Sestrin2 levels in patients with anxiety and depression myocardial infarction was up-regulated and suppressed inflammation and ferroptosis by LKB1-mediated AMPK activation

ABSTRACT

Although great progress has been made in the diagnosis and treatment of acute myocardial infarction (AMI) in recent years, its morbidity and mortality are still relatively high. In this study, we explain that the function of Sestrin2 gene in Anxiety and Depression Myocardial infarction and its possible mechanism. 26 patients with Anxiety and Depression Myocardial infarction (ADMI) and 26 normal volunteers were collected from our hospital. All mice anaesthetized using 50 mg/kg of pentobarbital sodium and the left anterior descending arteries (LAD) were ligated to induce myocardial infarction. H9c2 cells were stimulated with 5% oxygen (O2) and 5% carbon dioxide (CO2) and 90% N2 for 24 h. The serum expression of Sestrin2 in patients with ADMI was up-regulated. Sestrin2 gene up-regulation reduced collagen I/II and KEAP1 mRNA expressions, and increased GPX4 and Nrf2 mRNA expressions in vitro model of AMI. Down-regulation of Sestrin2 increased collagen I/II and KEAP1 mRNA expressions, and decreased GPX4 and Nrf2 mRNA expressions in vitro model of AMI. These data confirmed that Sestrin2 reduced inflammation and ferroptosis in model of ADMI by LKB1-mediated AMPK activation. This infers that Sestrin2 is potential target to be used in the treatment of premature AMI.

KEYWORDS:

• Sestrin2
• ferroptosis
• Anxiety
• myocardial infarction
• LKB1

Introduction

Acute myocardial infarction (AMI) refers to myocardial necrosis caused by acute persistent coronary ischemia and hypoxia, which is one of the common critical diseases in emergency department (1). In recent years, the level of AMI disease management, drug, and interventional therapy has been continuously improved, making the incidence rate of people over 45 years old decrease year by year (2). However, the incidence rate and mortality rate of AMI among people under 45 years old are increasing year by year (3).

The incidence of cardiovascular disease continues to rise, with nearly 300 million patients. In addition, the mortality rate of cardiovascular diseases is higher than that of other diseases such as tumors, ranking first (4). Among them, the overall mortality rate of AMI is increasing year by year (4). Percutaneous coronary intervention (PCI) is currently the preferred method for the treatment of AMI, which can significantly increase the survival rate of patients and improve the long-term quality of life (5). With the development of the bio-psychological-social medical model, researchers found that patients with AMI may have mental problems during coronary intervention or coronary artery bypass surgery, which makes the mortality rate of AMI higher than that of other patients with cardiovascular diseases and the prognosis worse (6,7). Therefore, the psychological problems of patients with AMI have also become a research hotspot in related fields (8).

Cardiac remodeling after AMI involves inflammatory response (9). Inflammatory cell phagocytizes dead cells and disrupted matrix, and the removal of dead cells triggers an anti-inflammatory effect (10). During this process, inflammatory cells synthesize IL-1, ROS, and chemokines through signaling pathways such as TGFβ and NF-κB, which promote the inflammatory process. Simultaneously, fibroblasts proliferate and transdifferentiate into myofibroblasts, which release large amounts of extracellular matrix proteins to maintain the structural integrity of the infarct site (11). With the apoptosis of granulation tissue cells and the formation of collagen network crosslinks, the scar gradually matures to complete the cardiac remodeling after AMI (12). However, the failure of inflammation inhibition and excessive fibrosis during repair may cause adverse cardiac remodeling, including adverse events such as heart failure, atrial fibrillation, and cardiac rupture (13).

There is a lot of evidence that AMPK plays an important role in the normal development of the heart and myocardial metabolism, as well as a protective role in myocardial ischemia/reperfusion injury. The activation of AMPK suppresses pathological cardiac hypertrophy (14,15). The loss of AMPK causes pathological cardiac hypertrophy and AMI under pressure overload (16).

As a stress-induced protein, Sestrin2 regulates oxidative stress response by activating AMPK, protects cells from stress-induced apoptosis, and maintains stable organ function (17,18). According to relevant literature, exercise can effectively improve skeletal muscle function and alleviate the loss of skeletal muscle mass, because it can promote the expression of Sestrin2 in skeletal muscle and activate AMPK and PGC-1α to improve skeletal muscle mass (19,20). In this study, we explain that the function of Sestrin2 gene in Anxiety and Depression Myocardial infarction and its possible mechanism.

Materials and methods

Clinical research model

26 patients with Anxiety and Depression Myocardial infarction (ADMI) and 26 normal volunteers were collected from our hospital. Serum was collected and saved at −80 °C. This experiment was performed in accordance with the Guide for the Care and Use of US National Institutes of Health. Experimental protocols were approved by our hospital. Each cancer patient provided their written informed consent for study participation.

Animals model

Mice were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health. All aspects of the animal care and experimental protocols were approved by the Animal Ethics Committee of our hospital.

All mice anaesthetized using 50 mg/kg of pentobarbital sodium and the left anterior descending arteries (LAD) were ligated to induce myocardial infarction. Mice were ventilated by a rodent ventilator (Shanghai Alcott Biotech Co., Shanghai, China), then LAD was ligated by an 8.0 suture followed by the thoracotomy. At 2 weeks of modeling, mice were sacrificed and executed other experiment. Left ventricular internal diameter, left ventricular ejection fraction, left ventricular fractional shortening and left ventricular stroke volume were obtained from Nillar pressure-volume system (MPVS-400).

Vitro experimental design

H9c2 rat cardiomyocytes (Cell Bank of the Chinese Academy of Sciences, Shanghai, China) were grown in Dulbecco’s modified Eagle medium (DMEM, Gibco, Grand Island, NY, United States), with 5% fetal bovine serum (FBS, Gibco, Grand Island, NY, United States), in a humidified 5% CO2 incubator at 37°C. HSMECs were performed transfections using Lipofectamine 2000 (Thermo Fisher Scientific). Plasmid or siRNAs were transfected in the serum-free and antibioticfree media. After 48 h of transfection, H9c2 cells were stimulated with 5% oxygen (O2) and 5% carbon dioxide (CO2) and 90% N2 for 24 h.

Histological analysis and immunohistochemistry

Colon tissue samples were fixed in 4% paraformaldehyde, and executed histological analysis and immunohistochemistry according to references (21,22).

Real-time PCR

Total RNAs were isolated with RNA isolator total RNA extraction reagent (Takara) and cDNA was synthesized using PrimeScipt RT Master Mix (Takara). qPCR was performed with the ABI Prism 7500 sequence detection system according to the Prime-ScriptTM RT detection kit. Relative levels of the sample mRNA expression were calculated and expressed as 2-DDCt.

ELISA kits

Tissue or cell samples in each group were collected at 2000 g for 10 min at 4°C. IL-1β, SOD, GSH, ROS production, MDA, GSH-px kits were used to measure the cytokine levels.

Western blot

Western blot was performed as previously described (23). Membranes were incubated with Sestrin2, LKB1, p-AMPK, AMPK, GPX4 and β-Actin (BS6007MH, 1:5000, Bioworld Technology, Inc.) at 4°C overnight. The membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (sc-2004 or sc-2005, 1:5000, Santa Cruz, USA) for 1 h at 37°C after washing with TBST for 15 min. Protein was measured using an enhanced chemiluminescence system (ECL, Beyotime) and analyzed using an Image Lab 3.0 (Bio-Rad Laboratories, Inc.).

Coimmunoprecipitation assay

The ChIP-qPCR experiment was performed as previously described (24). Cells were treated with RIP lysis buffer, supernatant was enriched with antibody- or rabbit IgG-conjugated protein A/G magnetic beads in IP buffer supplemented with RNase inhibitors and incubated overnight at 4°C.

Statistical analysis

Data were represented as mean ± standard error of the mean (SEM). Student’s t test and one way ANOVA test were used for statistical analyses of the data. A p-value less than 0.05 is considered with significant difference.

Results

Sestrin2 expression levels in patients with anxiety and depression myocardial infarction

This work examined the levels of Sestrin2 in patients with ADMI. The serum expression of Sestrin2 in patients with ADMI was up-regulated (Figure 1a). However, in patients with ADMI, the expression of Sestrin2 in patients with I-II was higher than that of patients with III or IV (Figure 1b). Meanwhile, the serum expression of Sestrin2 was negative correlation with collagen I/II in patients with ADMI (Figure 1c,d). The receiver operating characteristic (ROC) curve was constructed to assess diagnostic value of Sestrin2 in patients with ADMI (Figure 1e). Then, at mice model of AMI, the serum expression of Sestrin2 mRNA was increased at 15 min of induction AMI, compared with control sham group (Figure 1f). At the expression of serum Sestrin2 mRNA in 30 min of induction mice AMI was lower than that of 15 min of induction mice AMI (Figure 1f). At 15 min of induction AMI, Sestrin2 protein expression was increased in heat tissue, compared with Control sham group (Figure 1g,h). So, Sestrin2 was up-regulated in the early disease of ADMI.

Figure 1. Sestrin2 expression levels in patients with anxiety and depression myocardial infarction.

Sestrin2 mRNA expression (a and b), serum Sestrin2 was negative correlation with collagen I/II in patients (c and d), receiver operating characteristic (e) curve in patients;

Sestrin2 mRNA and protein expression (f, g and h) in mice model.

^##p < 0.01 compared with normal volunteers group or patients with I/II group.

Sestrin2 gene reduced fibrosis in vitro model of AMI

Next, we up-regulated the Sestrin2 expression in vitro model of AMI using Sestrin2 plasmid (Figure 2a). Sestrin2 gene up-regulation reduced collagen I/II and KEAP1 mRNA expressions, and increased GPX4 and Nrf2 mRNA expressions in vitro model of AMI (Figure 2b–f). Then, we conducted si-Sestrin2 mimics to reduce Sestrin2 expression in vitro model of AMI (Figure 2g). Then down-regulation of Sestrin2 increased collagen I/II and KEAP1 mRNA expressions, and decreased GPX4 and Nrf2 mRNA expressions in vitro model of AMI (Figure 2h–i).

Figure 2. Sestrin2 gene reduced fibrosis in vitro model of AMI.

Sestrin2 mRNA expression (a), collagen I/II (b and c), GPX4 (d), KEAP1 (e), Nrf2 (f) mRNA expressions in vitro model by Sestrin2 gene;

Sestrin2 mRNA expression (g), collagen I/II (h and i), GPX4 (j), KEAP1 (k), Nrf2 (l) mRNA expressions in vitro model by si-Sestrin2 mimics.

^##p < 0.01 compared with control group or si-nc group.

Sestrin2 gene reduced inflammation and oxidative stress in vitro model of AMI

Next, Sestrin2 gene up-regulation reduced IL-1β levels, inhibited ROS production and MDA levels, increased SOD and GSH levels, and promoted GSH-px levels (Figure 3a–f). However, down-regulation of Sestrin2 increased IL-1β levels, promoted ROS production and MDA levels, decreased SOD and GSH levels, and reduced GSH-px levels (Figure 3g–i).

Figure 3. Sestrin2 gene reduced inflammation and oxidative stress in vitro model of AMI.

IL-1β levels (a), ROS production (b), MDA (c), SOD (d), GSH (e), GSH-px (f) levels in vitro model by Sestrin2 gene;

IL-1β levels (g), ROS production (h), MDA (i), SOD (j), GSH (k), GSH-px (l) levels in vitro model by si-Sestrin2 mimics.

^##p < 0.01 compared with control group or si-nc group.

Sestrin2 gene reduced ferroptosis in vitro model of AMI

This work further explored the potential function of Sestrin2 on ferroptosis in vitro model of AMI. Sestrin2 gene up-regulation promoted cell viability in vitro model, compared with control group (Figure 4a). Down-regulation of Sestrin2 reduced cell viability in vitro model, compared with control group (Figure 4a). Conversely, Sestrin2 gene up-regulation reduced LDH activity level and IL-1α level, increased JC-1 disaggregation and calcien-AM/CoCl2 levels, and inhibited iron concentration in vitro model of AMI (Figure 4b–g). Down-regulation of Sestrin2 increased LDH activity level and IL-1α level, reduced JC-1 disaggregation and calcien-AM/CoCl2 levels, and advanced iron concentration in vitro model of AMI (Figure 4b-g). Furthermore, Sestrin2 gene up-regulation increased GPX4 protein expression, and down-regulation of Sestrin2 suppressed GPX4 protein expression in vitro model of AMI (Figure 4h-i).

Figure 4. Sestrin2 gene reduced ferroptosis in vitro model of AMI.

Cell viability (a), LDH activity level (b), IL-1α level (c), JC-1 disaggregation (d), calcien-AM/CoCl2 (e), PI (f), iron concentration (g), GPX4 protein expression (h and i).

^##p < 0.01 compared with control group or si-nc group.

The inhibition of Sestrin2 gene expanded AMI via the activation of inflammation and oxidative stress in mice model of AMI

Subsequently, the experiment evaluated that the effects of Sestrin2 gene in inflammation and oxidative stress in mice model of AMI using sh-Sestrin2 virus. Sh-Sestrin2 virus increased heart weight/body weight and infarct size (HE and MASS staining), heightened CK and LDH activity levels, reduced left ventricular ejection fraction and left ventricular fractional shortening, enhanced left ventricular internal diameter, and inhibited left ventricular stroke volume in mice model of AMI (Figure 5a-h). Afterwards, sh-Sestrin2 virus also increased collagen I/III and TGF-β1 mRNA expression levels in heart tissue of AMI model (Figure 5i-k).

Figure 5. The inhibition of Sestrin2 gene expanded AMI in mice model of AMI.

Heart weight/body weight (a), CK and LDH activity levels (b and c), infarct size (HE and MASS staining, d), left ventricular ejection fraction (e), left ventricular fractional shortening (f), left ventricular internal diameter (g), left ventricular stroke volume (h), collagen I/II (i and j), TGF-β1 mRNA expression levels (k).

^##p < 0.01 compared with model+nc group.

Next, sh-Sestrin2 virus enhanced IL-1β levels in serum and heart tissue of AMI model (Figure 6a). Sh-Sestrin2 virus increased MDA level, and reduced SOD, GSH and GSH-px levels in heart tissue of AMI model (Figure 6b–e). Sh-Sestrin2 virus induced KEAP1 mRNA expression, and suppressed Nrf2 mRNA expression in heart tissue of AMI model (Figure 6f-g). So, these results showed that the inhibition of Sestrin2 gene reduced inflammation and oxidative stress to expanded AMI.

Figure 6. The inhibition of Sestrin2 gene expanded AMI via the activation of inflammation and oxidative stress in mice model of AMI.

IL-1β levels (a), MDA (b), SOD (c), GSH (d) and GSH-px (e) levels, KEAP1 and Nrf2 mRNA expression (f and g).

^##p < 0.01 compared with model+nc group.

LKB1 was one target spot for Sestrin2 gene in mode of AMI

Thereafter, the study further investigated the mechanism of Sestrin2 on AMI using Microarray analysis. LKB1 was one target spot for Sestrin2 gene in vitro mode of AMI (Figure 7a–c). Sestrin2 gene up-regulation induced Sestrin2, LKB1 and p-AMPK protein expressions in vitro model (Figure 7d). Scanning confocal microscopy showed that Sestrin2 gene up-regulation increased Sestrin2 and LKB1 expressions in vitro mode of AMI (Figure 8a). Down-regulation of Sestrin2 suppressed Sestrin2, LKB1 and p-AMPK protein expressions in vitro model (Figure 8b). IP also showed that Sestrin2 protein catenated LKB1 protein (Figure 8c). Sh-Sestrin2 virus suppressed GPX4, LKB1 and p-AMPK protein expressions in heart tissue of mice AMI (Figure 8d).

Figure 7. LKB1 was one target spot for Sestrin2 gene in mode of AMI.

Heart map (a), Volcano map (b), KEGG terms (c), Sestrin2/LKB1/p-AMPK protein expressions (d).

^##p < 0.01 compared with control group.

Figure 8. Sestrin2 protein catenated LKB1 protein.

Scanning confocal microscopy (a), Sestrin2/LKB1/p-AMPK protein expressions (b) in vitro model, Sestrin2 protein catenated LKB1 protein (IP, c), GPX4/LKB1/p-AMPK protein expressions (d) in mice model.

^##p < 0.01 compared with control or model+nc group.

Next, LKB1 inhibitor (Pim1/AKK1-IN-1, 300 nM) reduced the effects of Sestrin2 on GPX4, LKB1 and p-AMPK protein expressions, increased ROS-induced oxidative stress, and ferroptosis in vitro model of AMI (Figure 9).

Figure 9. The inhibition of LKB1 reduced the effects of Sestrin2 in vitro model of AMI.

GPX4/LKB1/p-AMPK protein expressions (a), ROS production (b), MDA (c), SOD (d), GSH (e), GSH-px (f) levels, collagen I/II (g and h), TGF-β1 mRNA expression levels (i), cell viability (j), LDH activity level (k), JC-1 disaggregation (l), calcien-AM/CoCl2 (m), PI (n), iron concentration (o).

LKB1 i, LKB1 inhibitor, ##p < 0.01 compared with control group, **p < 0.01 compared with Sestrin2 group.

It was found that LKB1 Agonist (Gomisin J, 500 nM) also inhibited the effects of si-Sestrin2 on GPX4, LKB1 and p-AMPK protein expressions, increased ROS-induced oxidative stress, and ferroptosis in vitro model of AMI (Figure 10). So, these results showed that LKB1 was one target spot for Sestrin2 gene on ROS-induced oxidative stress and ferroptosis in mode of AMI.

Figure 10. The activation of LKB1 reduced the effects of si-Sestrin2 in vitro model of AMI.

GPX4/LKB1/p-AMPK protein expressions (a), ROS production (b), MDA (c), SOD (d), GSH (e), GSH-px (f) levels, collagen I/II (g and h), TGF-β1 mRNA expression levels (i), cell viability (j), LDH activity level (k), JC-1 disaggregation (l), calcien-AM/CoCl2 (m), PI (n), iron concentration (o).

LKB1, LKB1 agonist, ^##p < 0.01 compared with si-nc group, **p < 0.01 compared with si-Sestrin2 group.

Methylation control Sestrin2 stability

Moreover, Sestrin2 gene has multiple suspicious methylation modification sites near the stop codon (Figure 11a). m6A antibody suppressed Sestrin2 mRNA enrichment level in vitro model of AMI by si-METTL3 (Figure 11b). Si-METTL3 reduced the stability of Sestrin2 mRNA in vitro model of AMI (Figure 11c). Three m6A sites in the 3′-untranslated region (UTR) of Sestrin2 and m6A significantly enriched at sites 1, 2 and 3 (Figure 11d). Si- METTL3 reduced luciferase activity level by wild-type (WT) and 2 sites of Sestrin2 (Figure 11e). The m6A enrichment at sites 1 and 3 was decreased Sestrin2 levels (Figures 11f–g). These results revealed that METTL3 methylation control Sestrin2 stability

Figure 11. Methylation control Sestrin2 stability.

m6A modification site of Sestrin2 (a), METTL3-mediated Sestrin2 m6A modifications (b and c), the position of m6A motifs within Sestrin2 transcript sequence (d), luciferase reporter activity level (e), m6A levels of Sestrin2 (f and g), **p < 0.01 compared with vector or negative or IgG group.

Discussion

AMI has become one of the diseases with the highest fatality rate in China and even in the world, with gradually younger disease age and increasing overall incidence rate year by year (25). This disease often leads to excessive psychological stress in patients (26,27). Some studies suggest that the psychological and emotional changes of patients with AMI will affect the disease outcome to a certain extent. Among them, most patients will have different degrees of anxiety and depression after the onset of the disease (28,29). Here, we found that the expression of serum Sestrin2 in patients with ADMI was up-regulated. Quan et al. showed that Sestrin2 prevents myocardial infarction to ischemic insults (30). Thus, these results suggest that Sestrin2 was up-regulated in the early disease of ADMI. So, Sestrin2 might demonstrate protection for ADMI. In this study, we only analyzed that 26 patients with ADMI, these samples were insufficient for this experiment. We will analyzed more samples in further experiment.

Cardiomyocyte death caused by AMI can trigger a series of cardiac pathophysiological reactions, involving pro-inflammatory response and extracellular matrix degradation, as well as anti-inflammatory responses and neo-scar formation (31). However, strong inflammatory response caused by tissue damage can promote excessive fibrosis of the heart (32). Adversarial cardiac remodeling can eventually develop into adverse cardiovascular events such as heart failure (33). The research on the regulation mechanism of inflammation after AMI is of great significance for the prevention and treatment of poor prognosis (33). We demonstrated that the inhibition of Sestrin2 gene expanded AMI via the activation of inflammation and oxidative stress in mice model of AMI. Ren et al. indicated that Sestrin2 reduced cardiac inflammatory response and oxidative stress during ischemia and reperfusion (34). Therefore, our results revealed that Sestrin2 protected ADMI via the inhibition of inflammatory response and oxidative stress.

Ferroptosis is a new type of iron-dependent programmed cell death, which is caused by the increase of fatty acid lipid peroxidation and the accumulation of reactive oxygen species induced by cellular iron overload (35). Ferroptosis is closely related to the occurrence and development of many major diseases such as tumor, neurodegenerative disease, metabolic disease, and aging disease (36,37). Many diseases, such as cardiomyopathy, AMI, heart failure, coronary atherosclerosis, myocardial ischemia-reperfusion injury, diabetes cardiomyopathy, are involved in ferroptosis (38). We revealed that Sestrin2 gene reduced inflammation and oxidative stress, inhibited ferroptosis in vitro model of AMI. Li et al. indicated that Sestrin2 protected against ferroptosis in model of sepsis (39). Therefore, Sestrin2 might contribute to the inhibition of ferroptosis in ADMI.

Myocardial ischemia is a common disease in which myocardial cells are hypoxic due to reduced blood flow, which in turn causes severe myocardial damage (40). With high morbidity and mortality, it is now one of the leading causes of death worldwide (15,41). Studies have shown that autophagy is involved in the occurrence and development of ischemic heart disease (15,41). Autophagy refers to a metabolic process in which substrates are transported to lysosomes by autophagosomes and then degraded to maintain the homeostasis of the body. Its classic pathway is mainly AMPK signaling pathway (38). AMPK is one of the sensors that maintain cellular energy metabolism. The myocardial hypoxic-ischemic phase is due to decreased intracellular ATP production (42,43). The imbalance of myocardial energy first activates AMPK (44). LKB1 protein is the upstream kinase of AMPK, which is mainly distributed in the nucleus and plays a role in regulating cellular energy metabolism (45). Under the conditions of nutritional deficiency and hypoxia, LKB1 negatively regulates mTOR by activating AMPK and inhibiting the activity of stimulatory proteins that activate mTOR, and further regulates cell cycle, growth and proliferation (46). In this study, LKB1 was one target spot for Sestrin2 gene in mode of AMI to control AMPK activity. METTL3 methylation control Sestrin2 stability. Morrison et al. demonstrated that Sestrin2 induced LKB1/AMPK activation in the ischemic heart (47). These findings suggested that Sestrin2 induced LKB1/AMPK activation to reduce ferroptosis in model of ADMI. In this study, we mainly analyzed the effects of Sestrin2 in model of ADMI using mice model or H9c2 cell, these results were only verified in mice model or H9c2 cell, we will verified these results in clinical samples.

In conclusion, Sestrin2 reduced inflammation and ferroptosis in model of ADMI by LKB1-mediated AMPK activation. This study provided a new mechanism for understanding the Sestrin2 presented ADMI, and indicated novel target for AMI treatment. This infers that Sestrin2 is potential target to be used in the treatment of premature AMI.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

Zhejiang Provincial Traditional Chinese Medicine Science and Technology Project (grant. 2021ZQ070).