ANXA1sp Protects against Sepsis-Induced Myocardial Injury by Inhibiting Ferroptosis-Induced Cardiomyocyte Death via SIRT3-Mediated p53 Deacetylation

Sepsis-induced myocardial injury (SIMI), a common complication of sepsis, may cause significant mortality. Ferroptosis, a cell death associated with oxidative stress and inflammation, has been identified to be involved in SIMI. This study sought to investigate the role of ANXA1 small peptide (ANXA1sp) in SIMI pathogenesis. In this study, the mouse cardiomyocytes (H9C2 cells) were stimulated with lipopolysaccharide (LPS) to imitate SIMI in vitro. It was shown that ANXA1sp treatment substantially abated LPS-triggered H9C2 cell death and excessive secretion of proinflammatory cytokines (TNF-α, IL-1β, and IL-6). ANXA1sp pretreatment also reversed the increase of ROS and MDA generation as well as the decrease of SOD and GSH activity in H9C2 cells caused by LPS treatment. In addition, ANXA1sp considerably eliminated LPS-caused H9C2 cell ferroptosis, as revealed by the suppression of iron accumulation and the increase in GPX4 and FTH1 expression. Furthermore, the ameliorative effects of ANXA1sp on LPS-induced H9C2 cell damage could be partially abolished by erastin, a ferroptosis agonist. ANXA1sp enhanced SIRT3 expression in LPS-challenged H9C2 cells, thereby promoting p53 deacetylation. SIRT3 knockdown diminished ANXA1sp-mediated alleviation of cell death, inflammation, oxidative stress, and ferroptosis of LPS-treated H9C2 cells. Our study demonstrated that ANXA1sp is protected against LPS-induced cardiomyocyte damage by inhibiting ferroptosis-induced cell death via SIRT3-dependent p53 deacetylation, suggesting that ANXA1sp may be a potent therapeutic agent for SIMI.


Introduction
Sepsis, a severe systemic inflammatory response syndrome featured with multiorgan dysfunction [1], mainly arises from excessive host response to infection [2]. Approximately, half of the sepsis patients develop myocardial injury [3], known as sepsis-induced myocardial injury (SIMI), which is associated with increased mortality [4]. In addition, SIMI, in turn, contributes to multiorgan dysfunction in sepsis [5]. As a result of a poor understanding of SIMI pathogenesis, there are no specific interventions for SIMI.
At the cellular level, cardiomyocyte death is a major manifestation of SIMI [6]. As a kind of iron-dependentprogrammed cell death distinguished from apoptosis, pyroptosis, necroptosis, or autophagy-dependent cell death [7], ferroptosis is featured with strong oxidative stress as well as accumulation of lipid peroxides and iron and possesses no typical morphological features [8]. Studies have shown that ferroptosis is engaged in sepsis occurrence and development [9]. Recent research has suggested that ferroptosis is crucial for sepsis-induced organ injury, such as SIMI. [10]. In addition, ferroptosis inhibitor can also improve SIMI and reduces cardiomyocyte death [11]. Consequently, inhibition of cardiomyocyte ferroptosis is an effective approach to protect against SIMI.
Annexin A1 (ANXA1), a glucocorticoid-regulated protein widely expressed in human tissues and cells [12], has been demonstrated to participate in a number of cellular processes, including cell proliferation, differentiation, and apoptosis [13]. As evidenced by prior studies, ANXA1 exerts potent antioxidative and anti-inflammatory effects [14]. Moreover, ANXA1 also plays an advantageous role in cardiovascular diseases [15]. ANXA1 small peptide (ANXA1sp), a bioactive peptide derived from ANXA1, exhibits biological effects similar to those of ANXA1 [16], hinting that ANXA1sp also plays a cardioprotective role in cardiovascular diseases. However, whether ANXA1sp can alleviate SIMI through inhibiting ferroptosis has not been identified yet.
Herein, we aimed to explore whether ANXA1sp alleviates LPS-induced cardiomyocyte damage and elucidates the underlying mechanism, hoping to find a novel therapeutic agent for SIMI treatment.

Cell Survival.
Cell survival was evaluated via CCK-8 and LDH assays. In brief, H9C2 cells were seeded into a 96-well plate (2 × 10 4 cells/well) and exposed to LPS for 24 h. Then, the CCK-8 reagent was added (10 μL/well). H9C2 cells were cultured with CCK-8 reagent at 37°C for another 2 h. The absorbance was measured with a microplate reader at 450 nm. LDH release was evaluated via an LDH assay kit (Beyotime, China) as per the standard instruction.

Intracellular ROS Assay.
To detect ROS content in H9C2 cells, a Reactive Oxygen Species Assay Kit (Beyo-time, China) was applied. In brief, H9C2 cells were plated into 6-well plates (1 × 10 5 cells/well) for 24 h. Then, H9C2 cells were incubated with a DCFH-DA probe, washed, harvested, and resuspended in ice-cold PBS. Finally, the ROS fluorescence was measured via flow cytometry (FACSCalibur, USA).

Measurement of Oxidative Stress-Related Indicators.
Briefly, H9C2 cells were lysed in PBS via sonication and centrifuged at 15000× g for 5 min at 4°C to acquire cell supernatants for further measurement. Malondialdehyde (MDA) level, superoxide dismutase (SOD) activity, and glutathione (GSH) level were measured via commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing City, China) as per standard instructions.
2.7. Iron Assay. Total iron content was measured with an iron assay kit (Sigma-Aldrich). Briefly, H9C2 cells were lysed in the iron assay buffer and centrifuged at 16,000 g for 10 min. Then, 5 μL iron reducer was added to 100 μL cell lysate for total iron (Fe 2+ and Fe 3+ ) assessment. Next, 100 μL iron probe was added. 1 h later, the absorbance was detected at 593 nm via spectrophotometry.

Western
Blotting. Proteins were extracted from H9C2 cells, separated by SDS-PAGE, and transferred onto PVDF membranes. Then, the membranes were blocked with 5% skimmed milk at room temperature. Next, the membranes were incubated with primary antibodies (anti- , and anti-GAPDH antibody (1 : 10000 dilution; ab9485; Abcam)) at 4°C overnight and HRP-conjugated secondary antibodies (1 : 5000 dilution; ab6721 and ab6728; Abcam) at room temperature for 1 h. An ECL chemiluminescence detection system was adopted to visualize the target bands.
2.9. Coimmunoprecipitation (Co-IP). Co-IP was performed using indicated antibodies and IgG (Invitrogen) as per standard instructions. Briefly, H9C2 cell lysates were incubated with antibody-conjugated beads at 4°C for 2 h. Then, the beads were collected, washed extensively, and boiled in an SDS loading buffer. Western blot was applied to study the immunoprecipitated proteins.
2.10. Statistical Analysis. GraphPad 6.0 software was utilized for statistical analysis. All data were displayed as mean ± SD. Comparisons were performed with Student's t-test (2 groups) and one-way analysis of variance (ANOVA) (>2 groups). Differences with p < 0:05 were deemed statistically significant.

ANXA1sp Inhibits LPS-Caused Oxidative Stress and
Ferroptosis in H9C2 Cardiomyocytes. ANXA1sp has been proven to protect against oxidative injury [18]. As illustrated in Figures 2(a)-2(d), LPS caused a substantial increase in ROS generation and MDA level but a significant decrease in SOD activity and GSH (Figure 2(d)) level, which was substantially reversed by ANXA1sp pretreatment, suggesting that ANXA1sp relieved LPS-induced oxidative stress damage in H9C2 cells.
In order to evaluate whether ANXA1sp affects LPSinduced ferroptosis of cardiomyocytes, iron accumulation and ferroptosis-related factors (GPX4 and FTH1) were assessed. It was shown that LPS increased iron content and decreased GPX4 and FTH1 protein levels in H9C2 cells; however, ANXA1sp partly eliminated such effects, indicating the inhibitory effect of ANXA1sp on LPS-induced ferroptosis in H9C2 cells (Figures 2(e) and 2(f)). Taken together, our data revealed that ANXA1sp repressed oxidative stress as well as ferroptosis in LPS-challenged H9C2 cardiomyocytes.

Erastin Eliminates the Protective Effect of ANXA1sp
against LPS-Induced H9C2 Cell Death, Inflammation, and Oxidative Stress. In order to confirm whether ANXA1sp relieves LPS-induced H9C2 cell damage by inhibiting ferroptosis, erastin (10 μM), a ferroptosis agonist, was applied to induce H9C2 cell ferroptosis 8 h before LPS treatment. Results of the CCK-8 assay exhibited that erastin eliminated ANXA1sp-mediated improvement of the cell viability of LPS-triggered H9C2 cells (Figure 3(a)). LDH assay further affirmed that the suppressive effect of ANXA1sp on LPSinduced H9C2 death was abated by erastin (Figure 3(b)).
As a deacetylase, SIRT3 has been identified as a regulator of p53 acetylation [25]. Therefore, we hypothesized that a similar mechanism might be involved in SIMI. To investigate whether ANXA1sp could affect p53 deacetylation via SIRT3, the binding of SIRT3 with p53 was first assessed by coimmunoprecipitation. As shown in Figure 4(c), SIRT3 could coimmunoprecipitate with p53 in H9C2 cells. Then, SIRT3 was knocked down in H9C2 cells with transfection efficiency assessed by western blotting (Figure 4(d)). Moreover, ANXA1sp pretreatment significantly reduced the acetylated p53 level in H9C2 cells, while SIRT3 knockdown substantially reversed such an effect (Figure 4(e)). Thus, it could be concluded that ANXA1sp coffered p53 deacetylation . Data were presented as mean ± SD. All statistical analyses were performed using Student's t-test or one-way ANOVA. * p < 0:05; * * p < 0:01; * * * p < 0:001.

Discussion
The effects of ANXA1sp on the suppression of inflammatory response [26] and oxidative injury [18] have been substantiated in previous research. Moreover, one study has demonstrated that ANXA1sp could also alleviate myocardial inflammation [27], implying that ANXA1sp plays a protective role in cardiac disorders. However, whether ANXA1sp could improve SIMI and the underlying mechanisms involved in LPS-caused H9C2 cardiomyocyte damage was still unclear. Our findings demonstrated that ANXA1sp inhibited ferroptosis-induced cell damage in LPS-challenged H9C2 cardiomyocytes via SIRT3-dependent p53 deacetylation, indicating that ANXA1sp could improve SIMI in vitro.
As a reversible form of cardiac depression, SIMI is featured with extravagant inflammatory response, oxidative stress, and cardiomyocyte death [28,29]. In this study, Data were presented as mean ± SD. All statistical analyses were performed using Student's t-test or one-way ANOVA. * p < 0:05; * * p < 0:01; * * * p < 0:001.  There is a growing body of evidence that ferroptosis has a role in a number of cardiovascular conditions, including heart failure, myocardial infarction, stroke, and arrhythmia [30], suggesting ferroptosis inhibition could be a potential therapeutic approach for cardiovascular diseases. Additionally, ferroptosis plays a significant role in controlling oxidative stress and inflammatory responses in cardiovascular diseases [31]. Also, ferroptosis plays a critical role in sepsis-associated organ injury, including SIMI [10]. Taking into account the anti-inflammatory and antioxidant effect of ANXA1sp, it was conjectured that the effects of ANXA1sp on LPS-caused H9C2 cardiomyocyte damage may involve ferroptosis. As expected, ANXA1sp partly relieved LPS-triggered H9C2 cell ferroptosis. Additionally, erastin, a ferroptosis agonist, could eliminate the protective effects of ANXA1sp on LPS-induced H9C2 cardiomyocyte death, inflammation, and oxidative stress, indicating that ANXA1sp-mediated protection against LPS-challenged H9C2 cells was achieved by inhibiting H9C2 cell ferroptosis.
SIRT3, a soluble protein located in the mitochondria, is a histone deacetylase that regulates cell metabolism through the deacetylation of a variety of protein substrates [25]. Previous research has demonstrated that SIRT3 expression considerably decreased in myocardial tissues of mice with LPSinduced myocardial injury, while restoration of SIRT3 expression could prevent LPS-induced myocardial injury [32]. The antioxidative and anti-inflammation effects on SIRT3 in LPS-challenged cardiomyocytes have also been demonstrated [33]. Intriguingly, Suliman et al. found that ANXA1sp could upregulate SIRT3 expression in the mitochondria of kidney tubular cells [18]. In line with the above studies, SIRT3 expression markedly declined in LPStriggered H9C2 cells; however, ANXA1sp treatment distinctly restored SIRT3 expression. Furthermore, SIRT3 silence could eliminate the impact of ANXA1sp pretreatment on LPS-triggered H9C2 cardiomyocyte death, inflammation, oxidative stress, and ferroptosis.
The p53 protein is widely recognized as a "guardian of the genome" for its essential role in maintaining genomic stability [34]. As a critical regulator of cellular processes, p53 is engaged in a variety of cellular processes, such as cell differentiation, senescence, proliferation, apoptosis, ferroptosis, and autophagy [35]. Former studies have proven that p53 deacetylation alleviates sepsis-induced organ damage [36,37]. Also, p53 deacetylation could inhibit ferroptosis [24]. Additionally, there is evidence that SIRT3 activates p53 deacetylation in H9C2 cells [38]. Consistently, it was found that SIRT3 could coimmunoprecipitate with p53 in H9C2 cells. Moreover, ANXA1sp pretreatment significantly attenuated the LPS-induced increase of p53 expression and acetylation in H9C2 cells, which was substantially reversed by SIRT3 silencing. Taken together, ANXA1sp contributed to p53 deacetylation in LPS-treated H9C2 cells by enhancing SIRT3 expression.

Conclusion
In summary, this study uncovered that ANXA1sp could inhibit LPS-induced cytotoxicity in H9C2 cardiomyocytes. Moreover, it was also demonstrated that ANXA1spmediated alleviation of LPS-triggered H9C2 cardiomyocyte death, inflammation, oxidative stress, and ferroptosis was closely related to SIRT3-dependent p53 deacetylation. These findings indicated that ANXA1sp might be a novel therapeutic agent for the treatment of SIMI. However, the protective effects of ANXA1sp against SIMI need further validation in vivo.

Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
The authors declare that they have no conflicts of interest.