNA damage, respectively. Western blot analysis was conducted to ascertain the expression of Senescence-Associated Secretory Phenotype (SASP) and NF-κB activation. Utilizing CRISPR/Cas9 technology, the RND3 gene was knocked out in H9C2 cells, followed by senescence analysis and sST2 level detection in the culture medium supernatant via ELISA. Post-AAV9 injection overexpressing RND3 in SD rats, IL33/ST2 and SASP expression in heart tissues, and serum IL33 and sST2 changes were evaluated using ELISA.

RESULTS: Exogenous IL-33 significantly induced IL-1α, IL6, and MCP1 expression, increased the p-p65/p65 ratio, and the proportion of SA-β-gal and γH2AX positive cells in AC16 cells. PDTC and Astegolimab application mitigated these effects. RND3 knockout in H9C2 cells led to increased intracellular IL33, ST2L, IL1 α, IL6, and MCP1 expression, decreased sST2 in the supernatant, and increased SA-β-gal and γH2AX positive cells. RND3 overexpression suppressed IL33, ST2L, IL-1α, IL6, and MCP1 expression in heart tissues, decreased serum IL33, and increased sST2 levels.

CONCLUSION: RND3 expression in cardiomyocytes modulates cell senescence by negatively regulating the IL33/ST2/NF-κB signaling pathway, underscoring its potential as a therapeutic target in cardiovascular senescence.

General Cell Biology & Physiology

RND3

IL33/ST2 signaling

NF-κB

Cardiomyocyte

Senescence

Aging cardiomyocytes are shortened and damaged, with increased pacing frequency, systolic dysfunction, and metabolic dysfunction [1], and can secrete senescence-associated secretory phenotype (SASP) factors to induce senescence of neighboring cell types, and eventually progress to heart failure [2]. Since inflammation is a determinant of aging, it also plays a central role in myocardial aging. Franceschi et al. proposed the term "inflammatory aging" to describe chronic, low-grade, systemic inflammation (i.e., aseptic inflammation) that develops with age in the absence of obvious infection. Systemic inflammation is closely related to cardiomyocyte failure [3], however, the molecular and mechanism mediating systemic inflammation and cardiomyocyte senescence is not well understood.

Interleukin 33 (IL33), a member of the IL-1 family, is widely expressed in immune cells, endothelial cells, epithelial cells and fibroblasts, and clinical studies have shown that it is significantly elevated during the occurrence of various inflammatory diseases [4, 5]. Relevant studies have also confirmed that exogenous IL33 is a key molecule in inducing respiratory, vascular, urinary, and nervous system inflammation [6–9]. However, the effect of exogenous IL33 on the heart is rarely reported, and the mechanism of regulating IL33 expression is still largely unknown.

The growth stimulating expression gene 2 (ST2) protein is a member of the IL-1 receptor family and consists of two important isoforms, the ST2 receptor (ST2L) and soluble ST2 (sST2). ST2L is a transmembrane receptor and sST2 is a soluble receptor that exists in the blood circulation. As a decoy receptor of IL33, sST2 binds to IL33 in circulation and then blocks its binding to the cell membrane ST2 receptor (ST2L) to play an inhibition role [10]. Although a large number of studies have shown that the increase of circulating sST2 is of great significance in the diagnosis and prognosis of myocardial infarction, hypertension, diabetes, pulmonary hypertension, myocardial hypertrophy and heart failure, the understanding of IL33/ST2 in the cardiovascular field is still controversial. In particular, there are completely different views on the pathophysiological role of cardiomyocytes [10–15].

RND3, also known as RhoE, is a member of the Rho GTP protein superfamily. Recent studies have found that abnormal expression of RND3 plays an important role in cardiovascular pathophysiological processes, including cardiac remodeling, arrhythmia, inflammation after myocardial infarction, and diabetic cardiac fibrosis [16–19], suggesting the potential value of restoring RND3 expression in the treatment of these cardiovascular diseases. Our group previously knocked out the RND3 gene in rat H9C2 cardiomyocytes using CRISPR/Cas9 technology, and found that IL33 was one of the potential downstream target genes [20].

In this study, we demonstrated that exogenous IL33 induces cardiomyocyte senescence. We found that the addition of high concentration of IL33 in the culture medium induced SA-β-gal accumulation and DNA damage in cardiomyocytes, and promoted the expression of SASP in cardiomyocytes by activating the NF-κB pathway. Blocking ST2 receptor can treat the effect of IL33 on cardiomyocyte senescence. Importantly, we found that RND3 plays a crucial role in the regulation of IL33/ST2 signaling. These results suggest that RND3 impacts cardiomyocyte senescence by mediating the IL33/ST2/NF-κB pathway.

2.1. Animal models

The 6-week-old male SD rats were purchased from SLAC Laboratory Animal Co. Ltd (Changsha, China), and the animal experiments were in accordance with the animal experimental ethics of Hainan Medical University (Approval number KYLL-2020-048). For establishing cardio-specific RND3 overexpressed rats, the exogenous RND3 overexpressing AAV9 (GeneChem, Shanghai, China) were infused to rats via one-time tail vein injection (10¹² vg/rat), and rats infused with control AAV9 particles were served as negative controls. After 12 weeks of routine feeding, the rats were anesthetized by isoflurane (#R510-22-10, RWD Life Science Co. Ltd, Guangdong, China), blood was collected from the abdominal aorta, centrifuged at 4℃ at 1300 rcf for 10 min, and the upper serum was used for ELISA to detect the levels of sST2 and IL33. Subsequently, the rats were killed by excessive anesthesia, the hearts were separated, and the left ventricular tissue was cut for total protein extraction and western blotting.

2.2 Cell culture

Human AC16 cardiomyocyte was purchased from ATCC. Rat H9C2 cardiomyocyte was from Cell bank of Chinese Academy of Sciences (Shanghai, China). AC16 and H9C2 cell line was cultured in Dulbecco’s modified eagle medium (DMEM) (#PM150210A, Procell, Wuhan, China) complete medium containing 10% fetal bovine serum (FBS) (#04-001-1ACS, BI, Iselin) and 1% Penicillin/Streptomycin at 37 °C with 5% CO₂. Medium was changed every two days. When reaching 80% − 90% confluence, the cells were detached with 0.25% trypsin and passaged at a 1:3 ratio. Cells were seeded in a 6-well plate or confocal dish at densities of 2 × 10⁵/ml. Cells planned for ELISA assay were replaced with FBS free culture medium 12 h in advance.

2.3 RND3 gene knockout in H9C2 cells

Using the previous method [20], H9C2 cells were infected with lentiviruses (# GCPL0357416, Genechem, Shanghai, China) carrying effective sgRNA targeting rat RND3 gene and recombinant Cas9 protein. Puromycin resistant cells were H9C2 cells with RND3 gene knockout (denoted as RND3_KO). The expression of RND3 protein was verified by western blotting.

2.4 IL33, Astegolimab and PDTC treatment

AC16 cells were treated with recombinant exogenous IL33 (0.5 µg/mL) (#C091, Nazyme, Nanjing, China), compared with PBS, and cells were harvested 45 min later for related detection. To determine the role of NF-κB signaling in IL33-induced downstream gene expression, NF-κB inhibitor PDTC (10 µmol/L) (#S1809, Beyotime Biotechnology, Nanjing, China) and ST2 monoclonal antibody Astegolimab (10 µmol/L) (#2173054-79-8; MedChemExpress, USA) treated 0.5 µg/mL IL33-stimulated AC16 cells and harvested the cells 48h later for subsequent relevant experiments.

2.5 Senescence associated β-galactosidase staining

Senescence associated (SA)-β-gal staining kit (#C0602, Beyotime) was used. The cells were inoculated with 6-well plates and treated when the cell growth fusion exceeded 60%. 48 h later, the culture medium was removed, washed once with PBS, and the β-galactosidase stationary liquid was added and fixed at room temperature for 15 min. The fixing solution was removed, and the SA-β-galactosidase staining solution was added after PBS washing for 3 times, and incubated overnight in a 37℃ incubator. The next day, the dye solution was removed, PBS was added, observed under an inverted microscope and photographed.

2.6 γH2AX immunofluorescence staining

DNA Damage Assay Kit by γ-H2AX Immunofluorescence (#C2035S, Beyotime) was used. The cells were inoculated in confocal dishes and treated when they grew to 60% fusion degree. After fixation, the cells were added with blocking solution, closed at room temperature for 20 min, added with γH2AX antibody, incubated at 4℃ overnight, washed with PBS, added with Alexa Fluor®488, incubated at room temperature for 1 h, and DAPI counterstaining the nuclei. The images were taken under confocal microscope.

2.7 Western blotting

Total protein was extracted from heart tissue and cardiomyocytes by RIPA (Beyotime) and quantified by BCA (Beyotime). The 40 µg protein was subjected to PAGE, transferred to PVDF membrane (#IPVH00010, Millipore, USA), blocked by BSA and washed by TBST, then added to primary antibody and incubated at 4℃ overnight. PVDF membrane was washed 3 times by TBST for 5 min each time, then HRP-IgGs was incubated at room temperature for 1 h, PVDF membrane was washed 3 times for 5 min each time, and finally immersed in ECL (#BL520A, Biosharp) developer gel imaging system for imaging and photographing. Primary antibodies used include RND3(1:1000; #66535-1-Ig, Proteintech, Wuhan, China); IL33 (1:1000; #DF8319, Affinity, Wuhan, China), p-p50(1:2000, #AF3219, Affinity), IL1α(1:2000; #DF6893, Affinity); ST2L (1:1000; (#PTM-6483; PTM, Hangzhou, China), p50(1:1000; #PTM-6483, PTM), β-actin (1:1000; #PTM-5706; #PTM-5028, PTM); MCP1 (1:1000; #bs-1955R, Bioss, Beijing, China) and IL6 (1:1000; #bs-4539R, Bioss).

2.8 ELISA

ELISA kit for Rat ST2 (#JM-10483R1; FankeW, Shanghai, China) and IL-33 (#DL-IL33-Ra, FankeW) were used to detect the cell culture solution supernatant and serum samples according to manufacturer's instructions.

2.9 Statistical processing

The experimental data were expressed as mean ± SD. The comparison between the two groups was performed by unpaired t-test, and the comparison between the multiple groups was performed by One-way ANOVA analysis and GraphPad Prism (version 9.4.1) for data analysis and plotting, P < 0.05 was statistically significant.

3.1 Exogenous IL33 induced cardiomyocyte senescence

To investigate the effect of exogenous IL33 on cardiomyocytes, we stimulated AC16 cardiomyocytes with recombinant human IL33 protein. Compared with the control group, exogenous IL33 can effectively induce the expression of MCP1, IL1α and IL6 in cardiomyocytes (Fig. 1A, B). However, cycle-related SASP such as p16 and p53 did not change significantly. The results of SA-β-gal staining showed that IL33 stimulation could significantly induce senescence of cardiomyocytes (Fig. 1C), and the results of γH2AX immunofluorescence staining showed that IL33 significantly induced DNA damage of cells (Fig. 1D).

3.2 IL33 induces SASP in cardiomyocytes by activating NF-κB signaling

Demyanets S et al reported that IL33 can induce inflammation in vascular endothelial cells by activating the NF-κB pathway [21]. In order to determine whether IL33 plays a role through NF-κB signaling in cardiomyocytes, we used NF-κB inhibitor PDTC to treat AC16 cells stimulated by IL33. The results showed that the phosphorylation level of p65 was increased in AC16 cardiomyocytes after exogenous IL33 stimulation for 45 min, while the phosphorylation level of p50 was not significantly increased. Further NF-κB-specific inhibitor PDTC was applied to treat IL33-stimulated AC16 cardiomyocytes, and it was found that PDTC could down-regulate the phosphorylation level of p65, and significantly inhibit the expression of MCP1, IL1α, and IL6 proteins. However, the protein levels of p16 and p53 were not affected by the above treatment factors (Fig. 2A, B).

3.3 Astegolimab blocks the effect of exogenous IL33 on cardiomyocyte senescence

To further confirm the role of membrane ST2L receptor in exogenous IL33 to cardiomyocyte senescence, we stimulated AC16 cardiomyocytes with Astegolimab, which is ST2 IgG2 monoclonal antibody, to block the binding of exogenous IL33 to ST2L. The results showed that blocking ST2 could down-regulate the phosphorylation level of p65, and significantly inhibit the expression of inflammatory SASP such as MCP1, IL1α, and IL6. Interestingly, the expression of p53 in cardiomyocytes stimulated by Astegolimab was significantly lower than the baseline level (Fig. 3A, B), suggesting that Astegolimab may have other pharmacological effects besides blocking IL33. The results of SA-β-gal and γH2AX staining suggest that Astegolimab can effectively treat cardiomyocyte senescence and DNA damage induced by IL33 (Fig. 3C, D).

3.4 RND3 knockout induced cardiomyocytes senescence and DNA injury

A mature lentivirus carrying sgRNA and recombinant Cas9 protein targeting rat RND3 gene was designed to infect H9C2 cells, and the experiment successfully obtained RND3_KO H9C2 cells with RND3 gene knockout. Western blot analysis showed that RND3_KO significantly increased the expression of MCP1, IL1, p53, p16 and IL6 proteins of SASP genes compared with the control group (Fig. 4A, B). Meanwhile, the results of SA-β-galactosidase staining also suggested that RND3_KO promoted cardiomyocyte senescence (Fig. 4C), accompanied by a significant increase in the degree of DNA damage (Fig. 4D).

3.5 Knocking out RND3 activates the IL33/ST2 signal

Subsequently, we further explored the regulatory role of RND3 on the IL-33/ST2 axis. Western blot results showed that after RND3 gene knockout, the expression levels of IL33 and ST2L proteins in H9C2 cells were increased, accompanied by increased phosphorylation levels of NF-κB p65 (Fig. 5A, B). At the same time, the level of the decoy receptor sST2 of IL33 in the cell culture supernatant significantly decreased with RND3 knockout (Fig. 5C).

3.6 Overexpression of RND3 inhibited the SASP of cardiomyocytes

In this study, AAV9 particles carrying the overexpression of exogenous RND3 was injected into the tail vein to achieve the overexpression of RND3 in the heart tissue. Western blot results showed that the level of RND3 protein in RND3_OE group was significantly increased, along with the inhibition of NF-κB pathway and the expression levels of SASP factor including MCP1, IL1α and IL6 were significantly decreased. Meanwhile, overexpression of RND3 inhibited the expression of IL33 and ST2L proteins (Fig. 6A, B). ELISA results showed that compared with the control group, plasma IL33 level of rats in RND3_OE group decreased, sST2 level increased (Figure. 6C), and plasma IL33/ sST2 ratio decreased (Figure. 6D).

RND3 belongs to the Rho GTP enzyme superfamily and has a variety of biological functions. In addition to receiving certain attention in the field of tumor [15], recently more attention has been paid to its role in cardiovascular system diseases [16–19, 23–25]. In the previous gene chip screening, we found that RND3 gene knockout was accompanied by IL33 signal activation in cardiomyocytes [20]. In view of the current controversy over the role of IL33/ST2 signaling in cardiovascular diseases, this study aims to explore the relationship between IL33/ST2 signaling and cardiomyocyte senescence. The results suggest that the down-regulation of RND3 gene in cardiomyocytes promotes the activation of IL33/ST2 signal, which further mediates the aging of cardiomyocytes through NF-κB signaling regulating the expression of SASP genes. This study proposed for the first time that the activation of IL33/ST2 signaling axis promotes cardiomyocyte senescence, expands the research on the relationship between RND3 and cardiovascular disease, and provides a new basis for elucidating the pathophysiological role of IL33/ST2 signaling system in cardiomyocytes.

IL33 is a member of the IL1 family and can be expressed in various tissue cells, including cardiomyocytes. Under normal circumstances, IL33 is expressed in the nucleus of the cell, and it is secreted to the outside of the cell in cases of infection, inflammation, tissue injury and cell necrosis [5, 26]. IL33 binds to its transmembrane receptor, ST2L, and the activated IL33/ST2 signal is involved in important pathophysiological processes including inflammation, fibrosis, and heart failure [27–30], so it is considered that the IL-33/ST2 pathway has extensive clinical transformation value. The mechanism of increased intracellular IL33 expression remains unclear, but some studies have suggested that Yap/Taz can directly regulate the IL33 promoter and promote its expression in cardiac fibroblasts [31]. In this study, it was found that the expression of IL33 and its receptor ST2L in cells increased significantly after RND3 gene knockout, suggesting that RND3 is the upstream regulator of IL33, but how RND3 regulates the expression of IL33 needs to be further clarified.

The biological role of IL33/ST2 signaling in cardiomyocytes is controversial. It has been reported that IL33 released after acute kidney injury can induce cardiomyocyte hypertrophy, showing a pathogenic effect [29], while in diabetic cardiomyopathy mice, IL33 administration can block autophagy and enhance cardiac diastolic function, thus having a protective effect [30]. In this study, we found that cardiomyocytes showed a significant SASP after administration of exogenous IL33, which was manifested as increased expression of SASP such as MCP1, IL1α and IL6, as well as increased number of SA-β-galactosidase active cells and the trend of cellular DNA damage, suggesting that the increase of IL33 has a damaging effect on cardiomyocytes. At the same time, we also found that administration of Astegolimab can effectively treat cardiomyocyte senescence and DNA damage caused by IL33, further confirming the role of IL33/ST2 signaling activation in cardiomyocyte senescence. In terms of mechanism, this study suggests that elevated IL33 increases the phosphorylation of p65 through myocardial cell membrane ST2L and activates NF-κB signaling to mediate the expression of aging-related inflammatory proteins, which is highly consistent with the results of recent studies on obstructive sleep apnea patients [32].

It has been reported that on the one hand, the expression of RND3 is reduced in the heart tissue of chronic heart failure [26], on the other hand, the decreased expression of RND3 promotes the inflammatory response in the local myocardium of myocardial infarction by activating NF-κB signaling [18]. Considering that some inflammatory factors such as IL1, IL6, TNF-α and MCP1 are consistent with secreted phenotypic factors related to cell aging in the inflammatory response after myocardial infarction, it is necessary to further elucidate the relationship between RND3 and IL33/ST2 signaling. In vitro experiments, it was found that RND3 gene knockout significantly increased the expression of IL33 and ST2L in cells, while reducing the level of sST2 in the cell supernatant. At the same time, the senescence -related factors MCP1, IL1α, p16, p53 and IL6 were also increased, suggesting that the decreased expression of RND3 activated IL33/ST2 signaling and promoted the age-related inflammatory response in cardiomyocytes. In vivo experiments, it was found that overexpression of RND3 in myocardial tissue inhibited the expression of IL33/ST2 signal and SASP such as MCP1, IL1α and IL6 in myocardial tissue, which confirmed the negative regulatory effect of RND3 on IL33/ST2 signal from another level.

It should be noted that in animal experiments, the serum sST2 level of rats in the overexpressed RND3 group was significantly increased compared with the control group, while the level of IL33 and the ratio of IL33/sST2 were also decreased. In this study, there was no statistical difference between the two groups in ejection fraction, short contraction fraction, and E/A peak 2 months after the administration of AAV9, but after induction into diabetic state, the over-expression of RND3 group had better cardiac function, and a significant negative correlation between plasma sST2 level and ejection fraction was also found (unpublished data). At present, whether sST2 is a marker of heart failure has aroused certain attention and controversy [13, 15, 34], and our results support the conclusion that sST2 level is a marker of heart failure. As for how cardiac overexpression of RND3 leads to a decrease in IL33 and an increase in sST2 levels in animal serum, the mechanism is still unclear.

Taken together, this study shows for the first time that IL33 can induce cardiomyocyte senescence. RND3 negatively regulates the IL33/ST2/NF-κB signaling axis in cardiomyocytes and is involved in the regulation of cardiomyocyte senescence related phenotypes. Our study provides experimental basis for follow-up intervention of heart senescence based on RND3/IL33/ST2/NF-κB signal axis.

Author Contributions

Linxu Wu, Xinglin Zhu, Cai Luo, Yangyang Zhao, Shanshan Pan and Kaijia Shi performed the experiments and collected data. Zhihua Shen searched literature and performed statistical analyses. Zhihua Shen, Junli Guo and Wei Jie conceived of and coordinated the study and drafted the manuscript. All of the authors read and approved the final manuscript.

Funding

This work was supported by Hainan Key Research and Development Project (ZDYF2022SHFZ038, ZDYF2020122), National Natural Science Foundation of China (82060053). The funders had no role in the design of the study, collection, analysis or interpretation of data, nor were they involved in writing the manuscript.

Disclosure

The authors have no conflicts of interest related to this work.

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Mechanistic role of RND3-regulated IL33/ST2 signaling on cardiomyocyte senescence

Status:

Journal Publication

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Abstract

Figures

1. Introduction

2. Materials and methods

2.1. Animal models

2.2 Cell culture

2.3 RND3 gene knockout in H9C2 cells

2.4 IL33, Astegolimab and PDTC treatment

2.5 Senescence associated β-galactosidase staining

2.6 γH2AX immunofluorescence staining

2.7 Western blotting

2.8 ELISA

2.9 Statistical processing

3. Results

3.1 Exogenous IL33 induced cardiomyocyte senescence

3.2 IL33 induces SASP in cardiomyocytes by activating NF-κB signaling

3.3 Astegolimab blocks the effect of exogenous IL33 on cardiomyocyte senescence

3.4 RND3 knockout induced cardiomyocytes senescence and DNA injury

3.5 Knocking out RND3 activates the IL33/ST2 signal

3.6 Overexpression of RND3 inhibited the SASP of cardiomyocytes

4. Discussion

Declarations

References

Status:

Journal Publication

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