Annexin A5 knockdown inhibits cardiomyocyte apoptosis and alleviates cardiac hypertrophy via activating the PI3K/AKT/Bcl-2 signaling pathway

Abstract

Annexin A5 (ANXA5) is a small calcium-dependent protein that binds specifically to negatively charged phosphatidylserine as a marker of apoptosis. Previous studies have shown that ANXA5 expression is elevated in hypertensive patients and is closely related to left ventricular systolic function in hypertensive patients, but its specific mechanism of action has not been clarified. GEO database analysis showed that ANXA5 expression was significantly upregulated in hypertensive myocardial hypertrophy. The expression of ANXA5 protein and mRNA was overexpressed, and knockdown of ANXA5 can effectively attenuate cardiomyocyte apoptosis and inflammatory response, ameliorate myocardial hypertrophy and cardiac dysfunction in ALD-induced hypertrophic cardiomyocytes and in SHR hypertrophic hearts. Mechanistically, ANXA5 has synergistic effect with intracellular calciumion level. In the meantime, ANXA5 knockdown inhibited cell apoptosis along with a decrease of Bax/Bcl-2 ratio, and induction of PI3K/AKT activation. It should be noted that LY290004 (PI3K/Akt signaling pathway inhibitor) can weaken the inhibitory effect of knockdown ANXA5 on cardiomyocyte apoptosis and myocardial protection. Therefore, ANXA5 knockdown improves hypertensive myocardial hypertrophy and cardiac function by inhibiting apoptosis and inflammatory response and activating PI3K/AKT/Bcl-2 pathway, ANXA5 may be a potential therapeutic direction for the treatment of hypertensive myocardial hypertrophy.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-024-83244-3.

Introduction

Hypertension is one of the most common diseases and a significant risk factor for Cardiovascular disease(CVD)¹. Left ventricular hypertrophy (LVH) is an indicator of target organ damage mainly due to hypertension. Long-term hypertension can lead to myocardial cell damage, myocardial hypertrophy and ventricular remodeling. Its pathological process is mainly due to myocardial intracellular calcium overload, apoptosis and necrosis of myocardial injury² lead to cardiac function and configuration of the disorder. Mammalian cardiomyocytes, a terminally differentiated organ, withdraw from the cell cycle and fail to carry out cell division and proliferation, resulting in the inability of cardiomyocytes to repair the damaged area³, thus leading to pathological changes in the size, volume and composition of cardiomyocytes, which participate in the pathophysiological process of cardiac hypertrophy and remodeling, and eventually progress to irreversible heart failure⁴. Aldosterone(ALD) is the main effector hormone at the end of Renin–angiotensin–aldosterone system (RAAS)⁵, abnormally elevated of ALD can cause elevate blood pressure and promote pathological cardiac remodeling and cardiac ionic homeostasis imbalance, resulting in cardiac hypertrophy, inflammation, remodeling and fibrosis⁶. Therefore, it is of great significance to study the specific pathological mechanism of ALD-induced myocardial hypertrophy for the prevention and treatment of hypertension.

Annexin A5 (ANXA5) is a Ca²⁺-related protein with a molecular weight of 32–35 kDa, a slightly curved three-dimensional molecule structure consisting of four homologous repeats of 70 amino acids forming a compact domain of five α-helices^7,8. These domains form hydrophilic pores at the center of the protein and have the ability to transport Ca²⁺ from high concentrations to low concentrations in the microenvironment^9–11. ANXA5 is closely related to cardiovascular diseases, and ANXA5 is increased in peripheral blood, coronary artery and myocardium of cardiovascular diseases such as myocardial infarction¹², heart failure, atherosclerosis¹³ and hypertension, and promotes myocardial remodeling^13–15, consistent with earlier studies¹⁶. SDF-1-AnxA5 effectively provides cardio protection after myocardial infarction¹⁷. ANXA5 may be an important regulator of hypertensive myocardial hypertrophy, but follow-up studies are required to understand the specific regulatory mechanism.

The PI3K/AKT signaling pathway participating in cell migration, translation response and cell survival, cell metabolism, vascular homeostasis and thrombosis¹⁸. Study have found that IGFBP3 induced by high glucose promotes cardiomyocyte apoptosis via inhibits PTEN-IGF1R-PI3K-AKT pathway^19–21. Qingda granule attenuates AngII-induced cardiac hypertrophy and apoptosis and modulates the PI3K/AKT pathway²². Some scholars have found that inhibition of ANXA5 induces the activation of downstream PI3K/AKT signaling pathway²³, and can also inhibit DLBCL cell invasion²⁴. Therefore, it is not surprising that ANXA5 improves myocardial hypertrophy and cardiac function by regulates PI3K/AKT signaling pathway.

The present study, based on the previous work, we found that ANXA5 is dramatically up-regulated in hypertensive cardiac hypertrophy. To further investigate its mechanism effects, we first determined the expression level of ANXA5 in cardiac hypertrophy by bioinformatic analysis of GEO database. After that, the expression level of ANXA5 and the biological effects of knockdown ANXA5 were verified in hypertensive myocardial hypertrophy and ALD-induced H9C2 myocardial hypertrophy, and further explored whether ANXA5 affects myocardial hypertrophy and cardiac function by activating PI3K/AKT/Bcl-2 signaling pathway, so as to provide a new therapeutic strategy for hypertensive myocardial hypertrophy. The design of this study was showed in the flowchart (Supplementary file 1: Fig. S1).

Materials and methods

Collection and processing of GEO data

Gene expression profile data were obtained from the Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geoprofiles/) is a public database for storing and accessing microarray datasets and high-throughput genomics data. To identify ANXA5 expression in a variety of diseases of the heart, we downloaded multiple microarray datasets from the NCBI GEO database and Twelve were obtained after screening, that samples origin at from Rattus norvegicus, Mus musculus, Homo sapiens, respectively. The following table lists the basic details of these datasets (Supplementary file 1: Table S1). The data were processed using the R language (R-4.2.1). In addition, ANXA5 protein expression levels in different normal human tissues were verified by human protein Altas (HPA, https://www.proteinatlas.org/).

Cell line culture and transfection

The H9C2 cell line of rat cardiomyocytes was obtained from the Cell Bank of the Shanghai Academy of Biosciences (Seria-gnr5, Shanghai, China). Cells were cultured atmosphere in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37 °C in a 5% CO₂. Cells were passaged when they reached 80% confluency. To mimic an in vitro model for the model of an ALD-induced based on our previous study. For PI3K inhibitor studies, cells were incubated with LY294002 (10 μM, Med Chem Express MCE, Shanghai, China) for 1 h.

The ANXA5-siRNA and NC siRNA were purchased from Yile Biotechnology (Shanghai, China). A plasmid vector that expresses the full-length ANXA5 was engineered by GenePharma (Shanghai, China) enhance ANXA5 expression and designated as pcDNA3.1-ANXA5. H9C2 cells were transfected with siRNA and a plasmid overexpressing while plated onto 6-well plates using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, United States) according to the manufacturer’s instructions. The medium was changed 6 h after the transfection. Knockdown efficiency was confirmed by qRT-PCR and western blot, and the siRNA sequence creating the highest knockdown efficiency was selected for all subsequent experiments. Information on the siRNA sequence is listed in Supplementary file 1: Table S2.

Ethics statement and animals

This study was approved by the Ethics of Animal Experimentation of Guizhou Medical University Institutional Animal Care and Use Committee (No.1900572). All animal experiments were conducted in strict adherence to relevant ethical and regulatory guidelines, including the ARRIVE guidelines (https://arriveguidelines.org) and the Guide for the Care and Use of Guizhou Medical University Laboratory Animals. Male Wistar (WT) and Spontaneously Hypertensive Rats (SHR) rats (200–250 g, 9 weeks) were bought from the experimental Animals Center at Guizhou Medical University. All rats were housed in an environment with free access to food, water and temperature (23–24 °C) and relative humidity (50–60%) control. Body weight and blood pressure were measured regularly. The tail systolic blood pressure (SBP) and diastolic blood pressure (DBP) of conscious rats were assessed regularly using a noninvasive computer tail-cuff system (Kent scientific Corporation, CT, USA). Each rat was placed in a heated tube (38 °C) for 10–15 min, and then the temperature rise was monitored. Each rat was measured 3 times, and each group of rats was measured simultaneously. The SHR heart gradually increases in size due to the chronic elevated blood pressure, leading to left ventricular hypertrophy.The value of this parameter for that animal was determined by averaging these three values. Echocardiography was used to examine the development of myocardial hypertrophy in all age groups. Transthoracic two-dimensional and M-mode echocardiography was performed on Mylab50 (Esaote, Genova, Italy) using a high-resolution transducer (SL3116) with a frequency of 22MHz. To inhibit the expression of ANXA5 in vivo, 40μg ANXA5 siRNA or negative control (Jima Biotechnology, Shanghai, China) were mixed with INVI DNA RNA Transfection reagent (Invigentech, CA, USA) for 15 min at room temperature (RT) according to the manufacturer’s instructions, and then was injected into the tail vein every alternate day for a total of four times. Then the animals were euthanized by intraperitoneal injection with pentobarbital sodium (100 mg/kg). Experiment was terminated two days after the last treatment, blood and hearts were collected for subsequent analysis.

In vitro cell viability assay

Cell viability was determined by CCK8 assay (Dojindo, Kumamoto, Japan). Cells were treated with ALD (0, 0.1, 1, 10, 100 and 1000 μmol/L) and cultured for 24 h, 48 h and 72 h, respectively. Subsequently, all cells were incubated with 10μl of CCK8 solution at 37 °C for 3h. The optical density (OD) value of each sample at the wavelength of 450nm was measured using a microplate reader (Thermo Fisher Scientific, Massachusetts, USA). The assay was repeated three times and the cell viability was calculated as following: Cell viability = (OD treated—OD blank) / (OD control − OD blank) × 100%.

Cardiomyocyte injury assessed by LDH release

To determine the extent of Myocardial injury, a lactate dehydrogenase (LDH) detection kit (Nanjing Jiancheng, Nanjing, China) was used to measure the level of LDH released from rat serum and H9C2 cells samples, according to the experimental protocols.

Immunofuorescence

The H9C2 cells were fixed with 4% paraformaldehyde at room temperature for 10 min, permeabilized with 0.5% Triton X-100 for 5 min, blocked with 5% bovine serum albumin (BSA) in PBS for 30 min at room temperature. Slides were later incubated with anti-ANXA5 primary antibody overnight at 4 °C Then were washed three times in PBS and then incubated with Cy3-labled goat anti-rabbitt antibody for 1 h at room temperature, and washed with PBS and fixed in anti-fading medium containing DAPI. The analysis was carried out using a fluorescence microscope (Olympus, VS200, Tokyo, Japan), with images attained under five different fields.

Calcium ion fluorescence measurement

The H9C2 cells were treated with 5 μM Fluo-3AM (Solarbio, Beijing, China) and 0.03% (W/V) Pluronic F-127 in culture medium for 20 min at 37 °C. Next, after adding HBSS with 1% fetal serum further incubated for 40 min at 37 °C, the cells were washed with HBSS 3 times. Fluorescence intensity was determined at an excitation wavelength of 488 nm, an emission wavelength of 530 nm under a fluorescent microscope (Olympus, VS200, Tokyo, Japan).

Rhodamine-phalloidin staining

The H9C2 cell hypertrophy was assessed by a Rhodaminephalloidin Kit (Beyotime Biotechnology, JiangSu, China), treated for 1 h at room temperature, and then blocked with anti-fluorescence quencher containing DAPI (Solarbio, Beijing, China), according to the manufacturer’s instructions. Fluorescence signal was observed under an Olympus fluorescence microscope (Olympus, VS200, Tokyo, Japan), and the staining was analysed quantitatively.

HE, WGA and IHC staining

Five-µm-thick sections were used for hematoxylin and eosin (HE, Solarbio, Beijing, China), Wheat germ agglutinin (WGA, Invitrogen) and Immunohistochemistry (IHC) staining. For HE staining was performed to evaluate pathological and morphological changes of myocardial tissue.The cardiac cell size was examined using WGA staining.IHC staining was performed according to the protocol in the SP9000 IHC reagents kit.

Protein isolation and Western blot

Total protein was extracted from H9C2 cells and myocardial tissue of SHR rats. After protein concentration was detected, protein was analyzed by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to PVDF membrane (Millipore, Billerica, MA, USA). The membranes were blocked by 5% non-fat milk for 1 h and incubated overnight in primary antibody diluted in TBST at 4 °C. The secondary antibody (Neobiology, shanghai, China) was incubated for 1 h at room temperature. Finally, protein signals were detected by Tanon 5200 chemiluminescence and the gray values of the bands were measured using ImageJ software (ImageJ 1.47, NIH, USA). Primary antibodies used in this study: ANXA5 (11060-1-AP, Proteintech, USA), β-actin (GTX109639, Genetex, USA), Caspase3 (GTX110543, Genetex, USA), Bax (GTX109683, Genetex, USA), Bcl-2 (26593-1-AP, Proteintech, USA), PI3K (26593-1-AP, Proteintech, USA), Akt (10176-2-AP,Proteintech, USA), p-PI3K (1C1088, p85, Proteintech, USA), p-Akt (Ser473, Proteintech, USA), C-caspase3 (9496,,Cell Signaling Technology, Germany).

RNA extraction and quantitative real-time PCR

Total RNA extracted using Trizol reagent (invitrogen, Carlsbad, CA, United States) for real-time qRT-PCR analysis. RNA reverse transcription was performed according to the kit instructions (YESEN, Shanghai, China). Then, qRT-PCR was performed using Talent qPCR Premix SYBR Green Master Mix (YESEN, Shanghai, China).Relative levels of β-actin mRNA were used as a reference. PCR primers of each gene refer to Supplementary fle 1: Table S3.

Inflammatory factor detection

The interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-a) ELISA Kit were purchased from Shanghai Enzyme-linked Biotechnology Co. Ltd. (Shanghai, China) as per the manufacturer’s instructions.

Cell apoptosis detection

Cell apoptosis was investigated through flow cytometry to detect intracellular Annexin V-APC/7-AAD apoptosis detection kit (Kaiji Biotechnology, Hangzhou, China) and Caspase-3 activity detection (Beyotime; Haimen, Jiangsu, China), in adherence to manufacturers’ instructions.

Statistical analyses

This study used SPSS Statistics version 25.0 (IBM, USA), GraphPad Prism 8 and R software(R Foundation for Statistical Computing, Vienna, Austria, v4.2.1) to process all the data. Quantitative results were expressed as the means ± SEM. Comparison between the two groups was performed by t-test, and comparison between multiple groups was performed by one-way analysis of variance, followed by LSD post hoc analysis, P < 0.05 indicates significant difference.

Result

Expression level of ANXA5 in differernt CVDs based on GEO database

To investigate the expression of ANXA5 in various cardiac diseases. First, ANXA5 expression levels was analyzed in 13 GEO datasets, including 1 dog, 1 human, 3 mice and 8 rats (Supplementary fle 1: Table S4). ANXA5 expression was significantly upregulated in hypoxia, TAC models, hypertension and hypertensive myocardial hypertrophy cardiovascular diseases, while the expression difference was low and statistically insignificant in diabetes mellitus, low salt diet, and heart failure cardiovascular diseases (Fig. 1a–j). The prominent upregulation of ANXA5 continued during hypertension progression from compensation to decompension phases (Fig. 1k). In the hypertension group, the expression of ANXA5 increased significantly with the increase of age, compared with normal group (Fig. 1l,m). Interestingly, ANXA5 expression was only upregulated in pathological cardiac hypertrophy, especially hypertension, and was not significantly changed in physiological changes (Fig. 1n,o). Moreover, the content of ANXA5 was significantly higher in myocardium tissue (Supplementary file 1: Fig. S2a,i). Heat maps indicating the expression level of ANXA5 was higher and stable compared with other annexin family proteins, while remained unchanged in physiological myocardial hypertrophy (Supplementary fle 1: Fig. S2b–h). These results suggest that ANXA5 is highly expressed in myocardium by hypertensive cardiac hypertrophy.

Fig. 1.

ANXA5 gene were expressed in differernt CVDs from GEO database. (a–o) Analysis of the mRNA level expression of ANXA5 in differernt CVDs (Cardiomyocytes hypoxia (a), Transverse aortic constriction (b,c,m), Low-salt induced hypertensive (d), Dilated cardiomyopathy (e), Hypertension (f,g,k,l), Cardiac hypertrophy (h), Heart failure (i), Diabetic (j), Exercise (n, o)) from the GEO database. Results shown are mean ± SEM. An unpaired t-test was used (a–j, n). One-way analysis of variance (ANOVA), LSD’s test were used (k,l,m,o). * P < 0.05 and ** P < 0.01, ns, not signifcant.

ANXA5 expression is elevated in vitro and in vivo models

To verify that ANXA5 is overexpressed in hypertensive cardiac hypertrophy, in vitro model of ALD-induce myocardial cell hypertrophy was established. H9C2 cells were treated with ALD at different concentrations (0, 0.1, 1, 10, 100 and 1000 μmol/l) for different times (24, 48 and 72 h). ALD treatment of the H9C2 cells inhibitory effect was enhanced in a concentration- and time-dependent manner, the cell inhibition rate decreased about 26% with the ALD 10 μmol/l for 48h, this was used for subsequent experiments(Fig. 2a). ANXA5 mRNA and protein expression were significantly increased in ALD-induced H9C2 model compared with NC group by Western blot and qRT-PCR (Fig. 2b,c). ANXA5 expression was significantly higher than normal cells by immunofluorescence, and positive in cytoplasm and some nuclei (Fig. 2d). The SHR were used as a hypertension cardiac hypertrophy model in this study. Western blot also confirmed that the expression of ANXA5 was significantly up-regulated in SHR (Fig. 2e). These results reconfirm that ANXA5 is overexpressed in hypertensive cardiac hypertrophy.

Fig. 2.

ANXA5 expression is elevated in vitro and in vivo models. (a) The CCK8 assay was used to determine the ALD-induced H9C2 cell viability (n = 3). (b,c) The expression of ANXA5 mRNA (b) and protein (c) were used qRT-PCR and western blot in vitro model (n = 3, ALD 10μmol/l for 48 h). (d) Immunofluorescence microscopy showing specific ANXA5 expression (n = 3, ALD 10 μmol/l for 48h). (e) Western blot assay for the protein expression Of ANXA5 in SHR heart (n = 6). Results shown are mean ± SEM. an unpaired t-test was used. Scale bar: 10 μm. * P < 0.05 and ** P < 0.01.

Effects of knockdown ANXA5 on myocardial function and structure

We analyzed the biological function of ANXA5 in vitro and in vivo using siRNA knockdown. First, the ANXA5-siRNA #2 led to a slightly greater knockdown by western blot (Fig. 3a). Therefore, we chose ANXA5-siRNA #2 for follow-up studies. Subsequently, knockdown of ANXA5 inhibited ANXA5 mRNA level and protein expression in H9C2 cells (Fig. 3b,c). Compared with NC group, LDH activity and the mRNA expression levels of ANP and BNP were significantly increased in the ALD group, while after knockdown ANXA5 the LDH and the mRNA expression levels of ANP and BNP were significantly decreased compared with ALD and SiNC group in H9C2 cell (Fig. 3d,e).Overexpression of ANXA5 was also successfully induced by transfecting with a pcDNA3.1-ANXA5 expression vector in H9C2 cells(Supplementary fle 2: Fig. S1a). Compared with ALD group, the mRNA expression levels of ANP and BNP were significantly increased in the pcDNA3.1-ANXA5 group (Supplementary fle 2: Fig. S1b). Rhodamine-phosphorus staining showed that the surface area of H9C2 cardiomyocytes significantly increased after ALD induction, while the surface area of H9C2 cardiomyocytes significantly decreased after ANXA5 knockdown compared with ALD-induced (Fig. 3f).

Fig. 3.

Silencing ANXA5 alleviated cardiac hypertrophy in ALD-induced H9C2 cell. (a) The transfected efficiencies of ANXA5-knockdown were confirmed by western blot analyses. (b,c) western blot (c) and qRT-PCR (b) assay for ANXA5 following ANXA5-knockdown in H9C2 Cell. (d) The extent of cardiomyocyte injury was measured by LDH release. (e) The mRNA levels of cardiomyocyte hypertrophy biomarkers markers (ANP, BNP and β-MHC) were detected by qRT‐PCR. (f) Cardiomyocytes 48 h after ANXA5-siRNA transfection following indicated treatments were stained with rhodamine–phalloidin for ALD‐induced H9C2 cardiomyocyte hypertrophy. H9C2 cells were treated with ALD (10 μmol/l) for 48 h. Results shown are mean ± SEM. One-way analysis of variance (ANOVA), LSD’s test were used. n = 3, Scale bar: 20 μm. * P < 0.05 and ** P < 0.01.

Next, the Western blot and IHC staining results showed that compared with the WT group, ANXA5 protein expression was increased in the SHR group; Compared with the SHR group, ANXA5 protein expression level was obviously inhibited in the Si-ANXA5 group(Fig. 4a). There was no significant changes in heart rate and body weight among the groups (Fig. 4b,c). Blood pressure increased gradually with age of SHR, and it is not significant trend after knockdown ANXA5 (Fig. 4d). Compared with WT group, left ventricular wall thickness(IVSD) signnificantly increased and ejection fraction (LVEF%) significantly decreased in SHR group, Conversely, the Si-ANXA5 group decreased IVSD and increased LVEF% as compared to SHR group (Fig. 4e). Concomitantly, Si-ANXA5 groups exhibited a reduced hypertrophic response, as evidenced by decreased IVSD, HW/BW, LDH and mRNA levels of ANP, and BNP compared with those of the SHR and Si-NC group (Fig. 4f–h). HE staining showed that the volume of myocardial cells increased, the arrangement was disordered, myocardial interstitial proliferation and a few inflammatory cells were observed in SHR group, while Si-ANXA5 group was significantly improved compared with SHR group. WGA staining showed that surface areas of cardiomyocytes were increased in SHR group compared to WT group, but knockdown of ANXA5 significantly decreased the surface areas which were enlarged by SHR (Fig. 4i). In conclusion, knockdown of ANXA5 can improve ALD induced cardiomyocyte hypertrophy and SHR myocardial hypertrophy.

Fig. 4.

Effects of knockdown ANXA5 on myocardial function and structure in SHR. (a) Western blotting assay for ANXA5 following ANXA5-knockdown in SHR heart. (b–d) The heart rate (b), weight (c), and blood pressure (d) of the SHR were regularly monitored. e Cardiac structures and functions were evaluated by echocardiography. (f) Heart-weight-to-body-weight ratio (HW/BW) of cardiomyocyte were quantitatively analyzed. (g) The extent of cardiomyocyte injury was measured by LDH release. (h) The mRNA levels of hypertrophic biomarkers including ANP and BNP were assess by qRT‐PCR. (i) HE-stained sections, IHC and WGA sections were shown. Results shown are mean ± SEM. One-way analysis of variance (ANOVA), LSD’s test were used. n = 6, Scale bar: 50 μm, 100 μm. *P < 0.05 and ** P < 0.01.

ANXA5 knockdown decrease apoptosis and inflammatory responses of cardiomyocytes by Ca²⁺ influx

Fluo-3am staining showed that ALD-induced H9C2 challenge dramatically increased calcium fluorescence intensity as compared to control group; while compared with the ALD group, the calcium fluorescence intensity significantly decreased in Si-ANXA5 group, and there was no significant difference in Si-NC group (Fig. 5). ANXA5 is positively correlated with Ca²⁺ levels during cardiomyocyte hypertrophy. Western blot assay showed that the expression levels of Bax and Caspase3 protein increased and the ratio of Bax/Bcl-2 decreased in ALD induced H9C2 cells compared with control group, and compared with ALD group, protein expression levels of Bax and Caspase3 decreased in Si-ANXA5 group (Fig. 6a). Consistent with in vitro results, Bax and Caspase3 protein expression significantly decreased, and myocardial apoptosis was significantly reduced in Si-ANXA5 group (Fig. 6b). Flow cytometry assays and Caspase3 activity assay proved that ALD treatment of H9C2 cells led to significantly increase the number of apoptotic cells, ANXA5 knockdown could inhibit cell apoptosis (Fig. 6c, Supplementary fle 2: Fig. S2a,b, S3a,b).The results in vitro and in vivo confirmed that the expression of pro-inflammatory factors TNF-α and IL-1β was significantly increased in the model group, and the expression level of pro-inflammatory factors decreased after knockdown ANXA5 (Fig. 6d). These results suggest that ANXA5 knockdown inhibits Ca²⁺ release, and plays an anti-apoptotic and anti-inflammatory role in cardiomyocyte hypertrophy.

Fig. 5.

Knockdown ANXA5 inhibited calcium influx. (a,b) The calcium levels were analyzed by fluorescence microscopy after green fluorescence probe (Fluo-3 AM) staining (a) and quantitative analysis (b). H9C2 cells were treated with ALD (10 μmol/l) for 48 h. Results shown are mean ± SEM. One-way analysis of variance (ANOVA), LSD’s test were used. n = 3, Scale bar: 20 μm. * P < 0.05 and ** P < 0.01.

Fig. 6.

The knockdown of ANXA5 inhibits apoptosis and inflammatory response in Vitro and in Vivo. (a,b) The expression of Bax, Bcl-2, Caspase3, C-caspase3 and β-actin protein in vitro (a) and in Vivo (b). (c) Flow cytometry to detect apoptosis rate. (d) Serum TNF-α and IL-1β level was measured by ELISA. H9C2 cells were treated with ALD (10 μmol/l) for 48h. Results shown are mean ± SEM. One-way analysis of variance (ANOVA), LSD’s test were used. n = 3 cells, 6 animals. * P < 0.05 and ** P < 0.01.

ANXA5 regulate apoptosis of cardiomyocytes by activating the PI3K/AKT signaling pathway

In vitro, the protein phosphorylation of PI3K and AKT decreased in ALD group, and ANXA5 knockdown significantly upregulated the protein phosphorylation of PI3K and AKT, indicating that PI3K/AKT signaling activity was affected by ANXA5 (Fig. 7a). The results of in vivo experiments were consistent with in vitro results. (Fig. 7b). To further determine whether the protective effect of ANXA5 silencing on cardiomyocytes is related to PI3K/AKT signaling pathway. LY2940002(LY), an inhibitor of PI3K/AKT, was applied to treat ALD induced cardiomyocytes. The results showed that compared with ALD group, ANXA5 protein expression levels in other groups were reduced, the order of the ANXA5 protein expression level was the ALD + LY group > ALD + Si-ANXA5 group > ALD + Si-ANXA5 + LY group. The order of Bax/Bcl-2 and Caspase3 protein expression level from highest to lowest was: the ALD + LY group, the ALD + Si-ANXA5 + LY group, the ALD + Si-ANXA5 group. The phosphorylation levels of PI3K and AKT protein obviously lower in the ALD + LY group and higher in ALD + Si-ANXA5 group than in the ALD group, and they were significantly decreased in the ALD + LY group and ALD + Si-ANXA5 + LY group, compared with ALD + Si-ANXA5 group (Fig. 7c). Rhodamine-phosphorus staining and caspase 3 activity assays showed that the surface area of H9C2 cardiomyocytes significantly increased in ALD group compared to NC group, but knockdown of ANXA5 and LY treatment significantly decreased the surface areas which were enlarged by ALD-induced H9C2. Meanwhile, cotreatment of LY and si-ANXA5 could partially counteract these effects (Supplementary fle 2: Fig. S4a,b).The present results suggest that knockdown ANXA5 protects myocardium by activating PI3K/AKT signaling, and the PI3K inhibitor LY294002 partially reversed the protective effects.

Fig. 7.

ANXA5 knockdown suppressed cell apoptotic through regulation of the activity of PI3K/AKT signaling pathway. (a,b) Western blotting assay for PI3K, p-PI3K, AKT, p-AKT and β-actin in H9C2 cell (a) and SHR heart (b). (c) LY2940002 reversed the protective effects of ANXA5 knockdown in ALD-induced H9C2 cell. H9C2 cells were treated with ALD (10 μmol/l) for 48h. Results shown are mean ± SEM. One-way analysis of variance (ANOVA), LSD’s test were used. n = 3 cells, 6 animals. * P < 0.05 and ** P < 0.01.

Discussion

Hypertension is one of the major public problems in cardiovascular disease and a major risk factor for morbidity and mortality from other cardiovascular diseases, doubling its prevalence²⁵. Apoptosis, hypertrophy and dysfunction of cardiomyocytes in long-term hypertension lead to changes in cardiac structure and function, which in turn lead to diastolic or systolic dysfunction²⁶. Early intervention of myocardial hypertrophy and delay of ventricular remodeling is particularly important. Since hypertensive myocardial hypertrophy can cause stress loading, resulting in enhances the membrane permeability that leads to excessive leaks out of LDH²⁷, and atrial ANP and ventricular BNP are up-regulated, as a major stimulators of cardiomyocyte stress release²⁸, HW/BW ratio and echocardiography are commonly used to quantify myocardial function an structure²⁶. Knockdown ANXA5 reduces impairment of myocardial function and abnormalities in cardiac structure, potently reduced the levels of LDH, obviously inhibited cardiomyocyte hypertrophy, as indicated by cardiomyocyte cross-sectional area, HW/BW ratio, and the expression of ANP and BNP, are consistent with Ravassa study⁷. In addition, we did not observe changes in weight, heart rate and blood pressure of rats, which may be due to the the relatively short duration of the treatment.

As a member of Annexin gene family, ANXA5 is a broad range of functions in controlling cell apoptosis, growth, development, signal transduction¹². In this study are consistent with previous studies, ANXA5 was one of the most abundant annexins in rat and human myocardium²⁹. ANXA5 is up-regulated in a variety of cardiovascular diseases^12,14, we showed that the expression level of ANXA5 was closely related to hypertrophic myocardium caused by hypertension and was positively correlated with the severity and duration of myocardial hypertrophy, while ANXA5 expression had no significant change in physiological hypertrophy.ANXA5 has a high affinity for PS in a calcium-dependent manner, forming a two-dimensional array with negatively charged PS⁹. In vitro experiments, a synergistic effect between ANXA5 expression and calcium influx in ALD induced H9C2 cardiomyocytes³⁰, ANXA5 knockdown significantly decreased intracellular Ca²⁺ levels, decreased Bax/Bcl-2 ratio and apoptosis was inhibited. The mechanism of this effect may be due to the increased affinity of ANXA5 to Ca²⁺, which drives the protein to reach a fully bound state to be able to migrate to the membrane in response to the increase of intracellular calcium ions³¹. When ANXA5 translocates and externalizes in cardiomyocytes, the externalization corresponds to the pro-apoptotic effect of ANXA5 related to Ca²⁺ channel activity^30,32, which makes ANXA5 excessively and strategically redistribute to sarcomere structure and intercalated disk in tissues where pathophysiology occurs, which may also change the processing of Ca²⁺ by heart, resulting in cytoplasmic protein loss, energetic changes and contractile dysfunction in some cardiomyocytes⁷. Therefore, the strategic redistribution of ANXA5 can influence Ca²⁺ levels and apoptosis.

Benzothiazine derivatives (K201 or JTV519) protect myocardium by binding to ANXA5, inhibiting its calcium channel activity and blocking the movement of Ca^2+7,33. These results are not necessarily due to cellular ANXA5 regulation, but may be due to exogenous ANXA5 action³⁴. These results in myocardium indicate that ANXA5, calcium levels and apoptosis interact, mainly ANXA5 dependent calcium regulation and then affect apoptosis. Moreover, the beneficial effects of ANXA5 are associated with a reduction in inflammation^12,35. Our founding knockdown ANXA5 decreased proinflammatory factors and inflammatory response in cardiomyocyte hypertrophy.

Many studies have demonstrated that ANXA5 induces apoptosis and inflammation by regulating multiple signaling pathways³⁶. Overexpression of ANXA5 affected phosphorylation of Akt and p38 MAPK, further inhibiting cell anti-apoptotic activity³⁷. Activation of PI3K/AKT pathway can protect the heart from ischemia reperfusion injury¹⁹. Similar to our results, downregulation of ANXA5 could inhibit apoptosis and improve myocardial injury by activate PI3K/AKT signaling pathway, while inhibition of PI3K/AKT signaling pathway reversed the protective effect of downregulation of ANXA5 on myocardium. In addition, previous studies by the team found that that calpain 1, 2 affects p38 expression by regulating ANXA5, while JNK and ERK 1, 2 protein expression levels did not effect, and induce cardiomyocyte apoptosis. In general, ANXA5-mediated myocardial hypertrophy and apoptosis are mainly related to p38MAPK and PI3K/AKT signaling pathways (Fig. 8).

Fig. 8.

The schematic describing the proposed signal pathway mechanism by which knockdown ANXA5 attenuates Hypertension myocardial apoptosis and cardiac hypertrophy.

This study has several limitations. First, although knockdown of ANXA5 had profound effective in the therapy of myocardial hypertrophy on cardiac hypertrophy, it would be more convincing if the same results were obtained by overexpression of ANXA5. Second, protects hypertension cardiac hypertrophy and by knockdown ANXA5 was confirmed in SHR and ALD-induced H9C2 cardiac hypertrophy, these results would be more convincing if also validated in additional other models.

Conclution

Knockdown of ANXA5 suppressed cardiac apoptosis, reduce myocardial hypertrophy and improve cardiac function by activating with the PI3K/AKT/Bcl-2 pathway in hypertensive myocardial hypertrophy. The present study provides novel evidence for ANXA5 in hypertensive myocardial hypertrophy and may also have potential significance as a therapeutic target.

Electronic Supplementary Material

Below is the link to the electronic supplementary material.

Author contributions

ZLN and CHJ contributed equally to this work.ZLN, LS and ZB conceived and designed the study, performed the experiments, and collected and analyzed of the data and wrote the manuscript.CHJ performed the experiments and analyzed and interpreted the data.YY, LCY and LXY participated analyzed of the data and performed experiments.ZYY, HD and HQF participated analyzed of the GEO data.

Funding

This work was supported by the National Natural Science Foundation of China (No.81960315, No.82360354); the Guizhou Science and Technology Department (qiankehejichu-ZK[2022]yiban359, qiankehejichu-ZK[2024]zhongdian045); the Guizhou Medical University Affiliated Hospital (2021-GMHCT-009); the Guizhou Provincial Key Laboratory of Pathogenesis and Drug Research of Common Chronic Diseases (KF-2022-1).

Data availability

The datasets during the current study are available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.

Ethics approval and consent to participate

All animal experiments were approved by the Animal Ethics Committee of the Guizhou Medical University (Permit No.1900572).

Consent for publication

Not applicable.