Ferroptosis exacerbates myocardial ischemia-reperfusion injury via worsening oxidative stress and inflammatory responses: the role of Ferritin/SLC7A11/GPX-4 signaling pathway

doi:10.21203/rs.3.rs-5312142/v1

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Ferroptosis exacerbates myocardial ischemia-reperfusion injury via worsening oxidative stress and inflammatory responses: the role of Ferritin/SLC7A11/GPX-4 signaling pathway

https://doi.org/10.21203/rs.3.rs-5312142/v1

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Ferroptosis is closely linked to pathological processes in cardiomyocytes. However, the role of ferroptosis in myocardial ischemia-reperfusion injury (MI/RI) and its underlying mechanisms are unknown. Transitional accumulation of iron ions, as well as oxidative stress and lipid peroxidation production were found in the MI/RI model. These were significantly inhibited by an iron death inhibitor. In MI/RI-induced tissue damage and inflammatory responses, inhibition of ferroptosis reduced cardiac infarct area and resisted inflammation. Mechanistic investigations show that inhibition of ferroptosis via the Ferritin/SLC7A11/GPX-4 axis can target MI/RI mitigation, highlighting the potential of inhibiting ferroptosis as a novel strategy for therapeutic of MI/RI.

Coronary heart disease

Myocardial ischemia/reperfusion injury

Ferroptosis

Inflammatory response

Mechanism

Myocardial ischemia/reperfusion injury (MI/RI) is the reestablishment of blood flow after occlusion of coronary arteries in patients with ischemic heart disease [1]. MI/RI exacerbates a series of injurious changes such as myocardial dysfunction and cardiomyocyte damage during ischemia, and in severe cases, it can lead to the occurrence of sudden death in patients [2]. Although the positive outcome of MI/RI after pharmacological intervention has been demonstrated, the search for new risk factors and therapeutic targets and the development of new therapeutic strategies are still necessary [3, 4].

MI/RI occurs through a complex mechanism with activation of multiple signaling molecules and pathways in the organism. Therein, the cellular oxidative stress caused by the accumulation of reactive oxygen species (ROS) is considered to be a primary cause of MI/RI [5]. Attributable to MI/RI-induced myocardial cell death may paradoxically impair cardiac function due to oxidative stress injury induced by excessive ROS accumulation, rapid pH correction, and intracellular Ca²⁺ imbalance [6, 7]. Furthermore, part of the mechanism is driven by the inflammatory response through multiple interaction pathways [8].

Ferroptosis is an iron-dependent form of regulated cell death in which the central event is the accumulation of lipid hydroperoxides to lethal levels induced by ROS and other relevant factors, leading to oxidative damage to the biofilm systems and inflammation of the organism [9]. It involves a unique combination of biological processes and pathophysiology, including elevated Fe²⁺, malondialdehyde (MDA), ROS and decreased antioxidant enzymes such as glutathione peroxidase 4 (GPX-4) and glutathione (GSH), as a non-apoptotic mode of cell death [10]. In addition, inflammatory pathways and their complex interactions are key mediators in the MI/RI process, which may be treatable by intervening in pathways associated with ferroptosis [8, 11]. Previous reports have shown that ferroptosis exhibits a high correlation with the development of various cardiac diseases such as myocardial infarction, MI/RI, and heart failure [12–14]. The occurrence and development of ferroptosis are closely related to the pathological process of cardiomyocytes, and ferroptosis is involved in the pathogenesis of MI/RI [15, 16]. GPX-4 mediates iron prolapse to regulate MIRI and is involved in the pathogenesis of MI/RI [17–19]. GPX-4 can regulate ferroptosis and oxidative stress by catalyzing the conversion of GSH to oxidative glutathione (GSSG) [16, 20]. Therefore, understanding the mechanisms and influencing factors of the occurrence of core events between ferroptosis and the MI/RI's might help the development of new therapeutic targets. For example, myocardial injury in rats with acute myocardial ischemia can be effectively ameliorated by controlling ROS levels in vivo [21]. Activation of the Nuclear factor E2-related factor 2 (Nrf-2)/ Ferroportin1 (FPN1) pathway by the antioxidant drug Piceatannol (PCT) can effectively inhibit the accumulation of Fe²⁺ and lipid peroxidation products in mouse cardiac cells and thus alleviate MI/RI [1].

Thus, in this study, we i) investigated the mechanisms and interactions of ferroptosis and MI/RI core events (tissue damage, oxidative stress, and inflammatory response) at the whole animal level; ii) The possibility and mechanism of iron death inhibitor Fer-1 regulating the occurrence of iron death in cardiomyocytes and thereby alleviating MI/RI were explored at the molecular level.

2.1. Experimental animals

SPF-grade male C57BL/6J mice (6–8 weeks old, body weight 22 ± 2 g) were selected and housed in a strictly controlled environment (temperature: 22 ± 3°C, humidity: 55 ± 15%, light/dark: 12/12), with unrestricted food and water supply. All procedures were in accordance with the Guide for the the National Institutes of Health (NIH), and all were approved by the ethics committee of Beichen hospital of Nankai university (IACUC of AMMS-04-2023-016).

2.2. MI/RI mice modelling

Before surgery, mice were anaesthetized by intraperitoneal injection of 1.2% tribromoethanol (0.2 ml/10 g, Aladdin, Shanghai, China), and were connected to a mouse ventilator for ventilation after tracheal intubation. The tidal volume of mice was maintained at 250–300 mL/min, and the respiratory rate was controlled at 110–130 breaths/min. Infrared body heaters were used to maintain the body temperature of mice at 36.5–37℃.

Model group (M): the left anterior descending coronary artery (LAD) was ligated for 30 min, followed by I/R-induced reperfusion for 2 h.

Treatment group (Z): 30 mg/kg of Ferrostatin-1 (Fer-1), an inhibitor of cellular ferroptosis, was intraperitoneally injected daily on the basis of the model group. mice were kept without dietary restriction for 21 d. Mice were treated with the same surgery as in the model group.

Sham group (J): the same operation without ligation of LAD.

2.3. TTC-Evans blue staining

The collected mice heart tissue samples were snap-frozen at -20°C for 30 min and sectioned to a thickness of 3 mm. Thereafter, they were stained with 2,3,5-triphenyltetrazolium (TTC, Sigma, Shanghai, China) and Evans blue dye (Aladdin, Shanghai, China). Myocardial necrosis was then observed and photographed with a Leica microscope (Wetzlar, Germany) and analyzed as a percentage of all areas using ImageJ software.

2.4. Hematoxylin-Eosin (H&E) staining

The collected mice heart tissues were fixed in 4% paraformaldehyde solution at 4°C for 24 h. Subsequently, the mice heart tissues were subjected to graded concentration ethanol dehydration (40 min each), dehydrated, clarified, dewaxed, and embedding procedures, and then sectioned at a set tissue section thickness of 3 µm. Then, hematoxylin and eosin (H&E) pathological staining was performed. The stained myocardial tissue sections were examined and imaged using a microscope (DP26, OLYMPUS).

2.5. Transmission electron microscopy (TEM) observation

Transmission electron microscopy (TEM) was performed to observe the pathological changes of heart tissues in each group. Briefly, for the collected mice heart tissues, they were washed three times with PBS and fixed in 2.5% glutaraldehyde solution at 4°C overnight. Subsequently, the washed tissues were subjected to 1% osmium tetroxide fixation, gradient ethanol dehydration, paraffin embedding and sectioning procedures to obtain ultrathin tissue sections (70–90 nm). After staining, changes in tissue structure were observed under a transmission electron microscope (Hitachi H-7000, Japan).

2.6. Detection of inflammatory factors

Determination of IL-1 (#ab197742, Abcam), IL-6 (#ab222503, Abcam), TNF-α (#ab208348, Abcam) and IFN-γ (#ab282874, Abcam) in mice serum were carried out using mouse enzyme linked immunosorbent assay (ELISA) kits, and all operations were performed in strict accordance with the manufacturer's instructions.

2.7. Lipid Peroxidation (MDA) and T-GSH/GSSG measurement

The MDA, GSH, T-GSH/GSSG assay kit (#ab118970, Abcam, USA; #S0053, Beyotime, China) was used to detect the MDA, T-GSH/GSSG content in mice heart tissues. All operations were performed in strict accordance with the manufacturer's instructions.

2.8. Reactive oxygen species (ROS)

The level of ROS in brain tissues was analyzed by using ROS assay kit (#S0033M, Beyotime, China) with 2',7'-Dichlorodihydrofluorescein diacetate (DCFH-DA) as the probe. All operations were performed in strict accordance with the instructions of the supplier.

2.9. Detection of iron content

Iron content in mice heart tissues was determined using an iron content assay kit (#ab83366, Abcam, USA), and all operations were performed in strict accordance with the instructions of the supplier.

2.10. Quantitative reverse transcription PCR (qRT-PCR)

Total RNA from mouse heart tissues was extracted with Trizol (Invitrogen) and reverse transcribed into cDNA using a cDNA synthesis kit (A5004, Promega, USA). Gene expression was detected using the Step one + Real-Time PCR system (Applied Biosystems, USA) and the amount of RNA was calculated by the comparative threshold cycling method. All primers were customized by Genscript as shown in Table S1. GAPDH was used as an internal standard for mRNA expression analysis.

2.11. Western blot (WB)

Total proteins were extracted using protease inhibitor-containing RIPA (Beyotime, China), and protein concentration was measured using a total protein concentration kit (BCA, Solarbio, China). Proteins were separated using 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene difluoride (PVDF) membrane. Appropriate dilutions of primary antibody were added, overnight at 4°C, and then incubated with HRP for 1 h, according to the instructions (manufacturer and dilutions were given in Table S2). The enhanced chemiluminescence kit (ECL, Thermo Fisher Scientific, USA) was used for color development, and a fully automated chemiluminescence image analysis system (Tanon 5200, China) was used to visualize the immunoreactive bands. Finally, the target protein content was quantified using Gelpro32 software.

2.12. Statistical Analyses

Without emphasis, all animal-related experiments were set up in six parallel groups. The results were expressed as mean ± standard deviation (SD). Comparisons between groups were made using one-way ANOVA and post hoc Tukey's multiple comparison test using IBM SPSS 25.0 software, p-value < 0.05 was considered to be significantly different.

3.1. Iron accumulation in MI/RI mice

Iron accumulation, lipid peroxidation, and ROS have been identified as key factors in the induction of ferroptosis, which is thought to be a regulated cellular necrosis caused primarily by iron-dependent oxidative damage [22]. Herein, we found that MI/RI modelling resulted in a significant elevation of iron content in the cardiac tissue (p < 0.01), which was 1.63 times higher than in the sham-operated group. In addition, by intraperitoneal injection of Fer-1, an iron death inhibitor, the iron level in the heart tissue of MI/RI mice was significantly reduced (p < 0.05).

3.2. Ferroptosis-mediated oxidative stress in MI/RI mice

Oxidative stress is considered to be an important pathological change characterizing the process of I/R injury, as well as an important factor inducing the production of ferroptosis [23]. In this study, we tested the oxide stress levels (key biomarkers: ROS, MDA, GSH, T-GSH and GSSG) in MI/IR mice under ferroptosis mediated (Fig. 2, Fig. S1). The effect of MI/RI-induced oxidative stress on cardiac tissues of mice was assessed by ROS content testing (Fig. 2a). Compared with the sham group (J), modelling resulted in a significant elevation of ROS levels (p < 0.05), which was significantly alleviated by treatment with the ferroptosis inhibitor Fer-1 (p < 0.01). This suggested that inhibition of ferroptosis can effectively alleviate the level of oxidative stress in model mice. The MDA content was also significantly reduced after Fer-1 treatment (p < 0.01), suggesting that inhibition of ferroptosis can attenuate tissue peroxidative damage (Fig. 2b). Detection of GSH/GSSG showed (Fig. 2c-d, Fig. S1) that MI/RI resulted in the conversion of GSH to GSSG in mice, and Fer-1 treatment inhibited this conversion, suggesting that inhibition of ferroptosis could alleviate redox metabolic abnormalities caused by MI/RI [24]. These results indicated that ferroptosis led to a disturbance of redox metabolism during MI/RI, mediating the generation of oxidative stress.

3.3. Role of ferroptosis in MI/RI modelling-guided tissue damage

The role of ferroptosis in MI/RI-induced cardiac tissue damage was assessed by HE and TTC staining of tissues and cellular ultrastructure observations [25]. Although the organ coefficients did not show significant differences between the groups (Fig. S2). The TEM observations showed that MI/RI disrupted cellular mitochondrial structure and reduced cristae density in mouse myocardial tissues compared to controls (Fig. 3a-b). However, Fer-1 treatment significantly reduced mitochondrial damage in cardiomyocytes of MI/RI model mice (Fig. 3c). The H&E staining showed (Fig. 3d) that cardiomyocytes in the sham group of mice were evenly distributed and fibers were regularly arranged. In contrast, MI/RI model mice had disrupted myocardial structure, disorganized fibers and significantly fewer cardiomyocytes. In addition, Fer-1 intervention significantly alleviated the pathological changes in cardiac tissue of MI/RI mice. To verify the effect of ferroptosis on cardiac tissue damage caused by MI/RI, the focal infarcted area was accessed by TTC staining. As shown in Fig. 3e and f, the infarct area was significantly larger in the model (M) and treatment (Z) groups compared with sham group (p < 0.05). The Fer-1 intervention treatment resulted in a reduction of the infarct area in the treatment group compared with the model group (p < 0.001). These results demonstrate that ferroptosis exacerbates cardiomyocyte damage during MI/RI and worsens infarction.

3.4. Ferroptosis-mediated inflammatory response in MI/RI mice

To further the proinflammatory effects of iron death chemotaxis induced MI/RI in vivo, we examined serum levels of inflammatory cytokines in mice. We investigated the effect of MI/RI modeling on the inflammatory response in mice (Fig. 4), and the levels of IL-1β, IL-6, TNF-α, and IFN-γ were significantly increased in the model mice (p < 0.001). In addition, the levels of IL-1β, IL-6, TNF-α, and IFN-γ in mouse heart tissue were decreased by Fer-1 (Fig. 4, p < 0.001). These results suggested that iron death induced inflammation by promoting the release of pro-inflammatory cytokines, and Fer-1 inhibition of iron death can reduce inflammation-mediated cardiac tissue damage.

3.5. Molecular mechanisms of ferroptosis regulation to MI/RI

In recent years, glutathione peroxidase 4 (GPX-4), transferrin receptor 1 (TFR-1), solute carrier family 7 member 11 (SLC7A11), and ferritin are the key genes involved in ferroptosis, which are associated with a number of key processes, such as metabolic disorders of iron ions, lipid peroxidation, and oxidative stress [10, 26]. Hereto, western blot (WB) and RT-PCR were employed to reveal the roles of these key proteins in the regulation of MI/RI by ferroptosis. The results of WB tests showed that the expression of GPX-4, SLC7A11, and Ferritin proteins were down-regulated in MI/RI model mice compared to the sham-operated group (Fig. 5a, 5c-e). Meanwhile, Fer-1 intervention ameliorated the downregulation of these proteins in MI/RI model mice. In addition, changes in TFR-1 showed the opposite trend (Fig. 5b, 5e). Furthermore, the expression of the corresponding mRNAs was examined. As shown in the results of Figs. 5f and h-i, MI/RI modelling resulted in significant expression down-regulation of GPX-4, SLC7A11, and Ferritin proteins in the model mice (p < 0.001), which was significantly attenuated by Fer-1 intervention (p < 0.001). Similarly, changes in TFR-1 expression showed the opposite trend (Fig. 5g, p < 0.001). These results demonstrated that ferroptosis can exacerbate MI/RI through inhibition of the GPX-4/SLC7A11/Ferritin pathway, as well as activation of TFR-1, and that modulation of the corresponding pathways may ameliorate the adverse consequences of MI/RI.

Myocardial ischemia/reperfusion injury (MI/RI) is a type of cardiovascular diseases (CVD) that involves complex pathophysiological mechanisms such as oxidative stress, calcium overload, inflammation, and mitochondrial dysfunction [27, 28]. Currently, percutaneous coronary intervention (PCI) and surgical coronary artery bypass grafting (CABG) are the most advanced techniques for hemodilution in acute myocardial infarction [29]. However, they have been shown to be ineffective in targeting reperfusion injury. Hence, revealing the pathophysiological mechanisms of MI/RI from new perspectives has been a windfall for exploring potential diagnostic and therapeutic strategies. In recent years, evidences from investigators sustain the notion that ferroptosis plays an essential role in the pathophysiology of MI/RI, emphasizing the modulation of ferroptosis as an emerging target for the therapy of MI/RI.

In the present study, mice exhibited Fe²⁺ accumulation, lipid peroxidation, and oxidative stress after MI/RI modelling. Elevated ferric ions, MDA, and ROS were all important features of ferroptosis, suggesting that ferroptosis occurs in the cardiac tissue of MI/RI mice [30, 31]. The level of oxidative stress in the cardiac tissues of model mice could be significantly suppressed by the intervention of Fer-1, an ferroptosis inhibitor (p < 0.05). Pathological indices showed that the intervention of Fer-1 could effectively reduce the cardiac infarct area ratio (p < 0.001). In addition, examination of pro-inflammatory factors showed that significant up-regulation of IL-1β, IL-6, TNF-α and IFN-γ levels occurred in MI/RI mice after modelling. Inhibition of ferroptosis significantly controlled the inflammatory response in the organism of model mice (p < 0.001). The results suggested that ferroptosis plays an important role in the pathophysiological occurrence of oxidative stress, inflammatory response and tissue damage during MI/RI. Therefore, inhibition of ferroptosis might play a protective role against MI/RI.

The mechanism of MI/RI is unclear, and multiple signaling pathways or molecular interactions are thought to promote MI/RI [32]. Ferroptosis has been reported to play an important role in ischemia-reperfusion-induced MI/RI [33, 34], which is consistent with the findings of the present study. Ferroptosis is dependent on the accumulation of iron, lipid ROS, and loss of activity of the lipid repair enzyme GPX-4. Therefore, inhibition of ferroptosis upregulates enhanced antioxidant expression can alleviate MI/RI. The researchers found that inhibition of iron death prevented acute kidney injury (AKI) [35, 36]. Herein, Fer-1, as a specific inhibitor of ferroptosis, ameliorated ischemia-reperfusion injury, consistent with the report of Li et al [33]. This was attributed to the improved accumulation of iron, lipid ROS, and lipid peroxidation in model mice after Fer-1 action.

In addition, the restoration of blood flow further increases tissue damage through the activation of pathways such as sudden oxidative stress. Damaged cardiomyocytes, extracellular matrix, and released substances act as danger signals, known as danger associated molecular patterns (DAMPs) [37]. DAMPs activate a range of inflammatory mediators through the innate immune system, thereby activating the inflammatory response. Thus, inhibition of the inflammatory response can be effective in alleviating MI/RI [38]. Given that the inflammatory response plays an essential role in MI/RI, we investigated the effect of ferroptosis in MI/RI-induced inflammatory response. Our results showed that inhibition of ferroptosis significantly suppressed the inflammatory response in vivo of MI/RI model mice (p < 0.001), suggesting that ferroptosis-induced inflammatory response exacerbates MI/RI.

SLC7A11, as a functional subunit of the cystine/glutamate transporter system (Xc-system), is highly specific for cystine and glutamate, and its role is to participate in the extracellular uptake of cystine and the release of glutamate, and to promote the synthesis of glutathione (GSH) [39, 40]. Down-regulation of SLC7A11 indirectly inhibits the activity of GPX-4, which, in turn, leads to the build-up of lipid peroxides and ultimately induces cellular to undergo ferroptosis [41]. Aside of being a major phenotype inducing iron death, GPX-4 deficiency can also mediate or accelerate inflammatory responses [42]. In addition, dysregulation of TFR-1 and Ferritin activities induces the onset of ferroptosis [22]. Herein, our results show that Fer-1 intervention reverses the dysregulated expression of TFR-1 and Ferritin, reduces Fe levels in tissues, and, upregulates the inhibited SLC7A11/GPX-4 signaling pathway. Our study demonstrated that by regulating the SLC7A11/GPX-4/Ferritin pathway in ferroptosis, oxidative stress, inflammatory response and tissue damage can be effectively alleviated, thereby reducing MI/RI.

In summary, we found that ferroptosis worsens MI/RI by exacerbating oxidative stress, inflammatory responses, and tissue damage. Intervention by Fer-1, an ferroptosis inhibitor, can provide multifaceted protection against MI/RI. Activation of the Ferritin/SLC7A11/GPX4 signaling pathway is an important molecular mechanism by which ferroptosis mediates worsening of MI/RI. These findings suggest that the targeted inhibition of ferroptosis is expected to be a new therapeutic strategy for MI/RI.

Author Contribution

Lihong Wang made contributions to data acquisition, analysis, and thesis. Dongfang Li made critical revision of important intellectual content. Jia Zhi made significant contributions to the conception or design of the work.

Acknowledgements

This work was financially supported by the Tianjin Beichen District Health and Wellness System Science and Technology Project (SHGY-2022009).

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Ferroptosis exacerbates myocardial ischemia-reperfusion injury via worsening oxidative stress and inflammatory responses: the role of Ferritin/SLC7A11/GPX-4 signaling pathway

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Abstract

Figures

1. Introduction

2. Materials and methods

2.1. Experimental animals

2.2. MI/RI mice modelling

2.3. TTC-Evans blue staining

2.4. Hematoxylin-Eosin (H&E) staining

2.5. Transmission electron microscopy (TEM) observation

2.6. Detection of inflammatory factors

2.7. Lipid Peroxidation (MDA) and T-GSH/GSSG measurement

2.8. Reactive oxygen species (ROS)

2.9. Detection of iron content

2.10. Quantitative reverse transcription PCR (qRT-PCR)

2.11. Western blot (WB)

2.12. Statistical Analyses

3. Results

3.1. Iron accumulation in MI/RI mice

3.2. Ferroptosis-mediated oxidative stress in MI/RI mice

3.3. Role of ferroptosis in MI/RI modelling-guided tissue damage

3.4. Ferroptosis-mediated inflammatory response in MI/RI mice

3.5. Molecular mechanisms of ferroptosis regulation to MI/RI

4. Discussion

5. Conclusion

Declarations

Author Contribution

Acknowledgements

References

Additional Declarations

Supplementary Files

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