{"paper_id":"3750fa2d-de9b-46aa-91e4-7900252d8b02","body_text":"Exercise training inhibits myocardial ischemia-reperfusion-induced ferroptosis via AMPK- dependent ACC phosphorylation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Exercise training inhibits myocardial ischemia-reperfusion-induced ferroptosis via AMPK- dependent ACC phosphorylation Yuxiang Xu, Jie Tang, Chenyang Wu, Ruizhen Li, Yifang Zhao, Mengxin Cai, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8034270/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Ferroptosis is critical in the pathogenesis of myocardial ischemia-reperfusion (I/R) injury. Exercise training (ET) protects against myocardial I/R injury, but its impact on ferroptosis and the mechanisms remain incompletely understood. This study explored whether AMP-activated protein kinase (AMPK)-regulated acetyl-CoA carboxylase (ACC) mediates ET-induced cardioprotection by suppressing ferroptosis in I/R mice. 3-month-old male C57BL/6J mice were divided into sedentary and ET groups. Mice in the ET group underwent six weeks of treadmill training before myocardial I/R induction. Cardiac function, oxidative stress, ferroptosis-related proteins expression were measured. H9C2 cardiomyocytes were exposed to hypoxia/reoxygenation (H/R) to simulate I/R. AMPK activity was modulated and ACC phosphorylation was blocked to explore the mechanism. In I/R mice, ET improved cardiac function, activated AMPKα, increased ACC phosphorylation, upregulated the expression of glutathione peroxidase 4 (GPX4) and solute carrier family 7 member 11 (SLC7A11), and downregulated acyl-CoA synthetase long chain family member 4 (ACSL4) expression. In vitro , H/R induced oxidative stress, reduced ACC phosphorylation and the expression of SLC7A11 and GPX4, and upregulated ACSL4 expression. AMPK inhibition reduced ACC phosphorylation, worsened H/R-induced oxidative stress and ferroptosis, while AMPK activation reversed these effects. Blocking ACC phosphorylation abolished the protective effects of AMPK activation and exacerbated H/R-induced ferroptosis. In conclusion, ET reduces I/R-induced ferroptosis, and AMPK-mediated ACC phosphorylation plays an important role in this process. Exercise training ferroptosis myocardial ischemia-reperfusion Acetyl-CoA carboxylase Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Acute myocardial infarction (AMI) remains a leading cause of mortality in the spectrum of cardiovascular diseases (CVDs). Although timely clinical interventions, notably interventional therapy, have significantly improved survival rates, the subsequent restoration of blood flow (reperfusion) after ischemia can paradoxically induce myocardial ischemia-reperfusion (I/R) injury. This exacerbates adverse cardiac structural remodeling, increases complication risk, and negatively impacts patient prognosis (Zhang et al. 2024 ; Heusch 2020 ; Hausenloy et al. 2015). Emerging evidence highlights ferroptosis, a regulated form of cell death driven by iron-dependent accumulation of lethal lipid peroxides (Dixon et al. 2012 ), as a pivotal contributor to the pathogenesis of myocardial I/R injury (Zhao et al. 2021 ). It has been reported that I/R can significantly induce ferroptosis through multiple mechanisms, including elevated free iron levels and the consequent generation of reactive oxygen species (ROS) via the Fenton reaction, dysregulated lipid metabolism and peroxidation of polyunsaturated fatty acids (PUFAs), inhibition of the cytoprotective System Xc⁻/glutathione (GSH)/ glutathione peroxidase 4 (GPX4) axis, mitochondrial damage and endoplasmic reticulum stress (ERS) in cardiomyocytes. Targeting ferroptosis represents a promising therapeutic strategy for the prevention and treatment of myocardial I/R injury (Zhang, Han et al. 2025 ; Yang et al. 2025 ; Hu et al. 2024 ; Deng et al. 2024 ; Yao et al. 2024 ). Exercise training (ET), a non-pharmacological intervention, performs significant cardioprotective effects against myocardial I/R injury. Studies report that ET enhances myocardial antioxidant defenses, reduces ROS levels, improves calcium handling, mitigates mitochondrial dysfunction, promotes efficient glycolipid metabolism, and reduces cardiomyocyte apoptosis, thereby protecting against myocardial I/R injury (Boulghobra et al. 2020 ; Rabinovich-Nikitin et al. 2018; Chen et al. 2024 ; Bei et al. 2024 ; Powers et al. 2008 ). It has been demonstrated that ET can reduce cellular ferroptosis and alleviate several diseases (He et al. 2022 ; Liu et al. 2022 ; Wang et al. 2023 ; Wang et al. 2024 ). In a rat model of cerebral I/R injury, ET can decrease the level of lipid peroxides in the cerebral cortex, upregulate the protein expression of solute carrier family 7 member 11 (SLC7A11) and GPX4, and inhibit ferroptosis (Liu et al. 2022 ). However, whether ET can reduce myocardial I/R-induced cardiomyocytes ferroptosis has not been verified. AMP-activated protein kinase (AMPK) is a conserved serine/threonine kinase that is composed of α catalytic subunit and β and γ regulatory subunits, could be activated by energy stress (Yan et al. 2021 ). The regulation of AMPK activity primarily depends on phosphorylation at the Thr172 site on the α subunit. ET induces substantial ATP consumption, which is a potent activator of AMPK signaling (Liu et al. 2020 ). It has been shown that AMPK can enhance mitochondrial biogenesis, improve electron transport chain efficiency, reduce ROS production, and mitigate oxidative stress (Steinberg et al. 2023). Furthermore, AMPK activation could promote fatty acid oxidation and reduce lipid accumulation, inhibited ferroptosis (Lee et al. 2020 ; Cheng et al. 2024 ; Zhong et al. 2023 ; Li et al. 2022 ; Huang et al. 2022 ). Acetyl-CoA Carboxylase (ACC) is a key enzyme in fatty acid synthesis, and its phosphorylation by AMPK leads to inhibits enzymatic activity, reducing de novo synthesis of PUFA-containing phospholipids (Hardie et al. 2002). Recently, one study indicated that ACC activation disrupts iron homeostasis to drive neuronal ferroptosis (Han et al. 2025 ). However, whether ET can activate AMPK to attenuate myocardial I/R-induced ferroptosis via regulating ACC remains untested. In the present study, we assessed the effects of ET on the activation of AMPK/ACC signaling pathway and ferroptosis markers using a myocardial I/R model in C57BL/6J mice. In vitro experiments, we employed a hypoxia/reoxygenation (H/R) model to simulate I/R in H9C2 cardiomyocytes, utilized the AMPK activator 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) to simulate the ET effect, AMPK activity and ACC phosphorylation were inhibited to explore the underlying mechanism of AMPK-regulated ACC in inhibiting H/R-induced ferroptosis. 2. Methods 2.1 Animals Sixty 3-month-old male C57BL/6J mice, weighing 25.0 ± 3.2 g, were obtained from the Laboratory Animal Center of Shaanxi Normal University. Mice were housed in the Institute of Sports Biology of Shaanxi Normal University under controlled conditions (25°C, 50% ± 10% humidity, 12 h light/dark cycle) with ad libitum access to food and water. After a one-week acclimatization period, mice were randomly divided into sedentary and ET groups (thirty mice per group). Mice in the ET group underwent six weeks of treadmill training. All mice subsequently received either sham surgery or myocardial I/R surgery. Mice with successful I/R induction were assigned to the following groups: I/R 2 h group (Sed-I/R 2 h), I/R 24 h group (Sed-I/R 24 h), ET + I/R 2 h group (ET-I/R 2 h) and ET + I/R 24 h group (ET-I/R 24 h). Sham controls included the sham-operated group (Sed-Sham) and ET + sham-operated group (ET-Sham), with six mice per group (Fig. 1 A). All experiments adhered to the Guide for Care and Use of Laboratory Animals and were approved by the Ethical Committee of Shaanxi Normal University (approval number: 202416015). Animal grouping and training were performed by a single observer to ensure that subsequent experimenters remained blinded to the group assignments. 2.2 ET protocol Mice in the ET group performed aerobic exercise on an eight-channel treadmill (Anhui Zhenghua Biological Instrument Equipment Co., Ltd., Anhui, China), following a previously published protocol with minor modifications (Massett et al. 2021 ). The first week was dedicated to adaptive training, starting at 8 m/min for 10 min, with incremental increases of 2 m/min in speed and 12 min in duration each day. By the fifth training day, the speed was raised to 16 m/min for 60 min. From the second to the seventh week, mice engaged in formal training at 16 m/min for 60 min per day, five days a week. 2.3 Myocardial I/R model On the day after the final ET intervention, mice were anesthetized via inhalation of a mixture of isoflurane and oxygen (3% for induction and 1% for maintenance) using a small animal anesthesia machine (Beijing Zhongshi Dichuang Technology Development Co., Ltd., Beijing, China) and placed in a supine position on the operating table. The depth of anesthesia was standardized by maintaining a heart rate of 450–500 beats/min. After fixation and dehairing, the chest was opened at the fourth intercostal space to expose the heart. The left anterior descending coronary artery was ligated for 30 min, followed by 2 h or 24 h of reperfusion under normal cage conditions. An Electrocardiogram (ECG) was used to monitor the surgery, and ST-segment elevation indicated cardiac ischemia. Sham controls underwent identical procedures without artery ligation. 2.4 Cardiac function measurement Cardiac function was evaluated using a small animal ultrasonic detector (Vinno 6 VET, VINNO, Suzhou, China) with M-mode ultrasound. The left ventricular internal dimension at diastole (LVIDd), left ventricular internal dimension at systole (LVIDs), ejection fraction (EF%) and fractional shortening (FS%) were measured or calculated using the ultrasound equipment. After measurement, mice were deeply anesthetized and euthanized by decapitation. Hearts were quickly removed on ice, and fixed in 4% paraformaldehyde (PFA) or liquid nitrogen for further analysis. 2.5 Histology staining PFA-fixed hearts were embedded in paraffin. Sections (5 µm thick) were prepared, and consecutive sections from the same anatomical position in each group were selected for Masson's trichrome staining to evaluate structural pathology. Enhanced Perls Prussian blue staining was performed using a staining kit (G1428, Solarbio, Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) to detect iron deposits. Paraffin sections were deparaffinized in xylene, rehydrated through a gradient of ethanol solutions, and rinsed in distilled water. Sections were incubated with freshly prepared Perls staining working solution at 37°C for 20 min, rinsed with distilled water. Then incubation solution was added to the slides and incubated at 37°C for 10 min, gently rinsed with PBS 1 min for three times. After that, sections were incubated with enhancement working solution at 37°C for 20 min, gently rinsed with PBS 5 s for three times. Hematoxylin was used to stain the nuclei and rinsed with distilled water. Sections were dehydrated through gradient ethanol, cleared in xylene, and mounted. Ferrohemoglobin and trivalent iron appeared as brownish yellow or brownish red, with nuclei appeared as blue-purple. 2.6 Bioassay Kit Testing Liquid nitrogen-fixed heart tissues were used to detect total glutathione (T-GSH) and lipid peroxidation (LPO) using biochemical assay kits (E-BC-K097-M, E-BC-K176-M, Elabscience Biotechnology Co., Ltd., Wuhan, China). Three samples were randomly selected from each group for tissue homogenization. 20 mg sample of myocardial tissues was homogenized in 200 µL of PBS. After centrifuging at 12,000 rpm for 10 min, the supernatant was collected. The assays were performed strictly according to the manufacturer’s instructions, and results were expressed as relative fold changes compared to the Sed-Sham group. 2.7 Cell culture H9C2 cells (SNL-029, Wuhan Sunncell Biotechnology Co., Ltd., Wuhan, China) were cultured in complete medium consisting of DMEM, FBS, and Penicillin/ Streptomycin (C100C5, NCM Biotech, Suzhou, China) at a ratio of 90:10:1 in a standard incubator set at 37°C with 5% CO 2 . When the cell density reached 70%–80%, cells were used for experiments. To induce the H/R model, H9C2 cells were initially incubated under normal conditions (37°C, 5% CO 2 ), then exposed to hypoxic conditions (37°C, 1% O 2 , 5% CO 2 , 94% N 2 ) for 8 h, followed by reoxygenation for 12 h (Han et al. 2021 ). To investigate the effect of AMPK activation, H9C2 cells were treated with the AMPK activator AICAR (HY-13417, MedChem Express, NJ, USA) at 1 mM for 20 h to simulate the effects of ET. In addition, Compound C (C-C, S7306, Selleck Chemicals LLC, TX, USA) at 4 µM for 20 h was used to inhibit AMPK activation, and Firsocostat (HY-16901, MedChem Express) at 500 nM for 20 h was employed to block ACC phosphorylation. 2.8 Dihydroethidium (DHE) staining DHE, a fluorescent probe for detecting intracellular superoxide anions (O₂·⁻), is oxidized by intracellular superoxide upon entering cells, forming a product that emits red fluorescence. In this study, frozen heart sections and H9C2 cells were incubated with DHE (S0063, Beyotime, Shanghai, China) in a dark environment at room temperature for 50 min. After washes with PBS, 5 min each for three times, sections were sealed and visualized using a fluorescence microscope (Nikon Eclipse 55i, Tokyo, Japan). Fluorescence intensity was quantified using Image-Pro Plus software. 2.9 Western blotting Total proteins from heart tissues and H9C2 cells were extracted in radio immunoprecipitation assay (RIPA, C500008, Sangon Biotech (Shanghai) Co., Ltd., Shanghai, China) buffer supplemented with phenylmethanesulfonyl fluoride (PMSF, P1081, Beyotime) and phosphatase inhibitors (P1005, Beyotime) at a ratio of 100:1:1. The mixture was homogenized on ice and centrifuged (12,000 × g , 15 min, 4°C) to collect the supernatant. Protein concentration was determined using the BCA assay (C503021, Sangon Biotech). A 20 µg sample of protein was separated on an 8%–12% SDS polyacrylamide gel and transferred onto nitrocellulose membranes. Membranes were blocked with 5% bovine serum albumin (BSA) for 1.5 h, and then incubated with primary antibodies at 4℃ overnight. The following antibodies were used: AMPKα, p-AMPKα, ACC and p-ACC (1:1000, #9957, Cell Signaling Technology, Inc. (CST), Danvers, MA, USA), ACC1 (1:1000, A19627, ABclonal Technology Co., Ltd, Wuhan, China), superoxide dismutase 1 (SOD1, 1:1000, ab308181, Abcam, Cambridge, UK), SOD2 (1:1000, ab68155, Abcam), GPX4 (1:2000, 67763-1-IG, Proteintech Group, Inc. Wuhan, CHN), acyl-CoA synthetase long-chain family member 4 (ACSL4, 22401-1-AP, 1:1000, Proteintech) and SLC7A11 (1:1000, 26864-1-AP, Proteintech). GAPDH (1:5000, 60004-1-IG, Proteintech) was used as the housekeeping protein. After washing with Tris-Buffered Saline Tween (TBST) 5 min for three times, membranes were incubated with goat anti-rabbit or mouse IgG, HRP conjugated secondary antibodies (1:5000, SA00001-2, SA00001-1, Proteintech) at room temperature for 1.5 h. Membranes were then washed with TBST and visualized using an enhanced chemiluminescence (ECL) reagent (C520045, Sangon Biotech) and an imaging system (ChemiDoc™ MP, Bio-Rad). 2.10 Cell malonyl CoA assay The Malonyl CoA (MCA) levels in H9C2 cells were detected by using Rat MCA ELISA Kit (CB10239-Ra, Shanghai Keaibo Biotechnology Co., Ltd, Shanghai, China). After intervention, cells were washed with pre-cooled PBS, trypsinized, and centrifuged at 1000×g for 5 min. The cell pellet was washed three times with cold PBS. For every 1×10⁶ cells, 150 µL of PBS was added. Cells were subjected to ultrasonic disruption at 20 kHz and 150 W in an ice bath, with three bursts (2 s on, 3 s off). The extract was centrifuged at 1500×g for 10 min, and the supernatant was analyzed using the kit according to the manufacturer’s instructions. 2.11 Statistical analysis Western blotting results were analyzed using Image J software (National Institutes of Health, MD, USA), and data were visualized with GraphPad Prism 8.0 software (Dotmatics, UK). All experimental results were expressed as mean ± standard error of the mean (SEM) to indicate the sampling error of the sample mean. Animal results were analyzed using two-factor ANOVA, while H9C2 cell results were analyzed using one-way ANOVA with Tukey’s post-hoc test. Significant differences between groups were defined as * P < 0.05, ** P < 0.01 levels. 3. Results 3.1 ET attenuated myocardial I/R-induced oxidative stress and preserved cardiac function Cardiac function was assessed by measuring LVIDd, LVIDs, EF% and FS%. Compared to the Sed-Sham group, myocardial I/R significantly increased LVIDs ( P < 0.05 at 2 h, P < 0.01 at 24 h) and LVIDd ( P < 0.01 at both 2 h and 24 h), while decreasing EF% and FS% ( P < 0.01 at both 2 h and 24 h). Compared to the Sed-I/R groups, ET significantly reduced LVIDs ( P < 0.05 at both 2 h and 24 h) and LVIDd ( P < 0.01 at 24 h), while increasing EF% ( P < 0.01 at both 2 h and 24 h) and FS% ( P < 0.05 at 2 h, P < 0.01 at 24 h) (Fig. 1 B-F). Masson’s trichrome staining revealed distinct alterations in cardiac structure. In the Sed-Sham and ET-Sham groups, myocardial fibers (stained red) were arranged regularly, with minimal collagen deposition (stained blue) surrounding vascular structures. In contrast, the Sed-I/R 24 h group exhibited disorganized myocardial fibers, extensive abnormal collagen deposition, and structural disruption. ET markedly improved tissue architecture in the ET-I/R 24 h group, showing relatively ordered fiber arrangement and significantly reduced collagen deposition (Fig. 1 G). DHE staining demonstrated that myocardial I/R increased O₂·⁻ levels compared to the Sed-Sham group ( P < 0.01). Notably, ET effectively suppressed I/R-induced ROS (O₂·⁻) overproduction ( P < 0.05, Fig. 1 H, I). These findings collectively indicated that myocardial I/R resulted in excessive ROS production and impaired cardiac function and structure, while, ET protected against these I/R-induced impairments. 3.2 ET attenuated I/R-induced ferroptosis and increased the phosphorylation of AMPKα and ACC Western blotting analysis revealed that compared to the Sed-Sham group, myocardial I/R significantly downregulated the expression of GPX4 ( P < 0.05 at 2 h) and SLC7A11 ( P < 0.01 at both 2 h and 24 h), increased ACSL4 expression ( P < 0.01 at both 2 h and 24 h). ET significantly increased the expression of GPX4 and SLC7A11, and downregulated ACSL4 expression ( P < 0.01 at both 2 h and 24 h) in the I/R hearts (Fig. 2 A-D). In addition, decreased T-GSH levels ( P < 0.05 at I/R 24 h) and increased LPO levels ( P < 0.01 at 2 h, P < 0.05 at 24 h) and iron deposition were shown in the Sed-I/R group when compared to the Sed-Sham group. ET significantly increased T-GSH levels ( P < 0.05 at 2 h and 24 h) and reduced LPO levels ( P < 0.01 at 2 h and 24 h) and iron deposition in the I/R heart (Fig. 2 E-G). Meanwhile, compared to the Sed-Sham group, myocardial I/R suppressed the phosphorylation of AMPKα ( P < 0.01 at 2 h) and ACC ( P < 0.01 at both 2 h and 24 h). ET significantly enhanced the phosphorylation of AMPKα ( P < 0.01 in the Sham, I/R 2 h and 24 h groups) and ACC ( P < 0.01 in the Sham group, P < 0.05 in the I/R 2 h group) (Fig. 2 H-J). These findings underscored the protective effect of ET against I/R-induced ferroptosis and confirmed that ET effectively activated AMPKα and increased ACC phosphorylation. 3.3 AMPK inhibition increased ACC phosphorylation and exacerbated H/R-induced ferroptosis To model the effects of I/R and AMPK inactivation, H9C2 cells were subjected to H/R and/or treated with the AMPK inhibitor Compound C(C-C). Results revealed that H/R treatment alone significantly reduced AMPK phosphorylation ( P < 0.01) and SOD2 expression ( P < 0.01), increased the ROS levels ( P < 0.01). C-C intervention under basal conditions also increased the ROS levels ( P < 0.01), decreased the antioxidant enzymes SOD1 and SOD2 expression (both P < 0.01). Additionally, C-C further inhibited AMPKα phosphorylation ( P < 0.01), elevated ROS levels ( P < 0.01) and suppressed SOD1 and SOD2 expression (both P < 0.01) in H/R-treated cells (Fig. 3 A-F), indicating AMPK inhibition severely compromises antioxidant capacity. Analysis of the expression of ACC and ferroptosis markers showed that C-C, H/R, and their combination all significantly reduced ACC phosphorylation ( P < 0.05 for C-C, P < 0.01 for H/R and combination). Both C-C or H/R treatment alone upregulated the expression of ACSL4 ( P < 0.01 for both) and ACC1 ( P < 0.01 for C-C, P < 0.05 for H/R), reduced the expression of SLC7A11 ( P < 0.01 for both) and GPX4 ( P < 0.01 for both). Combining C-C with H/R intensified these pro-ferroptotic changes, suppressed the expression of SLC7A11 ( P < 0.05) and GPX4 ( P < 0.01), and further upregulated the expression of ACSL4 ( P < 0.01) and ACC1 ( P < 0.01) compared to H/R alone (Fig. 3 D, G-K). Collectively, these data indicated that both H/R and AMPK inhibition could decrease ACC phosphorylation, trigger ferroptosis in H9C2 cells, and AMPK inhibition dramatically exacerbated this ferroptotic process. 3.4 AMPK activation induced ACC phosphorylation and attenuated H/R-induced oxidative stress and ferroptosis We verified that ET can activate AMPK and inhibit ferroptosis. To further explore the mechanism, we treated H9C2 cells with AICAR to simulate the effects of ET. Results revealed that AICAR treatment effectively activated AMPKα ( P < 0.01) and reduced ROS levels ( P < 0.01) in H/R-treated cells (Fig. 4 A-C). Under basal conditions, AICAR upregulated ACC phosphorylation ( P < 0.01) and the expression of SOD1 ( P < 0.01) and GPX4 ( P < 0.01), while reducing ACSL4 expression ( P < 0.01). In H/R condition, AICAR notably reversed H/R-induced alterations by increasing ACC phosphorylation ( P < 0.01) and upregulating the expression of SOD2 ( P < 0.01), SLC7A11 ( P < 0.01) and GPX4 ( P < 0.01), as well as reducing the expression of ACC1 ( P < 0.01) and ACSL4 ( P < 0.01) in H/R-treated cells (Fig. 4 D-K). These findings confirmed that AICAR-mediated AMPK activation protected against H/R injury by enhancing antioxidant defenses and regulating ferroptotic pathways. 3.5 Inhibition of ACC phosphorylation aggravated ferroptosis and abrogated the protective effects of AICAR To further elucidate the role of ACC in H/R-induced ferroptosis, and its interaction with AMPK activation, we treated H9C2 cells with Firsocostat, a specific inhibitor of ACC phosphorylation. Results revealed that Firsocostat intervention under basal conditions significantly reduced the phosphorylation of ACC ( P < 0.01) and AMPKα ( P < 0.01), downregulated the expression of SLC7A11 ( P < 0.05) and GPX4 ( P < 0.01), increased the expression of ACC1 ( P < 0.05) and ACSL4 ( P < 0.01) and MCA levels ( P < 0.01) (Fig. 5 A-H). In H/R-treated cells, Firsocostat similarly suppressed ACC phosphorylation ( P < 0.01) and the expression of SLC7A11 ( P < 0.01) and GPX4 ( P < 0.05), while increasing the expression of ACC1 ( P < 0.05) and ACSL4 ( P < 0.01) (Fig. 5 A-H). Co-treatment with AICAR and Firsocostat in H/R cells attenuated AICAR's protective effects. While AMPKα phosphorylation remained elevated compared to H/R alone ( P < 0.01), AICAR failed to increase the expression of SLC7A11 and GPX4 or decrease the expression of ACC1 and ACSL4 as well as MCA levels (Fig. 6 A-H). These findings demonstrated that blocking ACC phosphorylation (inactivating ACC) exacerbated H/R-induced ferroptosis by impairing the antioxidant defense system and promoting fatty acid synthesis. This indicated that ACC phosphorylation, not just AMPK activation, was essential for ferroptosis suppression. 4. Discussion Myocardial I/R injury remains a critical clinical challenge (Tong et al. 2024). ET has emerged as a promising non-pharmacological strategy for cardiovascular protection (Fiuza-Luces et al. 2018 ), however, its role in regulating ferroptosis is not fully elucidated. In this study, we demonstrated that ET activates AMPK, promotes ACC phosphorylation, which mitigates I/R-induced oxidative stress and ferroptosis. In vitro experiments further confirmed that AMPK inactivation inhibits ACC phosphorylation, exacerbates ferroptosis-related phenotypes, while, AMPK activation increased ACC phosphorylation​​, preserved GPX4/SLC7A11 and downregulated ACSL4. Critically, direct inhibition of ACC phosphorylation aggravates ferroptosis and abolishes AMPK’s benefits​​, proving ACC is a required effector of AMPK-activation anti-ferroptotic pathway in ET-induced I/R protection. Collectively, our findings provide experimental evidence supporting ​​ET could serve as a preconditioning strategy​​ for cardioprotection, and highlight ACC phosphorylation as a druggable target​​ for I/R injury. Myocardial I/R injury is characterized by excessive ROS production, which drives cellular damage, cardiac dysfunction, and structural remodeling (Zhang et al. 2024 ). Consistent with previous studies (Wu et al. 2018 ; Wang et al. 2025 ), our experiments demonstrated that myocardial I/R led to a marked decline in cardiac function at both 2 h (early reperfusion) and 24 h (subacute phase) post-reperfusion. Previous study has confirmed that ET also improves cardiac function three weeks after I/R (Xu et al. 2024 ), our data extend this finding by showing that ET exerts protective effects even in the immediate and subacute phases post-I/R. Functional deficits in I/R hearts were accompanied by structural abnormalities, including disorganized myocardial fibers and extensive collagen deposition, consistent with previous reports of I/R-induced cardiac remodeling (Domínguez-Rodríguez et al. 2018 ). I/R significantly elevated superoxide anion (O₂·⁻) levels, which further intensifies cardiomyocyte damage and death, and contributes to long-term remodeling by activating pro-fibrotic signaling (Zhang et al. 2024 ; Xiang et al. 2024 ). In this study, we confirmed that ET improved cardiac function and preserved myocardial architecture, with reduced collagen deposition and suppressed O₂·⁻ overproduction. Based on previous report, the protective effect of ET is involved with regulating myokines, enhancing antioxidant capacity and improving mitochondrial function (Boulghobra et al. 2020 ; Rabinovich-Nikitin et al. 2018). This aligns with studies showing that ET reduced ROS accumulation, preserved cardiac contractile function and pumping efficiency post-I/R injury (Zhang, Huang et al. 2025 ). Evidence suggests that ferroptosis is widely implicated in various oxidative stress-related diseases (Sun et al. 2023 ; Zhu et al. 2023 ; Zhang, Liu et al. 2025 ). The core mechanism of ferroptosis involves iron-dependent accumulation of lipid peroxides, which is driven by increased iron overload and impaired clearance of lipid peroxide (Pan et al. 2022 ). Key molecular regulators of ferroptosis include GPX4 (lipid peroxide scavenger), SLC7A11 (subunit of System Xc⁻, responsible for cystine import to synthesize GSH), and ACSL4 (enzyme promoting PUFA synthesis, a substrate for lipid peroxidation) (Yao et al. 2024 ; Doll et al. 2017 ). Previous studies have shown that myocardial I/R attenuates System Xc⁻ activity (consistent with reduced SLC7A11 expression), inhibits GPX4 activity as early as 2 h post-I/R, and upregulates ACSL4 expression (Yao et al. 2024 ; Fan et al. 2021 ). In this study, our study demonstrated that myocardial I/R in sedentary mice significantly downregulated GPX4 and SLC7A11 expression, increased ACSL4 expression, reduced total GSH (T-GSH) levels at 24 h post-I/R, and elevated LPO levels and iron deposition, collectively confirming that I/R enhances ferroptosis in the myocardium. In contrast, ET preconditioning upregulated GPX4 and SLC7A11 expression, reduced ACSL4 expression and LPO levels, and increased T-GSH levels, protected the heart against ferroptosis. These results are consistent with a recent study showing that pre-exercise improves ischemic stroke-induced ferroptosis by increasing SLC7A11 and GPX4 expression (Huang et al. 2024 ). In addition, studies have suggested that ferroptotic signals appear during ischemia, and ferroptosis occurs in the late reperfusion phase (Xiang et al. 2024 ). In the present study, the expression of certain ferroptosis-related proteins varied at different time points post-I/R, and the specific timing and duration of ferroptosis in myocardial I/R injury remain to be further clarified. The mechanism underlying ET’s anti-ferroptotic effect was linked to the AMPK/ ACC signaling pathway. AMPK is a master regulator of cellular energy homeostasis, which is activated by ET (via increased AMP/ATP ratio) and has been implicated in antioxidant and anti-ferroptotic responses in various tissues (Lee et al. 2020 ; Zhong et al. 2023 ; Carapeto et al. 2024 ). AMPK activation normally inhibits ACC by phosphorylating it, and reduces fatty acid synthesis, decreases PUFA production, and lowers lipid peroxidation, improves cellular energy supply (Lee et al. 2020 ; Pang et al. 2021 ). Our in vivo data confirmed that myocardial I/R in sedentary mice significantly suppressed the phosphorylation of both AMPKα and ACC, while ET preconditioning restored and enhanced AMPKα phosphorylation, which in turn specifically promoted ACC phosphorylation. We speculated that activated AMPKα phosphorylated and inactivated ACC, thereby reducing lipid substrate availability for peroxidation, which is a critical step in ferroptosis. Previous research has shown that swimming alleviates obesity and liver injury in non-alcoholic fatty liver disease by downregulating ACC1 protein expression in obese rats (Yang et al. 2024 ), suggesting that ET-induced regulation of ACC1 is a conserved mechanism across organs. To further validate the role of AMPK/ACC signaling in vitro , we used H9C2 cardiomyocytes subjected to H/R. Consistent with in vivo findings, H/R alone suppressed the phosphorylation of AMPKα and ACC, increased ROS production, and downregulated antioxidant enzymes SOD1 and SOD2, mirroring the oxidative stress phenotype observed in I/R hearts. Notably, treatment with C-C further reduced ACC phosphorylation, increased ACSL4 expression, and decreased GPX4 and SLC7A11 expression, confirming that AMPK is a key upstream regulator of ACC and ferroptosis during I/R. Conversely, activation of AMPK attenuated H/R-induced oxidative stress and ferroptosis, increased ACC phosphorylation and reduced MCA levels, which is crucial for fatty acid synthesis and lipid peroxidation (Ding et al. 2023 ). Based on these, we speculate that AMPK enhances cystine uptake and GSH synthesis via SLC7A11, strengthens lipid peroxide clearance via GPX4, and reduces the production of peroxidation substrates by inhibiting ACSL4 and ACC1. Given that ACC1-mediated fatty acid synthesis is the primary source of PUFAs, we hypothesized that ACC1, rather than ACC2, would be the critical isoform mediating AMPK’s anti-ferroptotic effect (Wang et al. 2022 ). To directly test whether ACC is a non-redundant effector of AMPK, we used Firsocostat, a specific ACC inhibitor that blocks ACC phosphorylation, thereby activating ACC enzymatic activity. We found that reducing ACC phosphorylation by Firsocostat promoted the expression of ACSL4 and ACC1, decreased the expression of SLC7A11 and GPX4, and increased MCA levels. In H/R-treated cells, Firsocostat exacerbated ferroptosis and abrogated the protective effects of AICAR. Despite sustained AMPK activation, AICAR failed to upregulate the expression of GPX4 and SLC7A11 or downregulate the expression of ACC1 and ACSL4 as well as MCA levels in the presence of Firsocostat. These data provide definitive evidence that ACC1 phosphorylation is indispensable for AMPK-mediated ferroptosis inhibition. By inactivating ACC, AMPK reduced MCA-driven lipid peroxidation, and modulates the expression of GPX4, SLC7A11 and ACSL4 through a metabolism-linked oxidative stress regulatory cascade. This finding is particularly significant when compared to previous studies on AMPK’s anti-ferroptotic mechanisms. For example, AMPK has been shown to suppress ferroptosis in tumor cells by phosphorylating BECN1 (Song et al. 2018 ), or in cerebral I/R via the AMPK/FoxO3a pathway (Zhong et al. 2023 ). Additionally, energy stress promotes AMPK to mediate GPX4 expression and inhibits erastin-induced ferroptosis in renal cancer through the JAK2/STAT3/P53 signaling (Li et al. 2022 ) or mTOR signaling (Kou et al. 2025 ). However, these mechanisms are cell-type or disease-specific, whereas our data demonstrate that in the myocardium, ACC phosphorylation is the dominant, non-redundant pathway mediating AMPK’s anti-ferroptotic effect. Despite the novel findings of this study, several limitations warrant consideration. Firstly, while our focus was on the AMPK/ACC1 signaling pathway, other AMPK substrates (e.g., FoxO3a, mTOR) may also contribute to the antioxidant and anti-ferroptotic effects of ET. Future studies using isoform-specific AMPK knockout mice or ACC1/ACC2 double knockout models will help clarify the relative contributions of ACC1 versus other AMPK substrates. Secondly, we used pharmacological inhibitors (C-C, Firsocostat) to modulate AMPK and ACC1 activity; genetic models will further strengthen the causal link between AMPK/ACC1 signaling and ET-induced I/R protection. Thirdly, ferroptosis interacts with other forms of cell death and cellular processes, exploring these interrelationships could reveal synergistic mechanisms of ET’s protective effects. For instance, does ACC phosphorylation also regulate autophagy to further suppress ferroptosis? Finally, the optimal ET protocol (e.g., exercise intensity, duration, frequency, and timing relative to I/R) was not evaluated in this study. Variables such as moderate vs. high-intensity exercise, or pre-I/R vs. post-I/R ET, may affect the magnitude of ACC phosphorylation and ferroptosis inhibition, limiting the clinical translatability of our findings. Future studies should address these variables to provide a basis for evidence-based exercise prescriptions in clinical practice. 5. Conclusion Our findings suggest that ET protects against myocardial I/R injury by activating AMPK, phosphorylating ACC, and inhibiting ferroptosis by upregulating the expression of GPX4 and SLC7A11, and downregulating ACSL4. These results extend previous studies by identifying ferroptosis as a key target of ET-mediated I/R protection, and by establishing the AMPK/ACC signaling as a critical regulator of this process. While ET is known to improve cardiac resilience, our work links ET to ferroptosis inhibition, offering new insights into potential therapeutic strategies for I/R injury. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-8034270\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":true,\"archivedVersions\":[],\"articleType\":\"Research Article\",\"associatedPublications\":[],\"authors\":[{\"id\":544653627,\"identity\":\"8e2f7e22-f588-420d-ba39-694de147ca49\",\"order_by\":0,\"name\":\"Yuxiang Xu\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Shaanxi Normal University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Yuxiang\",\"middleName\":\"\",\"lastName\":\"Xu\",\"suffix\":\"\"},{\"id\":544653632,\"identity\":\"be5a5059-df8a-45b3-bfd2-ed6fb3b3f77e\",\"order_by\":1,\"name\":\"Jie 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tissues; H: DHE staining of heart tissues indicating ROS levels (red fluorescence); I: Quantification of ROS fluorescence intensity. Data are presented as mean ± standard error of the mean (SEM), n = 6 in each group. Statistical analysis was performed by GraphPad Prism 7 using two-way ANOVA, *\\u003cem\\u003eP\\u003c/em\\u003e\\u0026lt;0.05, **\\u003cem\\u003eP\\u003c/em\\u003e\\u0026lt;0.01. Sed: Sedentary; ET: Exercise training; I/R: Ischemia-reperfusion.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8034270/v1/fd4e337c838b2a91613e8db7.png\"},{\"id\":96366548,\"identity\":\"250fa62c-8b86-4764-96d3-0253edeb9f24\",\"added_by\":\"auto\",\"created_at\":\"2025-11-20 10:11:33\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":45345760,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eET inhibits myocardial I/R-induced ferroptosis and regulates AMPKα/ACC signaling\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eA-D: Western blotting images (A) and quantitative analysis of the protein expression of GPX4 (B), SLC7A11 (C) and ACSL4 (D); E and F: T-GSH (E) and LPO (F) levels of the heart tissues; G: Enhanced Perls Prussian blue staining. Iron deposition appears as brown; H-J: Western blotting images (H) and quantitative analysis of the protein expression of p-AMPKα, AMPKα (I), p-ACC and ACC (J) in the heart tissues. GAPDH was used as a loading control. Results were presented as mean ± SEM, n = 3-6 in each group. Statistical analysis was performed by GraphPad Prism 7 using two-way ANOVA, *\\u003cem\\u003eP\\u003c/em\\u003e\\u0026lt;0.05, **\\u003cem\\u003eP\\u003c/em\\u003e\\u0026lt;0.01. Sed: Sedentary; ET: Exercise training; I/R: Ischemia-reperfusion.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8034270/v1/29d5786a09eaf7725d28a62c.png\"},{\"id\":96353454,\"identity\":\"0b0415c1-1271-41d2-abf6-493f91f9700d\",\"added_by\":\"auto\",\"created_at\":\"2025-11-20 07:54:45\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":19163039,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eInhibition of AMPK aggravates oxidative stress and ferroptosis in H/R-treated H9C2 cells.\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eA: Western blotting analysis of the expression of p-AMPKα and AMPKα in H9C2 cells with H/R or/and C-C treatment; B and C: DHE staining (red fluorescence) and analysis results of H9C2 cells with H/R or/and C-C treatment (scale bar=200 µm); D-K: Western blotting analysis of the expression of SOD1 (E) and SOD2 (F), p-ACC/ACC (G), ACC1 (H), ACSL4 (I), SLC7A11 (J) and GPX4 (K) in H9C2 cells with H/R or/and C-C treatment. GAPDH was used as a loading control. Results were presented as mean ± SEM, n = 3 in each group. Statistical analysis was performed by GraphPad Prism 7 using one-way ANOVA, *\\u003cem\\u003eP\\u003c/em\\u003e\\u0026lt;0.05, **\\u003cem\\u003eP\\u003c/em\\u003e\\u0026lt;0.01. CON: Normal cultured cells; C-C: Cells treated with AMPK inhibitor Compound C; H/R: Cells subjected to hypoxia/ reoxygenation.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure3.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8034270/v1/9f1ecc4a36ae1a61fa274e6f.png\"},{\"id\":96367514,\"identity\":\"2456a250-d336-4deb-8354-5375ae69c185\",\"added_by\":\"auto\",\"created_at\":\"2025-11-20 10:12:59\",\"extension\":\"png\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":77187046,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eActivation of AMPK inhibits oxidative stress and ferroptosis in H/R-treated H9C2 cells.\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eA: Western blotting analysis of the expression of p-AMPKα and AMPKα in H9C2 cells with AICAR or/and H/R treatment; B and C: DHE staining (red fluorescence) and analysis results of H9C2 cells with AICAR or/and H/R treatment (scale bar=200 µm); D-K: Western blotting analysis of the expression of SOD1 (E) and SOD2 (F), p-ACC/ACC (G), ACC1 (H), ACSL4 (I), SLC7A11 (J) and GPX4 (K) in H9C2 cells with AICAR or/and H/R treatment. GAPDH was used as a loading control. Results were presented as mean ± SEM, n = 3 in each group. Statistical analysis was performed by GraphPad Prism 7 using one-way ANOVA, *\\u003cem\\u003eP\\u003c/em\\u003e\\u0026lt;0.05, **\\u003cem\\u003eP\\u003c/em\\u003e\\u0026lt;0.01. CON: Normal cultured cells; AICAR: Cells treated with AMPK agonist AICAR; H/R: Cells subjected to hypoxia/reoxygenation.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure4.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8034270/v1/6495ad51494a751eeaec6693.png\"},{\"id\":96367547,\"identity\":\"be8604cc-18a1-485f-8d28-39522aa8749e\",\"added_by\":\"auto\",\"created_at\":\"2025-11-20 10:13:09\",\"extension\":\"png\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":13420208,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eInhibition of phosphorylated ACC aggravatesH/R-induced ferroptosis in H9C2 cells.\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eA: Effects of Firsocostat treatment on ferroptosis-related protein expression in H/R-treated H9C2 cells; B-G: Western blotting analysis of the expression of p-AMPKα/ AMPKα (B), p-ACC/ACC (C), ACC1 (D), SLC7A11 (E), ACSL4 (F), and GPX4 (G) in H9C2 cells with Firsocostat or/and H/R treatment. GAPDH was used as a loading control; H: MCA levels in H9C2 cells with Firsocostat or/and H/R treatment. Results were presented as mean ± SEM, n = 3 in each group. Statistical analysis was performed by GraphPad Prism 7 using one-way ANOVA, *\\u003cem\\u003eP\\u003c/em\\u003e\\u0026lt;0.05, **\\u003cem\\u003eP\\u003c/em\\u003e\\u0026lt;0.01. Firsocostat: Cells treated with ACC Inhibitor Firsocostat; H/R: Cells subjected to hypoxia/reoxygenation; MCA: Malonyl CoA.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure5.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8034270/v1/8476fdaefd8cad76f5c063ab.png\"},{\"id\":96353455,\"identity\":\"ea217d60-3bae-44aa-8bbc-edd9387ee5af\",\"added_by\":\"auto\",\"created_at\":\"2025-11-20 07:54:45\",\"extension\":\"png\",\"order_by\":6,\"title\":\"Figure 6\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":8490831,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eInhibition of phosphorylated ACC aggravates the protection of AICAR on H/R-induced ferroptosis in H9C2 cells.\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eA: Effects of combined treatment with AICAR and Firsocostat on ferroptosis-related protein expression in H/R-treated H9C2 cells; B-G: Western blotting analysis of the expression of p-AMPKα/AMPKα (B), p-ACC/ACC (C), ACC1 (D), SLC7A11 (E), ACSL4 (F), and GPX4 (G) in H9C2 cells with or without AICAR, Firsocostat and H/R treatment. GAPDH was used as a loading control; H: MCA levels in H9C2 cells with or without AICAR, Firsocostat and H/R treatment. Results were presented as mean ± SEM, n = 3 in each group. Statistical analysis was performed by GraphPad Prism 7 using one-way ANOVA, *\\u003cem\\u003eP\\u003c/em\\u003e\\u0026lt;0.05, **\\u003cem\\u003eP\\u003c/em\\u003e\\u0026lt;0.01. AICAR and Firsocostat: Cells treated with both AMPK agonist AICAR and ACC Inhibitor Firsocostat; H/R: Cells subjected to hypoxia/reoxygenation; MCA: Malonyl CoA\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure6.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8034270/v1/a4dff670d6292cac30774b77.png\"}],\"financialInterests\":\"No competing interests reported.\",\"formattedTitle\":\"Exercise training inhibits myocardial ischemia-reperfusion-induced ferroptosis via AMPK- dependent ACC phosphorylation\",\"fulltext\":[{\"header\":\"1. Introduction\",\"content\":\"\\u003cp\\u003eAcute myocardial infarction (AMI) remains a leading cause of mortality in the spectrum of cardiovascular diseases (CVDs). Although timely clinical interventions, notably interventional therapy, have significantly improved survival rates, the subsequent restoration of blood flow (reperfusion) after ischemia can paradoxically induce myocardial ischemia-reperfusion (I/R) injury. This exacerbates adverse cardiac structural remodeling, increases complication risk, and negatively impacts patient prognosis (Zhang et al. \\u003cspan citationid=\\\"CR48\\\" class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e; Heusch \\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e2020\\u003c/span\\u003e; Hausenloy et al. 2015). Emerging evidence highlights ferroptosis, a regulated form of cell death driven by iron-dependent accumulation of lethal lipid peroxides (Dixon et al. \\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e2012\\u003c/span\\u003e), as a pivotal contributor to the pathogenesis of myocardial I/R injury (Zhao et al. \\u003cspan citationid=\\\"CR51\\\" class=\\\"CitationRef\\\"\\u003e2021\\u003c/span\\u003e). It has been reported that I/R can significantly induce ferroptosis through multiple mechanisms, including elevated free iron levels and the consequent generation of reactive oxygen species (ROS) via the Fenton reaction, dysregulated lipid metabolism and peroxidation of polyunsaturated fatty acids (PUFAs), inhibition of the cytoprotective System Xc⁻/glutathione (GSH)/ glutathione peroxidase 4 (GPX4) axis, mitochondrial damage and endoplasmic reticulum stress (ERS) in cardiomyocytes. Targeting ferroptosis represents a promising therapeutic strategy for the prevention and treatment of myocardial I/R injury (Zhang, Han et al. \\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e2025\\u003c/span\\u003e; Yang et al. \\u003cspan citationid=\\\"CR44\\\" class=\\\"CitationRef\\\"\\u003e2025\\u003c/span\\u003e; Hu et al. \\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e; Deng et al. \\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e; Yao et al. \\u003cspan citationid=\\\"CR46\\\" class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e).\\u003c/p\\u003e\\u003cp\\u003eExercise training (ET), a non-pharmacological intervention, performs significant cardioprotective effects against myocardial I/R injury. Studies report that ET enhances myocardial antioxidant defenses, reduces ROS levels, improves calcium handling, mitigates mitochondrial dysfunction, promotes efficient glycolipid metabolism, and reduces cardiomyocyte apoptosis, thereby protecting against myocardial I/R injury (Boulghobra et al. \\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2020\\u003c/span\\u003e; Rabinovich-Nikitin et al. 2018; Chen et al. \\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e; Bei et al. \\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e; Powers et al. \\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e2008\\u003c/span\\u003e). It has been demonstrated that ET can reduce cellular ferroptosis and alleviate several diseases (He et al. \\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e; Liu et al. \\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e; Wang et al. \\u003cspan citationid=\\\"CR39\\\" class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e; Wang et al. \\u003cspan citationid=\\\"CR36\\\" class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e). In a rat model of cerebral I/R injury, ET can decrease the level of lipid peroxides in the cerebral cortex, upregulate the protein expression of solute carrier family 7 member 11 (SLC7A11) and GPX4, and inhibit ferroptosis (Liu et al. \\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e). However, whether ET can reduce myocardial I/R-induced cardiomyocytes ferroptosis has not been verified.\\u003c/p\\u003e\\u003cp\\u003eAMP-activated protein kinase (AMPK) is a conserved serine/threonine kinase that is composed of α catalytic subunit and β and γ regulatory subunits, could be activated by energy stress (Yan et al. \\u003cspan citationid=\\\"CR43\\\" class=\\\"CitationRef\\\"\\u003e2021\\u003c/span\\u003e). The regulation of AMPK activity primarily depends on phosphorylation at the Thr172 site on the α subunit. ET induces substantial ATP consumption, which is a potent activator of AMPK signaling (Liu et al. \\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e2020\\u003c/span\\u003e). It has been shown that AMPK can enhance mitochondrial biogenesis, improve electron transport chain efficiency, reduce ROS production, and mitigate oxidative stress (Steinberg et al. 2023). Furthermore, AMPK activation could promote fatty acid oxidation and reduce lipid accumulation, inhibited ferroptosis (Lee et al. \\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e2020\\u003c/span\\u003e; Cheng et al. \\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e; Zhong et al. \\u003cspan citationid=\\\"CR52\\\" class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e; Li et al. \\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e; Huang et al. \\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e). Acetyl-CoA Carboxylase (ACC) is a key enzyme in fatty acid synthesis, and its phosphorylation by AMPK leads to inhibits enzymatic activity, reducing de novo synthesis of PUFA-containing phospholipids (Hardie et al. 2002). Recently, one study indicated that ACC activation disrupts iron homeostasis to drive neuronal ferroptosis (Han et al. \\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e2025\\u003c/span\\u003e). However, whether ET can activate AMPK to attenuate myocardial I/R-induced ferroptosis via regulating ACC remains untested.\\u003c/p\\u003e\\u003cp\\u003eIn the present study, we assessed the effects of ET on the activation of AMPK/ACC signaling pathway and ferroptosis markers using a myocardial I/R model in C57BL/6J mice. \\u003cem\\u003eIn vitro\\u003c/em\\u003e experiments, we employed a hypoxia/reoxygenation (H/R) model to simulate I/R in H9C2 cardiomyocytes, utilized the AMPK activator 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) to simulate the ET effect, AMPK activity and ACC phosphorylation were inhibited to explore the underlying mechanism of AMPK-regulated ACC in inhibiting H/R-induced ferroptosis.\\u003c/p\\u003e\"},{\"header\":\"2. Methods\",\"content\":\"\\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003e2.1 Animals\\u003c/h2\\u003e\\u003cp\\u003eSixty 3-month-old male C57BL/6J mice, weighing 25.0\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;3.2 g, were obtained from the Laboratory Animal Center of Shaanxi Normal University. Mice were housed in the Institute of Sports Biology of Shaanxi Normal University under controlled conditions (25\\u0026deg;C, 50% \\u0026plusmn; 10% humidity, 12 h light/dark cycle) with \\u003cem\\u003ead libitum\\u003c/em\\u003e access to food and water. After a one-week acclimatization period, mice were randomly divided into sedentary and ET groups (thirty mice per group). Mice in the ET group underwent six weeks of treadmill training. All mice subsequently received either sham surgery or myocardial I/R surgery. Mice with successful I/R induction were assigned to the following groups: I/R 2 h group (Sed-I/R 2 h), I/R 24 h group (Sed-I/R 24 h), ET\\u0026thinsp;+\\u0026thinsp;I/R 2 h group (ET-I/R 2 h) and ET\\u0026thinsp;+\\u0026thinsp;I/R 24 h group (ET-I/R 24 h). Sham controls included the sham-operated group (Sed-Sham) and ET\\u0026thinsp;+\\u0026thinsp;sham-operated group (ET-Sham), with six mice per group (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eA). All experiments adhered to the Guide for Care and Use of Laboratory Animals and were approved by the Ethical Committee of Shaanxi Normal University (approval number: 202416015). Animal grouping and training were performed by a single observer to ensure that subsequent experimenters remained blinded to the group assignments.\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec4\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003e2.2 ET protocol\\u003c/h2\\u003e\\u003cp\\u003eMice in the ET group performed aerobic exercise on an eight-channel treadmill (Anhui Zhenghua Biological Instrument Equipment Co., Ltd., Anhui, China), following a previously published protocol with minor modifications (Massett et al. \\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e2021\\u003c/span\\u003e). The first week was dedicated to adaptive training, starting at 8 m/min for 10 min, with incremental increases of 2 m/min in speed and 12 min in duration each day. By the fifth training day, the speed was raised to 16 m/min for 60 min. From the second to the seventh week, mice engaged in formal training at 16 m/min for 60 min per day, five days a week.\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec5\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003e2.3 Myocardial I/R model\\u003c/h2\\u003e\\u003cp\\u003eOn the day after the final ET intervention, mice were anesthetized via inhalation of a mixture of isoflurane and oxygen (3% for induction and 1% for maintenance) using a small animal anesthesia machine (Beijing Zhongshi Dichuang Technology Development Co., Ltd., Beijing, China) and placed in a supine position on the operating table. The depth of anesthesia was standardized by maintaining a heart rate of 450\\u0026ndash;500 beats/min. After fixation and dehairing, the chest was opened at the fourth intercostal space to expose the heart. The left anterior descending coronary artery was ligated for 30 min, followed by 2 h or 24 h of reperfusion under normal cage conditions. An Electrocardiogram (ECG) was used to monitor the surgery, and ST-segment elevation indicated cardiac ischemia. Sham controls underwent identical procedures without artery ligation.\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec6\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003e2.4 Cardiac function measurement\\u003c/h2\\u003e\\u003cp\\u003eCardiac function was evaluated using a small animal ultrasonic detector (Vinno 6 VET, VINNO, Suzhou, China) with M-mode ultrasound. The left ventricular internal dimension at diastole (LVIDd), left ventricular internal dimension at systole (LVIDs), ejection fraction (EF%) and fractional shortening (FS%) were measured or calculated using the ultrasound equipment. After measurement, mice were deeply anesthetized and euthanized by decapitation. Hearts were quickly removed on ice, and fixed in 4% paraformaldehyde (PFA) or liquid nitrogen for further analysis.\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec7\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003e2.5 Histology staining\\u003c/h2\\u003e\\u003cp\\u003ePFA-fixed hearts were embedded in paraffin. Sections (5 \\u0026micro;m thick) were prepared, and consecutive sections from the same anatomical position in each group were selected for Masson's trichrome staining to evaluate structural pathology.\\u003c/p\\u003e\\u003cp\\u003eEnhanced Perls Prussian blue staining was performed using a staining kit (G1428, Solarbio, Beijing Solarbio Science \\u0026amp; Technology Co., Ltd., Beijing, China) to detect iron deposits. Paraffin sections were deparaffinized in xylene, rehydrated through a gradient of ethanol solutions, and rinsed in distilled water. Sections were incubated with freshly prepared Perls staining working solution at 37\\u0026deg;C for 20 min, rinsed with distilled water. Then incubation solution was added to the slides and incubated at 37\\u0026deg;C for 10 min, gently rinsed with PBS 1 min for three times. After that, sections were incubated with enhancement working solution at 37\\u0026deg;C for 20 min, gently rinsed with PBS 5 s for three times. Hematoxylin was used to stain the nuclei and rinsed with distilled water. Sections were dehydrated through gradient ethanol, cleared in xylene, and mounted. Ferrohemoglobin and trivalent iron appeared as brownish yellow or brownish red, with nuclei appeared as blue-purple.\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec8\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003e2.6 Bioassay Kit Testing\\u003c/h2\\u003e\\u003cp\\u003eLiquid nitrogen-fixed heart tissues were used to detect total glutathione (T-GSH) and lipid peroxidation (LPO) using biochemical assay kits (E-BC-K097-M, E-BC-K176-M, Elabscience Biotechnology Co., Ltd., Wuhan, China). Three samples were randomly selected from each group for tissue homogenization. 20 mg sample of myocardial tissues was homogenized in 200 \\u0026micro;L of PBS. After centrifuging at 12,000 rpm for 10 min, the supernatant was collected. The assays were performed strictly according to the manufacturer\\u0026rsquo;s instructions, and results were expressed as relative fold changes compared to the Sed-Sham group.\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec9\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003e2.7 Cell culture\\u003c/h2\\u003e\\u003cp\\u003eH9C2 cells (SNL-029, Wuhan Sunncell Biotechnology Co., Ltd., Wuhan, China) were cultured in complete medium consisting of DMEM, FBS, and Penicillin/ Streptomycin (C100C5, NCM Biotech, Suzhou, China) at a ratio of 90:10:1 in a standard incubator set at 37\\u0026deg;C with 5% CO\\u003csub\\u003e2\\u003c/sub\\u003e. When the cell density reached 70%\\u0026ndash;80%, cells were used for experiments.\\u003c/p\\u003e\\u003cp\\u003eTo induce the H/R model, H9C2 cells were initially incubated under normal conditions (37\\u0026deg;C, 5% CO\\u003csub\\u003e2\\u003c/sub\\u003e), then exposed to hypoxic conditions (37\\u0026deg;C, 1% O\\u003csub\\u003e2\\u003c/sub\\u003e, 5% CO\\u003csub\\u003e2\\u003c/sub\\u003e, 94% N\\u003csub\\u003e2\\u003c/sub\\u003e) for 8 h, followed by reoxygenation for 12 h (Han et al. \\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e2021\\u003c/span\\u003e).\\u003c/p\\u003e\\u003cp\\u003eTo investigate the effect of AMPK activation, H9C2 cells were treated with the AMPK activator AICAR (HY-13417, MedChem Express, NJ, USA) at 1 mM for 20 h to simulate the effects of ET. In addition, Compound C (C-C, S7306, Selleck Chemicals LLC, TX, USA) at 4 \\u0026micro;M for 20 h was used to inhibit AMPK activation, and Firsocostat (HY-16901, MedChem Express) at 500 nM for 20 h was employed to block ACC phosphorylation.\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec10\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003e2.8 Dihydroethidium (DHE) staining\\u003c/h2\\u003e\\u003cp\\u003eDHE, a fluorescent probe for detecting intracellular superoxide anions (O₂\\u0026middot;⁻), is oxidized by intracellular superoxide upon entering cells, forming a product that emits red fluorescence. In this study, frozen heart sections and H9C2 cells were incubated with DHE (S0063, Beyotime, Shanghai, China) in a dark environment at room temperature for 50 min. After washes with PBS, 5 min each for three times, sections were sealed and visualized using a fluorescence microscope (Nikon Eclipse 55i, Tokyo, Japan). Fluorescence intensity was quantified using Image-Pro Plus software.\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec11\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003e2.9 Western blotting\\u003c/h2\\u003e\\u003cp\\u003eTotal proteins from heart tissues and H9C2 cells were extracted in radio immunoprecipitation assay (RIPA, C500008, Sangon Biotech (Shanghai) Co., Ltd., Shanghai, China) buffer supplemented with phenylmethanesulfonyl fluoride (PMSF, P1081, Beyotime) and phosphatase inhibitors (P1005, Beyotime) at a ratio of 100:1:1. The mixture was homogenized on ice and centrifuged (12,000 \\u0026times; \\u003cem\\u003eg\\u003c/em\\u003e, 15 min, 4\\u0026deg;C) to collect the supernatant. Protein concentration was determined using the BCA assay (C503021, Sangon Biotech). A 20 \\u0026micro;g sample of protein was separated on an 8%\\u0026ndash;12% SDS polyacrylamide gel and transferred onto nitrocellulose membranes. Membranes were blocked with 5% bovine serum albumin (BSA) for 1.5 h, and then incubated with primary antibodies at 4℃ overnight. The following antibodies were used: AMPKα, p-AMPKα, ACC and p-ACC (1:1000, #9957, Cell Signaling Technology, Inc. (CST), Danvers, MA, USA), ACC1 (1:1000, A19627, ABclonal Technology Co., Ltd, Wuhan, China), superoxide dismutase 1 (SOD1, 1:1000, ab308181, Abcam, Cambridge, UK), SOD2 (1:1000, ab68155, Abcam), GPX4 (1:2000, 67763-1-IG, Proteintech Group, Inc. Wuhan, CHN), acyl-CoA synthetase long-chain family member 4 (ACSL4, 22401-1-AP, 1:1000, Proteintech) and SLC7A11 (1:1000, 26864-1-AP, Proteintech). GAPDH (1:5000, 60004-1-IG, Proteintech) was used as the housekeeping protein. After washing with Tris-Buffered Saline Tween (TBST) 5 min for three times, membranes were incubated with goat anti-rabbit or mouse IgG, HRP conjugated secondary antibodies (1:5000, SA00001-2, SA00001-1, Proteintech) at room temperature for 1.5 h. Membranes were then washed with TBST and visualized using an enhanced chemiluminescence (ECL) reagent (C520045, Sangon Biotech) and an imaging system (ChemiDoc\\u0026trade; MP, Bio-Rad).\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec12\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003e2.10 Cell malonyl CoA assay\\u003c/h2\\u003e\\u003cp\\u003eThe Malonyl CoA (MCA) levels in H9C2 cells were detected by using Rat MCA ELISA Kit (CB10239-Ra, Shanghai Keaibo Biotechnology Co., Ltd, Shanghai, China). After intervention, cells were washed with pre-cooled PBS, trypsinized, and centrifuged at 1000\\u0026times;g for 5 min. The cell pellet was washed three times with cold PBS. For every 1\\u0026times;10⁶ cells, 150 \\u0026micro;L of PBS was added. Cells were subjected to ultrasonic disruption at 20 kHz and 150 W in an ice bath, with three bursts (2 s on, 3 s off). The extract was centrifuged at 1500\\u0026times;g for 10 min, and the supernatant was analyzed using the kit according to the manufacturer\\u0026rsquo;s instructions.\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec13\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003e2.11 Statistical analysis\\u003c/h2\\u003e\\u003cp\\u003eWestern blotting results were analyzed using Image J software (National Institutes of Health, MD, USA), and data were visualized with GraphPad Prism 8.0 software (Dotmatics, UK). All experimental results were expressed as mean\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;standard error of the mean (SEM) to indicate the sampling error of the sample mean. Animal results were analyzed using two-factor ANOVA, while H9C2 cell results were analyzed using one-way ANOVA with Tukey\\u0026rsquo;s post-hoc test. Significant differences between groups were defined as *\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05, **\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01 levels.\\u003c/p\\u003e\\u003c/div\\u003e\"},{\"header\":\"3. Results\",\"content\":\"\\u003cdiv id=\\\"Sec15\\\" class=\\\"Section2\\\"\\u003e\\n\\u003ch2\\u003e3.1 ET attenuated myocardial I/R-induced oxidative stress and preserved cardiac function\\u003c/h2\\u003e\\n\\u003cp\\u003eCardiac function was assessed by measuring LVIDd, LVIDs, EF% and FS%. Compared to the Sed-Sham group, myocardial I/R significantly increased LVIDs (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05 at 2 h, \\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01 at 24 h) and LVIDd (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01 at both 2 h and 24 h), while decreasing EF% and FS% (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01 at both 2 h and 24 h). Compared to the Sed-I/R groups, ET significantly reduced LVIDs (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05 at both 2 h and 24 h) and LVIDd (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01 at 24 h), while increasing EF% (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01 at both 2 h and 24 h) and FS% (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05 at 2 h, \\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01 at 24 h) (Fig.\\u0026nbsp;\\u003cspan class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eB-F).\\u003c/p\\u003e\\n\\u003cp\\u003eMasson\\u0026rsquo;s trichrome staining revealed distinct alterations in cardiac structure. In the Sed-Sham and ET-Sham groups, myocardial fibers (stained red) were arranged regularly, with minimal collagen deposition (stained blue) surrounding vascular structures. In contrast, the Sed-I/R 24 h group exhibited disorganized myocardial fibers, extensive abnormal collagen deposition, and structural disruption. ET markedly improved tissue architecture in the ET-I/R 24 h group, showing relatively ordered fiber arrangement and significantly reduced collagen deposition (Fig.\\u0026nbsp;\\u003cspan class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eG).\\u003c/p\\u003e\\n\\u003cp\\u003eDHE staining demonstrated that myocardial I/R increased O₂\\u0026middot;⁻ levels compared to the Sed-Sham group (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01). Notably, ET effectively suppressed I/R-induced ROS (O₂\\u0026middot;⁻) overproduction (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05, Fig.\\u0026nbsp;\\u003cspan class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eH, I).\\u003c/p\\u003e\\n\\u003cp\\u003eThese findings collectively indicated that myocardial I/R resulted in excessive ROS production and impaired cardiac function and structure, while, ET protected against these I/R-induced impairments.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n\\u003c/div\\u003e\\n\\u003cdiv id=\\\"Sec16\\\" class=\\\"Section2\\\"\\u003e\\n\\u003ch2\\u003e3.2 ET attenuated I/R-induced ferroptosis and increased the phosphorylation of AMPK\\u0026alpha; and ACC\\u003c/h2\\u003e\\n\\u003cp\\u003eWestern blotting analysis revealed that compared to the Sed-Sham group, myocardial I/R significantly downregulated the expression of GPX4 (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05 at 2 h) and SLC7A11 (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01 at both 2 h and 24 h), increased ACSL4 expression (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01 at both 2 h and 24 h). ET significantly increased the expression of GPX4 and SLC7A11, and downregulated ACSL4 expression (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01 at both 2 h and 24 h) in the I/R hearts (Fig.\\u0026nbsp;\\u003cspan class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eA-D). In addition, decreased T-GSH levels (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05 at I/R 24 h) and increased LPO levels (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01 at 2 h, \\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05 at 24 h) and iron deposition were shown in the Sed-I/R group when compared to the Sed-Sham group. ET significantly increased T-GSH levels (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05 at 2 h and 24 h) and reduced LPO levels (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01 at 2 h and 24 h) and iron deposition in the I/R heart (Fig.\\u0026nbsp;\\u003cspan class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eE-G).\\u003c/p\\u003e\\n\\u003cp\\u003eMeanwhile, compared to the Sed-Sham group, myocardial I/R suppressed the phosphorylation of AMPK\\u0026alpha; (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01 at 2 h) and ACC (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01 at both 2 h and 24 h). ET significantly enhanced the phosphorylation of AMPK\\u0026alpha; (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01 in the Sham, I/R 2 h and 24 h groups) and ACC (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01 in the Sham group, \\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05 in the I/R 2 h group) (Fig.\\u0026nbsp;\\u003cspan class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eH-J). These findings underscored the protective effect of ET against I/R-induced ferroptosis and confirmed that ET effectively activated AMPK\\u0026alpha; and increased ACC phosphorylation.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n\\u003c/div\\u003e\\n\\u003cdiv id=\\\"Sec17\\\" class=\\\"Section2\\\"\\u003e\\n\\u003ch2\\u003e3.3 AMPK inhibition increased ACC phosphorylation and exacerbated H/R-induced ferroptosis\\u003c/h2\\u003e\\n\\u003cp\\u003eTo model the effects of I/R and AMPK inactivation, H9C2 cells were subjected to H/R and/or treated with the AMPK inhibitor Compound C(C-C). Results revealed that H/R treatment alone significantly reduced AMPK phosphorylation (\\u003cem\\u003eP\\u0026thinsp;\\u0026lt;\\u003c/em\\u003e\\u0026thinsp;0.01) and SOD2 expression (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01), increased the ROS levels (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01). C-C intervention under basal conditions also increased the ROS levels (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01), decreased the antioxidant enzymes SOD1 and SOD2 expression (both \\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01). Additionally, C-C further inhibited AMPK\\u0026alpha; phosphorylation (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01), elevated ROS levels (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01) and suppressed SOD1 and SOD2 expression (both \\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01) in H/R-treated cells (Fig.\\u0026nbsp;\\u003cspan class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eA-F), indicating AMPK inhibition severely compromises antioxidant capacity.\\u003c/p\\u003e\\n\\u003cp\\u003eAnalysis of the expression of ACC and ferroptosis markers showed that C-C, H/R, and their combination all significantly reduced ACC phosphorylation (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05 for C-C, \\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01 for H/R and combination). Both C-C or H/R treatment alone upregulated the expression of ACSL4 (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01 for both) and ACC1 (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01 for C-C, \\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05 for H/R), reduced the expression of SLC7A11 (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01 for both) and GPX4 (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01 for both). Combining C-C with H/R intensified these pro-ferroptotic changes, suppressed the expression of SLC7A11 (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05) and GPX4 (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01), and further upregulated the expression of ACSL4 (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01) and ACC1 (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01) compared to H/R alone (Fig.\\u0026nbsp;\\u003cspan class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eD, G-K). Collectively, these data indicated that both H/R and AMPK inhibition could decrease ACC phosphorylation, trigger ferroptosis in H9C2 cells, and AMPK inhibition dramatically exacerbated this ferroptotic process.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n\\u003c/div\\u003e\\n\\u003cdiv id=\\\"Sec18\\\" class=\\\"Section2\\\"\\u003e\\n\\u003ch2\\u003e\\u003cstrong\\u003e3.4 AMPK activation induced ACC phosphorylation and attenuated H/R-induced oxidative stress and ferroptosis\\u003c/strong\\u003e\\u003c/h2\\u003e\\n\\u003cp\\u003eWe verified that ET can activate AMPK and inhibit ferroptosis. To further explore the mechanism, we treated H9C2 cells with AICAR to simulate the effects of ET. Results revealed that AICAR treatment effectively activated AMPK\\u0026alpha; (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01) and reduced ROS levels (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01) in H/R-treated cells (Fig.\\u0026nbsp;\\u003cspan class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eA-C). Under basal conditions, AICAR upregulated ACC phosphorylation (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01) and the expression of SOD1 (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01) and GPX4 (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01), while reducing ACSL4 expression (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01). In H/R condition, AICAR notably reversed H/R-induced alterations by increasing ACC phosphorylation (\\u003cem\\u003eP\\u0026thinsp;\\u0026lt;\\u003c/em\\u003e\\u0026thinsp;0.01) and upregulating the expression of SOD2 (\\u003cem\\u003eP\\u0026thinsp;\\u0026lt;\\u003c/em\\u003e\\u0026thinsp;0.01), SLC7A11 (\\u003cem\\u003eP\\u0026thinsp;\\u0026lt;\\u003c/em\\u003e\\u0026thinsp;0.01) and GPX4 (\\u003cem\\u003eP\\u0026thinsp;\\u0026lt;\\u003c/em\\u003e\\u0026thinsp;0.01), as well as reducing the expression of ACC1 (\\u003cem\\u003eP\\u0026thinsp;\\u0026lt;\\u003c/em\\u003e\\u0026thinsp;0.01) and ACSL4 (\\u003cem\\u003eP\\u0026thinsp;\\u0026lt;\\u003c/em\\u003e\\u0026thinsp;0.01) in H/R-treated cells (Fig.\\u0026nbsp;\\u003cspan class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eD-K). These findings confirmed that AICAR-mediated AMPK activation protected against H/R injury by enhancing antioxidant defenses and regulating ferroptotic pathways.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n\\u003c/div\\u003e\\n\\u003cdiv id=\\\"Sec19\\\" class=\\\"Section2\\\"\\u003e\\n\\u003ch2\\u003e3.5 Inhibition of ACC phosphorylation aggravated ferroptosis and abrogated the protective effects of AICAR\\u003c/h2\\u003e\\n\\u003cp\\u003eTo further elucidate the role of ACC in H/R-induced ferroptosis, and its interaction with AMPK activation, we treated H9C2 cells with Firsocostat, a specific inhibitor of ACC phosphorylation. Results revealed that Firsocostat intervention under basal conditions significantly reduced the phosphorylation of ACC (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01) and AMPK\\u0026alpha; (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01), downregulated the expression of SLC7A11 (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05) and GPX4 (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01), increased the expression of ACC1 (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05) and ACSL4 (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01) and MCA levels (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01) (Fig.\\u0026nbsp;\\u003cspan class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eA-H). In H/R-treated cells, Firsocostat similarly suppressed ACC phosphorylation (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01) and the expression of SLC7A11 (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01) and GPX4 (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05), while increasing the expression of ACC1 (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05) and ACSL4 (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01) (Fig.\\u0026nbsp;\\u003cspan class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eA-H).\\u003c/p\\u003e\\n\\u003cp\\u003eCo-treatment with AICAR and Firsocostat in H/R cells attenuated AICAR's protective effects. While AMPK\\u0026alpha; phosphorylation remained elevated compared to H/R alone (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.01), AICAR failed to increase the expression of SLC7A11 and GPX4 or decrease the expression of ACC1 and ACSL4 as well as MCA levels (Fig.\\u0026nbsp;\\u003cspan class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eA-H). These findings demonstrated that blocking ACC phosphorylation (inactivating ACC) exacerbated H/R-induced ferroptosis by impairing the antioxidant defense system and promoting fatty acid synthesis. This indicated that ACC phosphorylation, not just AMPK activation, was essential for ferroptosis suppression.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n\\u003c/div\\u003e\"},{\"header\":\"4. Discussion\",\"content\":\"\\u003cp\\u003eMyocardial I/R injury remains a critical clinical challenge (Tong et al. 2024). ET has emerged as a promising non-pharmacological strategy for cardiovascular protection (Fiuza-Luces et al. \\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e), however, its role in regulating ferroptosis is not fully elucidated. In this study, we demonstrated that ET activates AMPK, promotes ACC phosphorylation, which mitigates I/R-induced oxidative stress and ferroptosis. \\u003cem\\u003eIn vitro\\u003c/em\\u003e experiments further confirmed that AMPK inactivation inhibits ACC phosphorylation, exacerbates ferroptosis-related phenotypes, while, AMPK activation increased ACC phosphorylation​​, preserved GPX4/SLC7A11 and downregulated ACSL4. Critically, direct inhibition of ACC phosphorylation aggravates ferroptosis and abolishes AMPK\\u0026rsquo;s benefits​​, proving ACC is a required effector of AMPK-activation anti-ferroptotic pathway in ET-induced I/R protection. Collectively, our findings provide experimental evidence supporting ​​ET could serve as a preconditioning strategy​​ for cardioprotection, and highlight ACC phosphorylation as a druggable target​​ for I/R injury.\\u003c/p\\u003e\\u003cp\\u003eMyocardial I/R injury is characterized by excessive ROS production, which drives cellular damage, cardiac dysfunction, and structural remodeling (Zhang et al. \\u003cspan citationid=\\\"CR48\\\" class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e). Consistent with previous studies (Wu et al. \\u003cspan citationid=\\\"CR40\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e; Wang et al. \\u003cspan citationid=\\\"CR37\\\" class=\\\"CitationRef\\\"\\u003e2025\\u003c/span\\u003e), our experiments demonstrated that myocardial I/R led to a marked decline in cardiac function at both 2 h (early reperfusion) and 24 h (subacute phase) post-reperfusion. Previous study has confirmed that ET also improves cardiac function three weeks after I/R (Xu et al. \\u003cspan citationid=\\\"CR42\\\" class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e), our data extend this finding by showing that ET exerts protective effects even in the immediate and subacute phases post-I/R. Functional deficits in I/R hearts were accompanied by structural abnormalities, including disorganized myocardial fibers and extensive collagen deposition, consistent with previous reports of I/R-induced cardiac remodeling (Dom\\u0026iacute;nguez-Rodr\\u0026iacute;guez et al. \\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e). I/R significantly elevated superoxide anion (O₂\\u0026middot;⁻) levels, which further intensifies cardiomyocyte damage and death, and contributes to long-term remodeling by activating pro-fibrotic signaling (Zhang et al. \\u003cspan citationid=\\\"CR48\\\" class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e; Xiang et al. \\u003cspan citationid=\\\"CR41\\\" class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e). In this study, we confirmed that ET improved cardiac function and preserved myocardial architecture, with reduced collagen deposition and suppressed O₂\\u0026middot;⁻ overproduction. Based on previous report, the protective effect of ET is involved with regulating myokines, enhancing antioxidant capacity and improving mitochondrial function (Boulghobra et al. \\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2020\\u003c/span\\u003e; Rabinovich-Nikitin et al. 2018). This aligns with studies showing that ET reduced ROS accumulation, preserved cardiac contractile function and pumping efficiency post-I/R injury (Zhang, Huang et al. \\u003cspan citationid=\\\"CR50\\\" class=\\\"CitationRef\\\"\\u003e2025\\u003c/span\\u003e).\\u003c/p\\u003e\\u003cp\\u003eEvidence suggests that ferroptosis is widely implicated in various oxidative stress-related diseases (Sun et al. \\u003cspan citationid=\\\"CR34\\\" class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e; Zhu et al. \\u003cspan citationid=\\\"CR53\\\" class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e; Zhang, Liu et al. \\u003cspan citationid=\\\"CR47\\\" class=\\\"CitationRef\\\"\\u003e2025\\u003c/span\\u003e). The core mechanism of ferroptosis involves iron-dependent accumulation of lipid peroxides, which is driven by increased iron overload and impaired clearance of lipid peroxide (Pan et al. \\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e). Key molecular regulators of ferroptosis include GPX4 (lipid peroxide scavenger), SLC7A11 (subunit of System Xc⁻, responsible for cystine import to synthesize GSH), and ACSL4 (enzyme promoting PUFA synthesis, a substrate for lipid peroxidation) (Yao et al. \\u003cspan citationid=\\\"CR46\\\" class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e; Doll et al. \\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e2017\\u003c/span\\u003e). Previous studies have shown that myocardial I/R attenuates System Xc⁻ activity (consistent with reduced SLC7A11 expression), inhibits GPX4 activity as early as 2 h post-I/R, and upregulates ACSL4 expression (Yao et al. \\u003cspan citationid=\\\"CR46\\\" class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e; Fan et al. \\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e2021\\u003c/span\\u003e). In this study, our study demonstrated that myocardial I/R in sedentary mice significantly downregulated GPX4 and SLC7A11 expression, increased ACSL4 expression, reduced total GSH (T-GSH) levels at 24 h post-I/R, and elevated LPO levels and iron deposition, collectively confirming that I/R enhances ferroptosis in the myocardium. In contrast, ET preconditioning upregulated GPX4 and SLC7A11 expression, reduced ACSL4 expression and LPO levels, and increased T-GSH levels, protected the heart against ferroptosis. These results are consistent with a recent study showing that pre-exercise improves ischemic stroke-induced ferroptosis by increasing SLC7A11 and GPX4 expression (Huang et al. \\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e). In addition, studies have suggested that ferroptotic signals appear during ischemia, and ferroptosis occurs in the late reperfusion phase (Xiang et al. \\u003cspan citationid=\\\"CR41\\\" class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e). In the present study, the expression of certain ferroptosis-related proteins varied at different time points post-I/R, and the specific timing and duration of ferroptosis in myocardial I/R injury remain to be further clarified.\\u003c/p\\u003e\\u003cp\\u003eThe mechanism underlying ET\\u0026rsquo;s anti-ferroptotic effect was linked to the AMPK/ ACC signaling pathway. AMPK is a master regulator of cellular energy homeostasis, which is activated by ET (via increased AMP/ATP ratio) and has been implicated in antioxidant and anti-ferroptotic responses in various tissues (Lee et al. \\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e2020\\u003c/span\\u003e; Zhong et al. \\u003cspan citationid=\\\"CR52\\\" class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e; Carapeto et al. \\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e). AMPK activation normally inhibits ACC by phosphorylating it, and reduces fatty acid synthesis, decreases PUFA production, and lowers lipid peroxidation, improves cellular energy supply (Lee et al. \\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e2020\\u003c/span\\u003e; Pang et al. \\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e2021\\u003c/span\\u003e). Our \\u003cem\\u003ein vivo\\u003c/em\\u003e data confirmed that myocardial I/R in sedentary mice significantly suppressed the phosphorylation of both AMPKα and ACC, while ET preconditioning restored and enhanced AMPKα phosphorylation, which in turn specifically promoted ACC phosphorylation. We speculated that activated AMPKα phosphorylated and inactivated ACC, thereby reducing lipid substrate availability for peroxidation, which is a critical step in ferroptosis. Previous research has shown that swimming alleviates obesity and liver injury in non-alcoholic fatty liver disease by downregulating ACC1 protein expression in obese rats (Yang et al. \\u003cspan citationid=\\\"CR45\\\" class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e), suggesting that ET-induced regulation of ACC1 is a conserved mechanism across organs.\\u003c/p\\u003e\\u003cp\\u003eTo further validate the role of AMPK/ACC signaling \\u003cem\\u003ein vitro\\u003c/em\\u003e, we used H9C2 cardiomyocytes subjected to H/R. Consistent with \\u003cem\\u003ein vivo\\u003c/em\\u003e findings, H/R alone suppressed the phosphorylation of AMPKα and ACC, increased ROS production, and downregulated antioxidant enzymes SOD1 and SOD2, mirroring the oxidative stress phenotype observed in I/R hearts. Notably, treatment with C-C further reduced ACC phosphorylation, increased ACSL4 expression, and decreased GPX4 and SLC7A11 expression, confirming that AMPK is a key upstream regulator of ACC and ferroptosis during I/R. Conversely, activation of AMPK attenuated H/R-induced oxidative stress and ferroptosis, increased ACC phosphorylation and reduced MCA levels, which is crucial for fatty acid synthesis and lipid peroxidation (Ding et al. \\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e). Based on these, we speculate that AMPK enhances cystine uptake and GSH synthesis via SLC7A11, strengthens lipid peroxide clearance via GPX4, and reduces the production of peroxidation substrates by inhibiting ACSL4 and ACC1. Given that ACC1-mediated fatty acid synthesis is the primary source of PUFAs, we hypothesized that ACC1, rather than ACC2, would be the critical isoform mediating AMPK\\u0026rsquo;s anti-ferroptotic effect (Wang et al. \\u003cspan citationid=\\\"CR38\\\" class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e).\\u003c/p\\u003e\\u003cp\\u003eTo directly test whether ACC is a non-redundant effector of AMPK, we used Firsocostat, a specific ACC inhibitor that blocks ACC phosphorylation, thereby activating ACC enzymatic activity. We found that reducing ACC phosphorylation by Firsocostat promoted the expression of ACSL4 and ACC1, decreased the expression of SLC7A11 and GPX4, and increased MCA levels. In H/R-treated cells, Firsocostat exacerbated ferroptosis and abrogated the protective effects of AICAR. Despite sustained AMPK activation, AICAR failed to upregulate the expression of GPX4 and SLC7A11 or downregulate the expression of ACC1 and ACSL4 as well as MCA levels in the presence of Firsocostat. These data provide definitive evidence that ACC1 phosphorylation is indispensable for AMPK-mediated ferroptosis inhibition. By inactivating ACC, AMPK reduced MCA-driven lipid peroxidation, and modulates the expression of GPX4, SLC7A11 and ACSL4 through a metabolism-linked oxidative stress regulatory cascade. This finding is particularly significant when compared to previous studies on AMPK\\u0026rsquo;s anti-ferroptotic mechanisms. For example, AMPK has been shown to suppress ferroptosis in tumor cells by phosphorylating BECN1 (Song et al. \\u003cspan citationid=\\\"CR32\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e), or in cerebral I/R via the AMPK/FoxO3a pathway (Zhong et al. \\u003cspan citationid=\\\"CR52\\\" class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e). Additionally, energy stress promotes AMPK to mediate GPX4 expression and inhibits erastin-induced ferroptosis in renal cancer through the JAK2/STAT3/P53 signaling (Li et al. \\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e) or mTOR signaling (Kou et al. \\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e2025\\u003c/span\\u003e). However, these mechanisms are cell-type or disease-specific, whereas our data demonstrate that in the myocardium, ACC phosphorylation is the dominant, non-redundant pathway mediating AMPK\\u0026rsquo;s anti-ferroptotic effect.\\u003c/p\\u003e\\u003cp\\u003eDespite the novel findings of this study, several limitations warrant consideration. Firstly, while our focus was on the AMPK/ACC1 signaling pathway, other AMPK substrates (e.g., FoxO3a, mTOR) may also contribute to the antioxidant and anti-ferroptotic effects of ET. Future studies using isoform-specific AMPK knockout mice or ACC1/ACC2 double knockout models will help clarify the relative contributions of ACC1 versus other AMPK substrates. Secondly, we used pharmacological inhibitors (C-C, Firsocostat) to modulate AMPK and ACC1 activity; genetic models will further strengthen the causal link between AMPK/ACC1 signaling and ET-induced I/R protection. Thirdly, ferroptosis interacts with other forms of cell death and cellular processes, exploring these interrelationships could reveal synergistic mechanisms of ET\\u0026rsquo;s protective effects. For instance, does ACC phosphorylation also regulate autophagy to further suppress ferroptosis? Finally, the optimal ET protocol (e.g., exercise intensity, duration, frequency, and timing relative to I/R) was not evaluated in this study. Variables such as moderate vs. high-intensity exercise, or pre-I/R vs. post-I/R ET, may affect the magnitude of ACC phosphorylation and ferroptosis inhibition, limiting the clinical translatability of our findings. Future studies should address these variables to provide a basis for evidence-based exercise prescriptions in clinical practice.\\u003c/p\\u003e\"},{\"header\":\"5. Conclusion\",\"content\":\"\\u003cp\\u003eOur findings suggest that ET protects against myocardial I/R injury by activating AMPK, phosphorylating ACC, and inhibiting ferroptosis by upregulating the expression of GPX4 and SLC7A11, and downregulating ACSL4. These results extend previous studies by identifying ferroptosis as a key target of ET-mediated I/R protection, and by establishing the AMPK/ACC signaling as a critical regulator of this process. While ET is known to improve cardiac resilience, our work links ET to ferroptosis inhibition, offering new insights into potential therapeutic strategies for I/R injury.\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003ch2\\u003eDeclaration of competing interest\\u003c/h2\\u003e\\u003cp\\u003eThe authors declare no potential conflict of interest.\\u003c/p\\u003e\\u003c/p\\u003e\\u003ch2\\u003eAuthor Contribution\\u003c/h2\\u003e\\u003cp\\u003eYX, MC and ZT conceived and designed the experiments. YX, JT, CW, RL and YZ performed the experiments and analyzed the data. MC and YX contributed reagents, materials, and analytical tools. MC, YX and ZT wrote and edited the manuscript.\\u003c/p\\u003e\\u003ch2\\u003eAcknowledgement\\u003c/h2\\u003e\\u003cp\\u003eThis work was supported by the Natural Science Foundation of Shaanxi Province of China (Grant Number 2023-JC-YB-204) to MC.\\u003c/p\\u003e\\u003ch2\\u003eData Availability\\u003c/h2\\u003e\\u003cp\\u003eAll data supporting the findings of this study are available within the paper.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\u003cli\\u003e\\u003cspan\\u003eBei Y, Zhu Y, Zhou J, Ai S, Yao J, Yin M, Hu M, Qi W, Spanos M, Li L, Wei M, Huang Z, Gao J, Liu C, van der Kraak PH, Li G, Lei Z, Sluijter JPG, Xiao J (2024) Inhibition of Hmbox1 promotes cardiomyocyte survival and glucose metabolism through Gck activation in ischemia/reperfusion injury. 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Redox Biol 62:102707\\u003c/span\\u003e\\u003c/li\\u003e\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":true,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":false,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":false,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true},\"keywords\":\"Exercise training, ferroptosis, myocardial ischemia-reperfusion, Acetyl-CoA carboxylase\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-8034270/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-8034270/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003eFerroptosis is critical in the pathogenesis of myocardial ischemia-reperfusion (I/R) injury. Exercise training (ET) protects against myocardial I/R injury, but its impact on ferroptosis and the mechanisms remain incompletely understood. This study explored whether AMP-activated protein kinase (AMPK)-regulated acetyl-CoA carboxylase (ACC) mediates ET-induced cardioprotection by suppressing ferroptosis in I/R mice. 3-month-old male C57BL/6J mice were divided into sedentary and ET groups. Mice in the ET group underwent six weeks of treadmill training before myocardial I/R induction. Cardiac function, oxidative stress, ferroptosis-related proteins expression were measured. H9C2 cardiomyocytes were exposed to hypoxia/reoxygenation (H/R) to simulate I/R. AMPK activity was modulated and ACC phosphorylation was blocked to explore the mechanism. In I/R mice, ET improved cardiac function, activated AMPKα, increased ACC phosphorylation, upregulated the expression of glutathione peroxidase 4 (GPX4) and solute carrier family 7 member 11 (SLC7A11), and downregulated acyl-CoA synthetase long chain family member 4 (ACSL4) expression. \\u003cem\\u003eIn vitro\\u003c/em\\u003e, H/R induced oxidative stress, reduced ACC phosphorylation and the expression of SLC7A11 and GPX4, and upregulated ACSL4 expression. AMPK inhibition reduced ACC phosphorylation, worsened H/R-induced oxidative stress and ferroptosis, while AMPK activation reversed these effects. Blocking ACC phosphorylation abolished the protective effects of AMPK activation and exacerbated H/R-induced ferroptosis. In conclusion, ET reduces I/R-induced ferroptosis, and AMPK-mediated ACC phosphorylation plays an important role in this process.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Exercise training inhibits myocardial ischemia-reperfusion-induced ferroptosis via AMPK- dependent ACC phosphorylation\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2025-11-20 07:54:40\",\"doi\":\"10.21203/rs.3.rs-8034270/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"a947ae5d-3ec2-42c5-ab42-89f11e708574\",\"owner\":[],\"postedDate\":\"November 20th, 2025\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"posted\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2026-03-02T16:40:41+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2025-11-20 07:54:40\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-8034270\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-8034270\",\"identity\":\"rs-8034270\",\"version\":[\"v1\"]},\"buildId\":\"8U1c8b4HqxoKbykW_rLl7\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}