Sirt1 deficiency promotes age-related heart failure through enhancing ferroptosis via GATA4-HADHA-GPX4 axis

Sirt1 deficiency promotes age-related heart failure through enhancing ferroptosis via GATA4-HADHA-GPX4 axis | 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 Article Sirt1 deficiency promotes age-related heart failure through enhancing ferroptosis via GATA4-HADHA-GPX4 axis Yun Zhang, Yu Duan, Yingchun Luo, Xuejie Han, Hui Yu, Yun Zhou, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7140279/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 23 Mar, 2026 Read the published version in Cell Death & Disease → Version 1 posted 9 You are reading this latest preprint version Abstract Aging is a major contributor to the escalating prevalence of heart failure (HF). Ferroptosis has been implicated in age-related disorders and cardiovascular diseases. The role of ferroptosis in age-related HF and the underlying mechanisms remain ambiguous. Herein, we found that aging rats displayed compromised cardiac function, with molecular characteristics indicative of ferroptosis, including diminished glutathione peroxidase 4 (GPX4) levels and heightened lipid peroxidation. Notably, a high-iron diet exacerbated ferroptosis and promoted cardiac dysfunction, while ferrostatin-1, a specific ferroptosis inhibitor, rescued this phenotype. Proteomic data analysis uncovered that the expression of hydroxyacyl-CoA dehydrogenase subunit A (HADHA) was significantly reduced in aging rats fed a high-iron diet. HADHA deficiency resulted in mitochondrial dysfunction and an accumulation of reactive oxygen species, leading to glutathione (GSH) exhaustion, causing the downregulation of GPX4 and subsequent ferroptosis. Furthermore, it was confirmed that the reduction of Sirt1 in the hearts of aging rats caused the downregulation of HADHA by binding and inhibited the expression of GATA4, a transcription factor of HADHA, as evidenced by co-immunoprecipitation experiments. Furthermore, resveratrol, a Sirt1 agonist, effectively shielded aging rats from HF by upregulating HADHA and mitigating ferroptosis. In conclusion, this study underscores the significance of ferroptosis in age-related HF, and suggests that targeting HADHA or Sirt1 may present potential strategies for the prevention and treatment of age-related HF. Biological sciences/Cell biology/Senescence Biological sciences/Cell biology/Cell death Heart failure Aging Ferroptosis HADHA Sirt1 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introdcution Heart failure (HF) is a leading cause of morbidity and mortality worldwide, affecting an estimated 56 million individuals globally [ 1 ] . Advanced age has been identified as a major risk factor for HF. By 2050, the global population of individuals aged 65 and over is projected to reach approximately 2 billion, and the number of people 80 years or older is projected to reach 425 million [ 2 ] , further exacerbating the burden of HF. Over the past few decades, the prevalence of HF has steadily increased due to the advancing age of the population and extended life [ 3 ] . Among the general adult population, HF prevalence ranges from 1–3%. This rate dramatically rises to 8% in individuals aged between 65 to 74 years, and it further surges to a staggering 16.1% in those aged over 74 years [ 4 , 5 ] . Given the significant growth of the aging population, age-related HF has emerged as a formidable challenge in the realm of global healthcare. Despite the urgency for effective therapeutic interventions, the precise mechanisms that driving age-related HF continue to be enigmatic. This presents a critical knowledge gap in our current understanding, underscoring the necessity for further research in this field. The pathophysiology of age-related HF is a complex interplay of various factors, many of which are not yet fully understood. Extensive research has explored the role of free radicals in the aging process. As the body ages, its ability to produce free radicals escalates, while its capacity to clear these potentially harmful molecules diminishes. This imbalance can lead to an accumulation of reactive oxygen species (ROS), disrupting cellular redox homeostasis and potentially triggering a cascade of age-related diseases [ 6 , 7 ] . It was reported that excessive ROS are likely to disrupt cellular redox homeostasis and induce ferroptosis [ 8 ] . Ferroptosis, a novel form of regulated cell death, is characterized by the lethal accumulation of iron-dependent lipid peroxides [ 9 ] . The role of ferroptosis in age-related diseases has been the subject of increasing scientific interest. Mounting studies have implicated ferroptosis in a range of age-related conditions, including Alzheimer's disease [ 10 ] , Parkinson's disease [ 11 ] , osteoporosis, and osteoarthritis [ 12 ] . More recently, the spotlight has turned to the potential role of ferroptosis in cardiovascular diseases [ 13 ] , including diabetic cardiomyopathy [ 14 ] , doxorubicin-induced cardiomyopathy [ 15 ] , abdominal aortic aneurysm [ 16 ] , myocardial ischemia-reperfusion injury [ 17 ] , and myocardial infarction [ 18 ] . Despite these advances, the role of ferroptosis in age-related HF and the underlying mechanisms remains a mystery, and warrant further exploration. In our present study, we elucidated the role of ferroptosis in age-related HF, and deciphered underlying mechanisms. Our findings found that aging rats displayed aggravated ferroptosis in the left ventricle, which was exacerbated by a high-iron diet. Interestingly, ferroptosis inhibitor (ferrostatin-1) improved cardiac function of aging rats. Mechanically, HADHA deficiency in the heart of aging rats leads to mitochondrial dysfunction, which in turn, results in an accumulation of reactive oxygen species (ROS), triggering a depletion of glutathione (GSH) and a downregulation of GPX4 expression. These changes increased the level of ferroptosis in cardiomyocytes, ultimately causing cardiac structural remodeling and the onset of HF. Furthermore, we found that the downregulation of Sirt1 reduced the expression of HADHA through restraint of GATA4 expression. A Sirt1 agonist, resveratrol, effectively reduced the level of ferroptosis in the hearts of aging rats and improved cardiac function. In summary, these findings illuminate the fundamental mechanisms of HF within the context of aging, particularly from the perspective of ferroptosis. It opens up a new avenue of research and intervention strategies, suggesting that modulation of ferroptosis could be a viable approach in the prevention and treatment of HF in the aging population. 2. Materials and Methods 2.1 Experimental animals The animal experiments in this study were performed in accordance with the Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee at the Harbin Medical University ( Ethical approval number:2020123 ). Male SD adult rats (200-250g) were purchased from Beijing Vital River Laboratory Animal Technology Co, Ltd (Beijing, China) and raised at the Experimental Animal Center of Harbin Medical University (Harbin, China).Cardiomyocyte-specific knockout GPX4 mice (Myh6-cre + GPX4 fl/fl ) were generated by Cyagen Biosciences, Inc (Suzhou,China). Briefly, Myh6-cre + GPX4 fl/fl were generated by crossing Myh6-Cre + mice with GPX4 flox/flox mice. Myh6-cre + GPX4 fl/fl offspring were born at the expected Mendelian ratio and were viable. Control mice for this group are sex- and age-matched wild type control. The animals were kept in cages with light/dark cycles of 12 h and fed with food and water available ad libitum in SPF conditions. 2.2 Treatment with high-iron diet To evaluate the effect of iron supplementation on age-related HF, 18-month old rats were randomly divided into two groups, one group received standard diet, another group received high-iron diet (1.5g/kg) for 4 months. Furthermore, 2-month old rats were randomly divided into two groups, one group received standard diet, another group received high-iron diet (1.5g/kg) for 4 months. 2.3 Treatment with ferrostatin-1 To testify the role of ferroptosis in age-related HF, 18-month old rats were randomly divided into two groups, one group were intraperitoneal injected with ferroptosis inhibitor ferrostatin-1 (0.8mg/kg), once a week for 4 months, the Vehicle control group were treated with saline of equal volume. 2.4 Treatment with acetylcysteine For acetylcysteine intervention experiment, 18-month old rats were randomly divided into two groups, one group received acetylcysteine dissolved in water (free drinking: 600mg/L), another group received water (free drinking) for 4 months. Furthermore, we used D-galactose to establish aging rat model via subcutaneous injection at a dose of 150mg/kg/day. Then, rats were randomly divided into two groups, one group received acetylcysteine dissolved in water (free drinkin: 600mg/L), another group received water (free drinking) for 12 weeks. 2.5 Treatment with resveratrol To assess the potential of resveratrol in age-related HF, 18-month old rats were randomly divided into two groups, one group received resveratrol (10mg/kg/d) through gavage for 4 months, another group received control solvent of equal volume. 2.6 Echocardiography Transthoracic echocardiography was performed on anesthetized rats using a Vivid 7 echo machine (GE Healthcare, Milwaukee, WI, USA) with two-dimensional M-mode analysis. The rats were anesthetized by 1% sodium pentobarbital (30mg/kg) intraperitoneal injection, then the left ventricular ejection fraction (LVEF), and left ventricular fractional shortening (LVFS) were tested for at least five nonstop cardiac cycles. 2.7 Histopathology The ventricular tissue of rats were collected, fixed overnight in 4% paraformaldehyde, embedded in paraffin, and serially sectioned at 4µm thickness. The tissues were then stained with hematoxylin and eosin (H&E) for routine histological examination. To measure collagen deposits, select sections were stained with Masson’s trichrome staining. Fibrotic area was quantified using ImageJ software and collagen volume fraction was calculated as collagen area/total area×100%. 2.8 Analysis of oxylipins The ventricular tissue samples were collected for oxylipidomics. Through targeted metabolomics using UPHLC-MS/MS with a EXIONLC System (SCIEX) connected to a SCIEX 6500 QTRAP + MS/MS system equipped equipped with an IonDrive Turbo V electrospray ionization (ESI) interface. Lipids were separated using a 1.7 µm C18 column (150*2.1mm). SCIEX Analyst Work Station Software (Version 1.6.3) and Multiquant 3.03 software were employed for MRM data acquisition and processing. 2.9 Culture of primary rat cardiomyocytes and fibroblasts Primary cardiomyocytes were isolated from the hearts of neonatal Sprague-Daw rats (1–3 days old). Briefly, the hearts was aseptically dissected and cut to small pieces. Then digested in 0.25% tryspin with gently shaking, and digestive fluid was collected in DMEM supplemented with 10% fetal bovine serum, centrifuged at 1200 rpm for 5 minutes. The cell pellet was then resuspended in DMEM containing 10% FBS and 1% penicillin-streptomycin. The cells were transferred to a culture dish and allowed to adhere for 90 minutes. Non-adherent cells, which primarily contain cardiomyocytes, were then transferred to new six-well plates and incubated at 37°C in a 5% CO 2 atmosphere. Upon reaching a fusion rate of 70%-80% and exhibiting good condition, the treatment was initiated. We used H 2 O 2 (50µmol/L) to establish a senescent cell model. 2.10 MTT assay Cell viability was assessed utilizing the MTT Cell Proliferation Assay (Beyotime, Shanghai, China), following the manufacturer's instructions. Briefly, cells were cultured in 96-well plates. Subsequent to the designated treatment, 10µL of MTT reagent was incorporated into each well containing phenol red-free culture medium, followed by an incubation period of 4 hours at 37°C. Then, DMSO was added to the cells, and the absorbance was measured by a microplate reader (Thermo, Massachusetts, USA). 2.11 JC-1 staining The mitochondrial membrane potential (MMP) was detected by JC-1 staining kit (Beyotime) in accordance to the manufacturer’s instructions. Briefly, primary rat cardiomyocytes were subjected to incubation with JC-1 staining solution (5pg/ml) at 37°C for 20 min. Subsequently, the cells were rinsed meticulously with JC-1 staining buffer to remove excess dye. Mitochondrial depolarization is indicated by an increase in the green/red fuorescence intensity ratio. 2.12 Intracellular reactive oxygen species (ROS) detection Intracellular levels of ROS were determined using Dihydroethidium (S0063, Beyotime). In brief, primary rat cardiomyocytes cultured were incubated with 5µM Dihydroethidium in serum-free medium at 37℃ for 30min away from light. Post incubation, cells were washed thoroughly to remove unincorporated dye, followed by examination using an immunofluorescence microscope (Zeiss, Jena, Germany). 2.13 Western blot The total protein samples were extracted from tissues of rats or primary cultured cardiomyocytes and cardiac fibroblasts. Briefly, approximately 30 ~ 50 µg of proteins were fractionated by 8 ~ 12% SDS-PAGE. Proteins were transferred to PVDF membranes (Millipore, Billerica, MA, USA). The samples were then incubated with primary antibodies for Tf (1:500, 17435-1-AP, Proteintech, Wuhan, China), FTH1 (1:500, bs-5907R, Bioss, Beijing, China), GPX4 (1:500, ab125066, Abcam, Cambridge, UK), HADHA (1:2000, 10758-1-AP, Proteintech, Wuhan, China) and Sirt1 (IP-1:500, 60303-1-Ig, Proteintech, Wuhan, China; IB-1:500, ab189494, Abcam, Cambridge, UK), GATA4 (1:500, 19530-1-AP, Proteintech, Wuhan, China), and GAPDH (1:10000, ab 128915, Abcam, Cambridge, UK) at 4℃ overnight. After washing, the membrane was incubated with anti-IgG horseradish peroxidase-conjugated secondary antibody (Jackson Immuno Research, West Grove, PA, USA). The membranes were exposed to ECL buffer and detected by ChemiDoc XRS gel documentation system (Bio-Rad, Hercules, CA, USA). Protein bands were analyzed by Bio-rad software and standardized with internal reference. 2.14 Co-immunoprecipitation The cardiomyocytes were transfected with negative control or si-Sirt1 plasmid. Total of 200µL 1x IP lysis buffer (containing protease inhibitor) was added to the collected cardiomyocytes, and the cells were lysed on ice for 15 min, and then centrifuged at 13500 g for 15 min to obtain supernatant. After that, the precleaned lysates were mixed with primary anti-Sirt1. The mixture was shaken gently at 4℃ and incubated overnight. The protein A/G beads were washed one time with lysis buffer and collected by magnetic separation. Then, 20 µL protein A/G beads were added to the mixture and gently shaken at 4℃ for 2 h. The samples were washed 3 times with lysis buffer and the supernatant was carefully removed by magnetic separation. Total of 24 µL of lysis buffer and 6 ul of 5x SDS sample buffer were added, and the samples were boiled for 10 min. The protein A/G beads were discarded by magnetic separation and supernatant was collected. Finally, 15 µL of each sample was separated by SDS-PAGE for Western blot analysis. 2.15 Statistical analysis Statistical analysis was performed using GraphPad Prism 8.0 software (GraphPad Software, Inc, La Jolla, CA). Shapiro-Wilk test was used for normality test. Continuous variables were expressed as mean ± standard error of mean (SEM) or median and interquartile range. Categorical variables were represented as numbers and percentages. Two-group comparisons were performed using non-paired Student’s t-test or Mann-Whitney U test for continuous variables. Variables with more than two groups were analyzed by one-way ANOVA, followed by Tukey tests. Two-tailed and P < 0.05 were considered statistically significant. Results 3.1 GPX4 Deficiency Predisposes Mice to Age-Related HF Aging is an important risk factor for heart failure. We observed cardiac function of 6-month-old and 22-month-old rats through echocardiography. Compared to the young rats, the aging rats demonstrated a significant reduction in cardiac ejection fraction (EF) and fractional shortening (FS) ( Fig. 1 A-C), accompanied by an increase in plasma NT-proBNP levels ( Fig. 1 D). These findings substantiate a decline in cardiac function in aging rats, while the underlying mechanisms remain elusive. Mounting research suggests that oxidative stress has been implicated in various age-related diseases [ 19 , 20 ] . We found that the levels of reactive oxygen species (ROS) was also significantly increased in aging rats (Fig. 1 E). To explore the role of oxidative stress in age-related HF, we examined the expression of a wide range of genes involved in the ROS detoxification system in the left ventricles of young and aging rats. We observed a significant reduction in the levels of antioxidant genes glutathione peroxidase 4 (GPX4) and superoxide dismutase (Sod1) in the left ventricles of aging rats (Fig. 1 F). GPX4 is a pivotal enzyme that perform the essential function of mitigating lipid peroxidation [ 21 , 22 ] . A decrease in GPX4 can heighten the susceptibility to ferroptosis [ 23 , 24 ] , increase the vulnerability to age-related diseases [ 25 , 26 ] . To further validate the pivotal role of GPX4 in age-related HF, we generated cardiomyocyte-specific GPX4 knockout mice (Myh6-cre + GPX4 fl/fl ) and established an aging model using D-galactose on both wild type mice and Myh6-cre + GPX4 fl/fl mice(Fig. 1 G). As expected, GPX4 conditional knockout significantly downregulated EF and FS ( Fig. 1 H-J ) , increased NT-proBNP ( Fig. 1 K ) , and aggravated cardiac structural disorder, and increased cardiac fibrosis levels in aging model(Fig. 1 L-N). Taken together, those findings confirmed the crucial role of GPX4 in age-related HF. Representative M-mode images of left ventricular wall motion in the hearts of young rats and aging rats. The statistical data of cardiac ejection fraction (EF) of young rats and aging rats (n = 8 per group). The statistical data of cardiac fraction shortening (FS) of young and aging rats (n = 8 per group). Quantitative analysis of plasma NT-proBNP levels of young and aging rats (n = 8 per group). Representative images of ROS staining in the left ventricle of young and aging rats. The relative expression of the indicated antioxidation genes was measured using real-time PCR in the left ventricle of young rats and aging rats (n = 5 per group). Schematic illustration of the experimental design. We established an aging mouse model with D-galactose (200mg/kg/day) via subcutaneous injection for 6 weeks. Representative M-mode images of heart in from WT group and Myh6-cre + GPX4 fl/fl group. The statistical data of left ventricular ejection fraction (EF) of mice from WT group and Myh6-cre + GPX4 fl/fl group (n = 6 per group). The statistical data of left ventricular fraction shortening (FS) of mice from WT group and Myh6-cre + GPX4 fl/fl group (n = 6 per group). The levels of plasma NT-ProBNP in mice from WT group and Myh6-cre + GPX4 fl/fl group (n = 6 per group). Representative images of HE staining of the left ventricle of mice from WT group and Myh6-cre + GPX4 fl/fl group. Representative images of Masson staining of the left ventricle of mice from WT group and Myh6-cre + GPX4 fl/fl group. The collagen volume fraction of the left ventricle of mice from WT group and Myh6-cre + GPX4 fl/fl group (n = 6 per group). The data are given as mean ± SEM and compared by Student’s t test. 3.2 Aged rats displayed aggravated ferroptosis in the heart The induction of ferroptosis depends on the oxidation of polyunsaturated fatty acids (PUFAs), which serve as precursors for bioactive oxylipins. Intriguingly, the oxidative lipidomics revealed a remarkable increase in the levels of multiple arachidonic acid and linoleic acid derived oxidized fatty acid metabolites in the ventricular tissues of aging rats (Fig. 2 A-D). Consistently, we observed an increase in iron deposition (Fig. 2 E), and elevated expression of the ferroptosis-related protein TF, along with a decrease in FTH1 and GPX4 expression levels in the aging rats compared to the young rats (Fig. 2 F). More interestingly, we established a senescent cardiomyocyte model using H 2 O 2 ( Fig. S1 A-B ), and found that both the levels of ROS and iron deposition were higher in senescent cardiomyocytes than that in the control group ( Fig. S1 C , Fig. 2 G ) , along with a elevated expression of TF, and reduced FTH1 and GPX4 (Fig. 2 H). These results suggest that ferroptosis may be a crucial mechanism contributing to the decline in cardiac function observed in aging rats. Principal component analysis (PCA) of oxidized fatty acid metabolites in the left ventricle from young and aging rats (n = 8 per group). (B-D) Quantitative analysis of arachidonic acid (AA) metabolites, linoleic acid (LA) metabolites and docosahexaenoic acid (DHA) metabolites in young and aging rats (n = 8 per group). (E) Representative image of Perls’ Blue staining in the left ventricle of young and aging rats. (F) Representative bands and quantification of expressions of TF, FTH1 and GPX4 in heart of young and aging rats (n = 6 per group). (G) Representative images of FerroOrange detection in cardiomyocytes of Control group and H 2 O 2 group. (H) Representative bands and quantification of the expression of Tf, FTH1 and GPX4 in cardiomyocytes of Control group and H 2 O 2 group (n = 6 per group). The data are given as mean ± SEM and compared by Student’s t test. 3.3 Ferroptosis is responsible for the occurance of age-related HF. To ascertain the role of ferroptosis in age-related HF, we administered a high-iron diet to aging rats for 4 months (Fig. 3 A). Iron supplementation significantly upregulated the expression of TF, and decreased FTH1 and GPX4 expression in the ventricular tissues of aging rats (Fig. 3 B).Compared to the control group, the EF and FS were significantly reduced, and the level of plasma NT-proBNP was increased in the high-iron diet group (Fig. 3 C-F). Concurrently, the high-iron diet led to an aggravated cardiac structural disorder, and exacerbated fibrosis (Fig. 3 G-I). Interestingly, when administered a high-iron diet to young rats, we found that iron supplementation did not affect their cardiac function or ferroptosis levels ( Fig. S2 ). To further demonstrate that ferroptosis is a critical pathological mechanism mediating age-related HF, we administered a ferroptosis inhibitor (Ferrostatin-1, Fer-1) to aging rats ( Fig. S3A) . Notably, Fer-1 significantly reduced the expression of TF and increased the expression levels of FTH1 and GPX4 in the cardiac tissue of aging rats ( Fig. S3B ). Compared to the control group, the aging rats received Fer-1 exhibited improved cardiac function (Fig. 3 J-M). These findings suggest that ferroptosis is a critical pathological mechanism for age-related HF. Schematic illustration of the experimental design for HID. 18 months old rats were randomly divided into two groups, receiving a standard diet (ND) or a high-iron diet (HID) for 4 months. Representative bands and quantification of the expression of Tf, FTH1 and GPX4 in the heart of rats from Aging ND group and Aging HID group (n = 6 per group). Representative M-mode images of heart in ND-fed and HID-fed aging rats. The statistical data of left ventricular ejection fraction (EF) of rats in Aging ND group and Aging HID group (n = 6 per group). The statistical data of left ventricular fraction shortening(FS) of rats in Aging ND group and Aging HID group (n = 6 per group). The levels of plasma NT-proBNP in rats from Aging ND group and Aging HID group (n = 6 per group). Representative images of HE staining in ND-fed and HID-fed aging rats. Representative images of Masson staining in ND-fed and HID-fed aging rats. The collagen volume fraction of the left ventricle of rats from Aging ND and Aging HID group (n = 6 per group). Representative M-mode images of heart in Aging Control group and Aging Fer-1 group. The statistical data of cardiac ejection fraction (EF) of rats in Aging Control group and Aging Fer-1 group (n = 6 per group). The statistical data of cardiac fraction shortening (FS) of rats in Aging Control group and Aging Fer-1 group (n = 6 per group). The levels of plasma NT-proBNP in rats from Aging Control group and Aging Fer-1 group (n = 6 per group). The data are given as mean ± SEM and compared by Student’s t test. 3.4 HADHA deficiency in aging rats contributes to ferroptosis. To elucidate the mechanism underlying ferroptosis in aging rats, we conducted a proteomics on the hearts of aging rats received normal diet and high-iron diet, revealing a significant differences in the protein expression profiles between the two groups ( Fig. 4 A ) . Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of these differentially expressed proteins indicated that the fatty acid metabolism pathway was the most enriched term ( Fig. 4 B ) . Notably, the expression level of HADHA was significantly downregulated in the cardiac tissues of aging rats on a high-iron diet (Fig. 4 C). HADHA, the alpha subunit of the mitochondrial trifunctional protein, catalyzes the beta-oxidation of fatty acids, playing a crucial role in maintaining fatty acid oxidation in cardiomyocytes [ 27 ] . We corroborated the downregulation of HADHA expression in the hearts of aging rats received a high-iron diet as well as aging rats (Fig. 4 D-F). It has been reported that HADHA deficiency leads to mitochondrial dysfunction, which in turn triggers the accumulation of ROS [ 28 ] . Interestingly, we discovered that silencing HADHA led to an increase in ROS levels and a significant decrease in mitochondrial membrane potential (MMP) in H 2 O 2 -induced senescent cardiomyocytes ( Fig. S4A-C ). The cell viability and ATP levels were also reduced in senescent cardiomyocytes with HADHA deficiency ( Fig. S4D-E ).Cellular experiments revealed that silencing HADHA increased iron deposition (Fig. 4 G-H), upregulated Tf expression, and decreased the expression levels of FTH1 and GPX4 in H 2 O 2 -induced senescent cardiomyocytes (Fig. 4 J). GSH, a vital cellular antioxidant, which depletion can lead to the downregulation of GPX4, eventually heightening the cell's vulnerability to ferroptosis [ 29 ] . We observed a remarkable depletion of GSH in cardiomyocytes following the silencing of HADHA(Fig. 4 I).However, silencing HADHA has no effects on the level of ferroptosis in senescent cardiac fibroblasts ( Fig. S5A-B ). Additionally, overexpression of HADHA abrogated the increase in ROS levels, the decrease in cell viability, GSH and ATP induced by H 2 O 2 ( Fig. S6A-E ). Taken together, HADHA deficiency causes ferroptosis in cardiomyocytes. The volcano plot of proteomics data showed diferentially expressed genes between ND-fed and HID-fed aging rats. n = 3 biological replicates/group. Downregulation and up-regulation are shown in blue and red, respectively. KEGG analyses of proteomics data showing the top 8 enriched pathways in the heart between ND-fed and HID-fed aging rats. The expression of HADHA in ND-fed and HID-fed aging rats determined by proteomics. Representative bands showing the expression of HADHA in the heart of rats from Aging ND and Aing HID groups, and rats from Young and Aging groups. Quantification of expressions of HADHA in the heart of rats from Aging ND and Aing HID groups (n = 6 per group). Quantification of expressions of HADHA in the heart of rats from Young and Aging groups (n = 6 per group). (G-J) Cardiomyocytes were first transfected with plasmids to silence HADHA. After 24 hours, following a change of culture media,they were treated with H 2 O 2 for 24 hours before conducting assays for relevant markers. Representative image of FerroOrange detection in cardiomyocytes. Fluorescence intensity of FerroOrange detection in cardiomyocytes (n = 4 per group). The levels of GSH in cardiomyocytes (n = 5 per group). Representative bands and quantification of the expression of Tf, FTH1 and GPX4 in cardiomyocytes (n = 6 per group). The data are given as mean ± SEM and compared by Student’s t test or one way ANOVA. 3.5 HADHA deficiency induces ferroptosis through regulation of GSH-GPX4. In order to further substantiate that HADHA deficiency promotes ferroptosis via GSH depletion, we administered N-acetylcysteine (NAC), a precursor of GSH, to the HADHA-silenced cardiomyocytes. Notably, NAC supplementation reduced the accumulation of ROS, led to an increased in MMP levels, accompanied by decreased expression of Tf, and increased expression of FTH1 and GPX4 (Fig. 5 A-D). Those results indicated that HADHA deficiency induces ferroptosis through regulation of GSH-GPX4. To substantiate the potential of NAC in age-related HF, 18-month old rats were treated with NAC for 4 months (Fig. 5 E). We found that supplementation of NAC reduced the expression of TF, while increased FTH1 and GPX4 expression (Fig. 5 F). Notably, NAC ameliorated cardiac function of aging rats, supported by the elevated EF and FS, and reduced NT-proBNP (Fig. 5 G-J). Furthermore, NAC significantly alleviated cardiac structural disorder and fibrosis levels (Fig. 5 K-M). To further evalulate the therapeutic potential of NAC for age-related HF, we established an aging rat model using D-galactose, followed by intervention with either a control solvent or NAC (Fig. S7A) . As expected, NAC significantly downregulated TF expression, and upregulated FTH1 and GPX4 expression in the hearts of aging rats induced by D-galactose ( Fig. S7B ). NAC treatment significantly upregulated EF and FS (Fig. S7C-E) , reduced NT-proBNP (Fig. S7F) , and rectified cardiac structural disorder, and reduced cardiac fibrosis levels ( Fig. S7G-I ). Taken together, these findings provide evidence that NAC prevents the occurrence of age-related HF by reducing cardiac ferroptosis levels. (A-D) Cardiomyocytes were first transfected with plasmids to silence HADHA. After 24 hours, they were treated with H2O2 for 24 hours. Following a change of culture media, the cells were treated with control solvent or NAC for 24 hours before conducting assays for relevant markers. Representative images of ROS staining and JC-1 staining in cardiomyocytes. The fluorescence intensity of ROS staining of cardiomyocytes (n = 5 per group). The JC-1 polymer/monomer fluorescence ratio of cardiomyocytes (n = 5 per group). Representative bands and quantification of expressions of Tf, FTH1 and GPX4 in cardiomyocytes (n = 6 per group). Schematic illustration of the experimental design for NAC intervention. 18 months old rats were randomly divided into two groups, receiving control solvent (Aging Control group) or NAC (Aging NAC group) through drinking for 4 months. Representative bands and quantification of the expression of Tf, FTH1 and GPX4 in the heart of rats from Aging Control group and Aging NAC group (n = 4 per group). Representative M-mode images of heart in Aging Control group and Aging NAC group. The statistical data of left ventricular ejection fraction (EF) of rats from Aging Control group and Aging NAC group (n = 4 per group). The statistical data of left ventricular fraction shortening (FS) of rats from Aging Control group and Aging NAC group (n = 4 per group). The levels of plasma NT-ProBNP in rats from Aging Control group and Aging NAC group (n = 4 per group). Representative images of HE staining of the left ventricle of rats from Aging Control group and Aging NAC group. Representative images of Masson staining of the left ventricle of rats from Aging Control group and Aging NAC group. The collagen volume fraction of the left ventricle of rats from Aging Control group and Aging NAC group (n = 4 per group). The data are given as mean ± SEM and compared by Student’s t test. 3.6 Sirt1 deficiency downregulates HADHA expression by inhibiting GATA4 expression. Sirtuins have been recognized as crucial regulators in anti-aging processes. Among them, Sirt1 has garnered significant attention as a potential therapeutic target for the mitigation of age-related disorders, including Alzheimer's disease and Parkinson's disease [ 30 , 31 ] . In our previous studies, we found a correlation between the reduction of Sirt1 in the atria of aging rats and the incidence of age-related atrial fibrillation. However, whether Sirt1 is involved in age-related HF remians unclear. Intriguingly, we found that silencing Sirt1 in cardiomyocytes led to a downregulation of HADHA, accompanied by an increase in Tf expression and a decrease in GPX4 and FTH1 levels (Fig. 6 A). However, the result of protein interaction prediction analysis showed no binding sites between Sirt1 and HADHA, while the PCR data confirmed that the mRNA levels of HADHA were significantly decreased in cardiomyocytes with Sirt1 silencing, indicating that Sirt1 may affect HAHDA expression by regulating the transcription factor of HADHA. Database analysis revealed that HADHA expression can be regulated by GATA4 (Fig. 6 B-C), a transcription factor of HADHA. To confirm that Sirt1 can bind to GATA4 and regulate GATA4 expression, we performed co-immunoprecipitation experiments. As expected, the binding between Sirt1 and GATA4 was observed. Furthermore, silencing Sirt1 led to a downregulation of GATA4 (Fig. 6 D). These findings suggest that Sirt1 deficiency downregulates GATA4, thereby inhibiting HADHA expression and promoting the occurrence of ferroptosis. 3.7 Supplementation of resveratrol restrains ferroptosis and protects aging rats from heart failure. Resveratrol is a known activator of Sirt1. To confirm the role of Sirt1 deficiency is the key mechanism for age-related HF, we administrated resveratrol to 18-month-old aged rats for 4 months(Fig. 6 E). Compared to the control group, the resveratrol-treated group exhibited significantly upregulated expression levels of Sirt1, HADHA, and GATA4 in cardiac tissues(Fig. 6 F). Simultaneously, resveratrol notably reduced TF expression levels, while it upregulated FTH1 and GPX4 expression levels in the heart tissues of aging rats(Fig. 6 G-H), suggesting a significant alleviation of cardiac ferroptosis. Additionally, resveratrol markedly improved cardiac function ( Fig. 6 I-L ) , and alleviated cardiac structural disorder and cardiac fibrosis in aging rats (Fig. 6 M-O). These findings suggest that Sirt1 is a potential therapeutic target in aging-related HF. The use of resveratrol, as a Sirt1 activator, may represent a promising therapeutic strategy for the treatment and management of this age-related cardiac condition. Cardiomyocytes were transfected with plasmids to silence Sirt1.Representative bands and quantification of expressions of HADHA, Tf, FTH1 and GPX4 in cardiomyocytes (n = 5 per group). Molecular docking of Sirt1 and HADHA. The relative mRNA expression of HADHA in cardiomyocytes (n = 6 per group). Representative Western blots and quantification of protein expressions of Sirt1 and GATA4 (n = 6 per group). Fractions of cardiomyocytes were immunoprecipitated with anti-Sirt1 antibody, and immunoblotting was performed with Sirt1 antibody and GATA4 antibody. Schematic illustration of the experimental design for resveratrol intervention. 18 months old rats were randomly divided into two groups, receiving control solvent (Aging Control group) or resveratrol (Aging RES group) through gavage for 4 months. Representative bands of expressions of Sirt1, HADHA and GATA4 in the heart of rats from Aging Control group and Aging RES group. Representative bands of expressions of Tf, FTH1 and GPX4 in the heart of rats from Aging Control group and Aging RES group. Quantification of protein expressions of Sirt1, HADHA, GATA4, Tf, FTH1 and GPX4 in the heart of rats from Aging Control group and Aging RES group (n = 6 per group). Representative M-mode images of heart in from Aging Control group and Aging RES group. The statistical data of left ventricular ejection fraction (EF) of rats from Aging Control group and Aging RES group (n = 6 per group). The statistical data of left ventricular fraction shortening (FS) of rats from Aging Control group and Aging RES group (n = 6 per group). The levels of plasma NT-ProBNP in rats from Aging Control group and Aging RES group (n = 6 per group). Representative images of HE staining of the left ventricle of rats from Aging Control group and Aging RES group. Representative images of Masson staining of the left ventricle of rats from Aging Control group and Aging RES group. The collagen volume fraction of the left ventricle of rats from Aging Control group and Aging RES group (n = 6 per group). The data are given as mean ± SEM and compared by Student’s t test. Discussion This research presents groundbreaking pathophysiological perspectives on the intricate link between ferroptosis and age-related HF, thereby paving the way for a novel therapeutic approach in managing heart failure associated with aging. Herein, we revealed that ferroptosis is the predominant mechanism for age-related HF. In the heart of aging rats, the expression of HADHA was significantly decreased, which led to mitochondrial dysfunction, causing the accumulation of reactive oxygen species, following the exhaustion of GSH and remarkable downregulation of GPX4, inducing ferroptosis. Furthermore, we found that Sirt1 deficiency contributed to the downregulation of HADHA in aging rats through binding GATA4. Supplementation of resveratrol, an agonist of Sirt1, effectively mitigated ferroptosis and protected aging rats from HF. Taken together, targeting ferroptosis may be a potential therapeutic target for age-related HF. Heart failure is a significant health threat, characterized by high morbidity and mortality rates. Its incidence notably escalates with age, and the pathophysiology of heart failure in the elderly remains complex and not fully understood. In the present study, we observed a remarkable increase in ferroptosis levels in the cardiac tissues of aging rats. Ferroptosis is an iron-dependent form of cell death characterized by lipid peroxidation. Mounting studies have linked ferroptosis with various age-related disorders [ 32 , 33 ] . Recently, an increasing body of research has demonstrated the involvement of ferroptosis in the development and progression of various cardiovascular diseases, such as myocardial ischemia-reperfusion injury and cardiomyopathy [ 34 , 35 ] . Targeting ferroptosis has emerged as a potential therapeutic strategy for various diseases, such as age-related diseases [ 10 ] , cancer [ 36 , 37 ] , and cardiovascular diseases [ 38 , 39 ] . Our findings revealed that a high-iron exacerbated ferroptosis in the cardiac tissues of aging rats, leading to a decline in cardiac function. Conversely, ferroptosis inhibitors can effectively prevent this decline in cardiac function. These results underscore ferroptosis as a critical mechanism mediating aging-related HF. HADHA is one of the subunits of hydroxyacyl CoA dehydrogenase trifunctional multienzyme complex, which catalyzes the last three reactions of the mitochondrial fatty acid β-oxidation [ 40 ] . HADHA plays a essential role in mitochondrial fatty acid oxidation and as an acyltransferase in cardiolipin remodeling for cardiac homeostasis [ 27 ] . Impairment of lipid oxidation can cause accumulation of lipid peroxides, which is an important mechanism for ferroptosis. We found that the expression of HADHA was significantly decreased in the heart of aging rats, and further decreased in high-Iron diet fed aging rats. HADHA deficiency led to mitochondrial dysfunction and generation of large amounts of reactive oxygen species in cardiomyocytes. The accumulation of reactive oxygen species caused GSH depletion and downregulation of GPX4, which eventually triggering ferroptosis. We used NAC, a precursor of GSH, comfirmed that HADHA deficiency induced accumulation of reactive oxygen species and GSH depletion is the key mechanism for ferroptosis. We firstly revealed the causal relationship between HADHA deficiency and ferroptosis in age-related HF. Sirt1, is a highly conserved NAD + -dependent class III histone/protein deacetylase of sirtuins family, has been identified as important anti-aging regulatory factors [ 41 ] . Targeting Sirt1 has emerged as a novel measures for age-related disorders [ 42 , 43 ] . Our previous study demonstrated that Sirt1 deficiency contributed to age-related atrial fibrillation through regulation of necroptosis. Berger also revealed the downregulation of Sirt1 in senescence and ageing [ 44 ] . In our present study, silencing Sirt1 led to a decrease in the expression of HADHA through binding and downregulating the levels of GATA4, a transcription factor of HADHA. Furthermore, Sirt1 deficiency aggravated ferroptosis of cardiomyocytes. Administration of resveratrol, an agonist of Sirt1, exhibited a potential to alleviate ferroptosis and prevented aging rats from cardiac dysfunction. It was reported that melatonin MT1 receptors prevents α-syn-induced ferroptosis in Parkinson's disease through enhancement of the Sirt1/Nrf2/Ho1/Gpx4 pathway [ 45 ] , consistent with our study that indicating the potential of Sirt1 activation in suppression of ferroptosis. In summary, our study provides novel insights into the crucial role of ferroptosis in the progression of age-related HF. We demonstrated that aging rats displayed accumulated lipid peroxides and aggravated ferroptosis in the heart, while administration of ferroptosis inhibitor effectively prevented the decline of cardiac function in aging rats. Specifically, we found that the decline of Sirt1 in aging rats causing the downregulation of HADHA through binding its transcription factor GATA4. HADHA deficiency caused mitochondrial dysfunction and accumulation of ROS, which inducing exhaustion of GSH and downregulation of GPX4, leading to ferroptosis and age-related HF. These findings highlight that targeting HADHA or Sirt1 may be a novel measures for the prevention and treatment of age-related HF. Aged-related Sirt1 deficiency leads to the downregulation of HADHA through inhibiting GATA4. The decreased HADHA induces mitochondrial dysfunction, resulting in an accumulation of reactive oxygen species, which in turn depletes GSH, causing a downregulation of GPX4, ultimately triggers cardiac ferroptosis and aged-related cardiac dysfunction. Limitations Several limitations are acknowledged in our study. Firstly, due to the inherent challenges in obtaining human cardiac tissue, our findings related to the expression levels of HADHA and related mechanism lack validation in human samples. Secondly, our assertion of the protective effects of resveratrol against age-related HF is based solely on animal models. Further studies, particularly clinical trials in human subjects, are needed to validate the therapeutic potential of resveratrol in the context of age-related HF. Finaly, our study primarily focused on the role of HADHA deficiency-induced ferroptosis and did not comprehensively explore the contributions of other proteins or other forms of cell death. CRediT authorship contribution statement Yun Zhang and Yu Duan conducted the statistical analyses, interpreted the results, and prepared the manuscript with input from all authors. Yingchun Luo analyzed the proteomics databases. Xuejie Han drafted the manuscript. Yue Li and Yong Zhang designed the experiments. Yun Zhou, Yunlong Gao and Hui Yu contributed to the animal experiments, while Qian Xu and Ying Wei performed the cell experiments and qRT-PCR experiments. Ruoxin Min, Yong Hong, and Xuanrui Ji supervised and performed sample data collection. Yun Zhang and Yu Duan have reviewed and verified the underlying data. All authors have read and approved the final version of the manuscript. Declarations Declaration of competing interest The authors declare no competing interests. Acknowledgements This work was supported by grants from the National Key R&D Program of China (2024YFA1307001) to Y. L, the National Natural Science Foundation of China (82200413) to Y.Z, the State Key Program of National Natural Science Foundation of China (No.82330014) to Y. L, the National Natural Science Foundation of China (No.82070336) to Y. L, and the National Natural Science Foundation of China (No. 82400375) to XJ. H. Data availability Data will be made available on request. References Haddad F, Saraste A, Santalahti KM, et al. Smartphone-Based Recognition of Heart Failure by Means of Microelectromechanical Sensors. JACC Heart Fail. 2024: S2213-1779(24)00165-3 [pii]. Zhang ZD, Milman S, Lin JR, et al. Genetics of extreme human longevity to guide drug discovery for healthy ageing. Nat Metab. 2020. 2(8): 663–672. Newman JD, O'Meara E, Böhm M, et al. Implications of Atrial Fibrillation for Guideline-Directed Therapy in Patients With Heart Failure: JACC State-of-the-Art Review. J Am Coll Cardiol. 2024. 83(9): 932–950. Roger VL. Epidemiology of Heart Failure: A Contemporary Perspective. 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Additional Declarations (Not answered) Supplementary Files supplementfigure.docx supplemental figure Uncroppedblotsusedfrothestudy.docx uncropped blots used for the study Cite Share Download PDF Status: Published Journal Publication published 23 Mar, 2026 Read the published version in Cell Death & Disease → Version 1 posted Editorial decision: revise 18 Aug, 2025 Review # 1 received at journal 11 Aug, 2025 Review # 2 received at journal 10 Aug, 2025 Reviewer # 2 agreed at journal 07 Aug, 2025 Reviewer # 1 agreed at journal 27 Jul, 2025 Reviewers invited by journal 27 Jul, 2025 Submission checks completed at journal 17 Jul, 2025 Editor assigned by journal 16 Jul, 2025 First submitted to journal 16 Jul, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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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-7140279","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":491558519,"identity":"d75396bb-49e0-47fd-a31d-a8786b84b0fe","order_by":0,"name":"Yun 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dysfunction.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eRepresentative M-mode images of left ventricular wall motion in the hearts of young rats and aging rats.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B) \u003c/strong\u003eThe statistical data of cardiac ejection fraction (EF) of young rats and aging rats (n=8 per group).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C) \u003c/strong\u003eThe statistical data of cardiac fraction shortening (FS) of young and aging rats (n=8 per group).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(D) \u003c/strong\u003eQuantitative analysis of plasma NT-proBNP levels of young and aging rats (n=8 per group).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(E) \u003c/strong\u003eRepresentative images of ROS staining in the left ventricle of young and aging rats.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(F) \u003c/strong\u003eThe relative expression of the indicated antioxidation genes was measured using real-time PCR in the left ventricle of young rats and aging rats (n=5 per group).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(G) \u003c/strong\u003eSchematic illustration of the experimental design. We established an aging mouse model with D-galactose (200mg/kg/day) via subcutaneous injection for 6 weeks.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(H) \u003c/strong\u003eRepresentative M-mode images of heart in from WT group and Myh6-cre\u003csup\u003e+\u003c/sup\u003eGPX4\u003csup\u003efl/fl\u003c/sup\u003e group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(I) \u003c/strong\u003eThe statistical data of left ventricular ejection fraction (EF) of mice from WT group and Myh6-cre\u003csup\u003e+\u003c/sup\u003eGPX4\u003csup\u003efl/fl\u003c/sup\u003e group (n=6 per group).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(J) \u003c/strong\u003eThe statistical data of left ventricular fraction shortening (FS) of mice from WT group and Myh6-cre\u003csup\u003e+\u003c/sup\u003eGPX4\u003csup\u003efl/fl\u003c/sup\u003e group (n=6 per group).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(K) \u003c/strong\u003eThe levels of plasma NT-ProBNP in mice from WT group and Myh6-cre\u003csup\u003e+\u003c/sup\u003eGPX4\u003csup\u003efl/fl\u003c/sup\u003e group (n=6 per group).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(L) \u003c/strong\u003eRepresentative images of HE staining of the left ventricle of mice from WT group and Myh6-cre\u003csup\u003e+\u003c/sup\u003eGPX4\u003csup\u003efl/fl\u003c/sup\u003e group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(M) \u003c/strong\u003eRepresentative images of Masson staining of the left ventricle of mice from WT group and Myh6-cre\u003csup\u003e+\u003c/sup\u003eGPX4\u003csup\u003efl/fl\u003c/sup\u003e group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(N) \u003c/strong\u003eThe collagen volume fraction of the left ventricle of mice from WT group and Myh6-cre\u003csup\u003e+\u003c/sup\u003eGPX4\u003csup\u003efl/fl\u003c/sup\u003e group (n=6 per group).\u003c/p\u003e\n\u003cp\u003eThe data are given as mean ± SEM and compared by Student’s t test.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7140279/v1/ba69c5738a4ef7841b7739f3.png"},{"id":88012353,"identity":"e569a80f-99c2-41f0-bae1-81e73ca67766","added_by":"auto","created_at":"2025-07-31 12:14:01","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2064150,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAging rats displayed increased oxidative stress and aggravated ferroptosis .\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003ePrincipal component analysis (PCA) of oxidized fatty acid metabolites in the left ventricle from young and aging rats (n=8 per group).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B-D) \u003c/strong\u003eQuantitative analysis of arachidonic acid (AA) metabolites, linoleic acid (LA) metabolites and docosahexaenoic acid (DHA) metabolites in young and aging rats (n=8 per group).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(E) \u003c/strong\u003eRepresentative image of Perls’ Blue staining in the left ventricle of young and aging rats.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(F) \u003c/strong\u003eRepresentative bands and quantification of expressions of TF, FTH1 and GPX4 in heart of young and aging rats (n = 6 per group).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(G) \u003c/strong\u003eRepresentative images of FerroOrange detection in cardiomyocytes of Control group and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(H) \u003c/strong\u003eRepresentative bands and quantification of the expression of Tf, FTH1 and GPX4 in cardiomyocytes of Control group and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e group (n=6 per group).\u003c/p\u003e\n\u003cp\u003eThe data are given as mean ± SEM and compared by Student’s t test.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7140279/v1/e9fad84596f1b029a507a54f.png"},{"id":88011191,"identity":"26eafffc-9513-4ec7-88e6-0a22a2082d4c","added_by":"auto","created_at":"2025-07-31 12:06:02","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2098065,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHID-fed aging rats exhibited cardiac dysfunction, and ferroptosis inhibitor protected aging rats from cardiac structural remodeling and HF.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eSchematic illustration of the experimental design forHID. 18 months old rats were randomly divided into two groups, receiving a standard diet (ND) or a high-iron diet (HID) for 4 months.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B) \u003c/strong\u003eRepresentative bands and quantification of the expression of Tf, FTH1 and GPX4 in the heart of rats from Aging ND group and Aging HID group (n=6 per group).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C) \u003c/strong\u003eRepresentative M-mode images of heart in ND-fed and HID-fed aging rats.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(D) \u003c/strong\u003eThe statistical data of left ventricular ejection fraction (EF) of rats in Aging ND group and Aging HID group (n=6 per group).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(E) \u003c/strong\u003eThe statistical data of left ventricular fraction shortening(FS) of rats in Aging ND group and Aging HID group (n=6 per group).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(F) \u003c/strong\u003eThe levels of plasma NT-proBNP in rats from Aging ND group and Aging HID group (n=6 per group).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(G) \u003c/strong\u003eRepresentative images of HE staining in ND-fed and HID-fed aging rats.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(H) \u003c/strong\u003eRepresentative images of Masson staining in ND-fed and HID-fed aging rats.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(I) \u003c/strong\u003eThe collagen volume fraction of the left ventricle of rats from Aging ND and Aging HID group (n=6 per group).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(J) \u003c/strong\u003eRepresentative M-mode images of heart in Aging Control group and Aging Fer-1 group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(K) \u003c/strong\u003eThe statistical data of cardiac ejection fraction (EF) of rats in Aging Control group and Aging Fer-1 group (n=6 per group).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(L) \u003c/strong\u003eThe statistical data of cardiac fraction shortening (FS) of rats in Aging Control group and Aging Fer-1 group (n=6 per group).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(M) \u003c/strong\u003eThe levels of plasma NT-proBNP in rats from Aging Control group and Aging Fer-1 group (n=6 per group).\u003c/p\u003e\n\u003cp\u003eThe data are given as mean ± SEM and compared by Student’s t test.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7140279/v1/3deab9a92d692b9bf63a82b0.png"},{"id":88013460,"identity":"b58aa23b-5af2-49a9-96bf-cce17027f333","added_by":"auto","created_at":"2025-07-31 12:30:02","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1474060,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHADHA deficiency in aging rats contributes to ferroptosis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eThe volcano plot of proteomics data showed diferentially expressed genes between ND-fed and HID-fed aging rats. n = 3 biological replicates/group. Downregulation and up-regulation are shown in blue and red, respectively.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B) \u003c/strong\u003eKEGG analyses of proteomics data showing the top 8 enriched pathways in the heart between ND-fed and HID-fed aging rats.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C) \u003c/strong\u003eThe expression of HADHA in ND-fed and HID-fed aging rats determined by proteomics.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(D) \u003c/strong\u003eRepresentative bands showing the expression of HADHA in the heart of rats from Aging ND and Aing HID groups, and rats from Young and Aging groups.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(E) \u003c/strong\u003eQuantification of expressions of HADHA in the heart of rats from Aging ND and Aing HID groups (n=6 per group).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(F) \u003c/strong\u003eQuantification of expressions of HADHA in the heart of rats from Young and Aging groups (n=6 per group).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(G-J) \u003c/strong\u003eCardiomyocytes were first transfected with plasmids to silence HADHA. After 24 hours, following a change of culture media,they were treated with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 24 hours before conducting assays for relevant markers.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(G) \u003c/strong\u003eRepresentative image of FerroOrange detection in cardiomyocytes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(H) \u003c/strong\u003eFluorescence intensity of FerroOrange detection in cardiomyocytes (n=4 per group).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(I) \u003c/strong\u003eThe levels of GSH in cardiomyocytes (n=5 per group).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(J) \u003c/strong\u003eRepresentative bands and quantification of the expression of Tf, FTH1 and GPX4 in cardiomyocytes (n=6 per group).\u003c/p\u003e\n\u003cp\u003eThe data are given as mean ± SEM and compared by Student’s t test or one way ANOVA.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7140279/v1/f50e2b741c582e263814b9c9.png"},{"id":88011184,"identity":"00233dd9-8a33-4382-b069-c11a675c08f1","added_by":"auto","created_at":"2025-07-31 12:06:02","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1880427,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementation of NAC restrains ferroptosis in cardiomyocytes and protects aging rats from heart failure.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A-D) \u003c/strong\u003eCardiomyocytes were first transfected with plasmids to silence HADHA. After 24 hours, they were treated with H2O2 for 24 hours. Following a change of culture media, the cells were treated with control solvent or NAC for 24 hours before conducting assays for relevant markers.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eRepresentative images of ROS staining and JC-1 staining in cardiomyocytes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B) \u003c/strong\u003eThe fluorescence intensity of ROS staining of cardiomyocytes (n=5 per group).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C) \u003c/strong\u003eThe JC-1 polymer/monomer fluorescence ratio of cardiomyocytes (n=5 per group).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(D) \u003c/strong\u003eRepresentative bands and quantification of expressions of Tf, FTH1 and GPX4 in cardiomyocytes (n=6 per group).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(E) \u003c/strong\u003eSchematic illustration of the experimental design for NAC intervention. 18 months old rats were randomly divided into two groups, receiving control solvent (Aging Control group) or NAC (Aging NAC group) through drinking for 4 months.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(F) \u003c/strong\u003eRepresentative bands and quantification of the expression of Tf, FTH1 and GPX4 in the heart of rats from Aging Control group and Aging NAC group (n=4 per group).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(G) \u003c/strong\u003eRepresentative M-mode images of heart in Aging Control group and Aging NAC group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(H) \u003c/strong\u003eThe statistical data of left ventricular ejection fraction (EF) of rats from Aging Control group and Aging NAC group (n=4 per group).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(I) \u003c/strong\u003eThe statistical data of left ventricular fraction shortening (FS) of rats from Aging Control group and Aging NAC group (n=4 per group).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(J) \u003c/strong\u003eThe levels of plasma NT-ProBNP in rats from Aging Control group and Aging NAC group (n=4 per group).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(K) \u003c/strong\u003eRepresentative images of HE staining of the left ventricle of rats from Aging Control group and Aging NAC group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(L) \u003c/strong\u003eRepresentative images of Masson staining of the left ventricle of rats from Aging Control group and Aging NAC group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(M) \u003c/strong\u003eThe collagen volume fraction of the left ventricle of rats from Aging Control group and Aging NAC group (n=4 per group).\u003c/p\u003e\n\u003cp\u003eThe data are given as mean ± SEM and compared by Student’s t test.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7140279/v1/7e10d54f7fdf635f16dbdc6c.png"},{"id":88011186,"identity":"8d7eeb7f-6f5d-4534-adcc-db4b636d58fa","added_by":"auto","created_at":"2025-07-31 12:06:02","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2116531,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSirt1 deficiency downregulates HADHA expression by inhibiting GATA4 expression, and supplementation of resveratrol protects aging rats from cardiac dysfunction.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Cardiomyocytes were transfected with plasmids to silence Sirt1.Representative bands and quantification of expressions of HADHA, Tf, FTH1 and GPX4 in cardiomyocytes (n=5 per group).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B)\u003c/strong\u003e Molecular docking of Sirt1 and HADHA.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C)\u003c/strong\u003e The relative mRNA expression of HADHA in cardiomyocytes (n=6 per group).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(D)\u003c/strong\u003e Representative Western blots and quantification of protein expressions of Sirt1 and GATA4 (n = 6 per group). Fractions of cardiomyocytes were immunoprecipitated with anti-Sirt1 antibody, and immunoblotting was performed with Sirt1 antibody and GATA4 antibody.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(E)\u003c/strong\u003e Schematic illustration of the experimental design for resveratrol intervention. 18 months old rats were randomly divided into two groups, receiving control solvent (Aging Control group) or resveratrol (Aging RES group) through gavage for 4 months.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(F)\u003c/strong\u003e Representative bands of expressions of Sirt1, HADHA and GATA4 in the heart of rats from Aging Control group and Aging RES group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(G)\u003c/strong\u003e Representative bands of expressions of Tf, FTH1 and GPX4 in the heart of rats from Aging Control group and Aging RES group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(H)\u003c/strong\u003e Quantification of protein expressions of Sirt1, HADHA, GATA4, Tf, FTH1 and GPX4 in the heart of rats from Aging Control group and Aging RES group (n=6 per group).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(I)\u003c/strong\u003e Representative M-mode images of heart in from Aging Control group and Aging RES group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(J)\u003c/strong\u003e The statistical data of left ventricular ejection fraction (EF) of rats from Aging Control group and Aging RES group (n=6 per group).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(K)\u003c/strong\u003e The statistical data of left ventricular fraction shortening (FS) of rats from Aging Control group and Aging RES group (n=6 per group).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(L)\u003c/strong\u003e The levels of plasma NT-ProBNP in rats from Aging Control group and Aging RES group (n=6 per group).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(M)\u003c/strong\u003e Representative images of HE staining of the left ventricle of rats from Aging Control group and Aging RES group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(N)\u003c/strong\u003e Representative images of Masson staining of the left ventricle of rats from Aging Control group and Aging RES group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(O)\u003c/strong\u003e The collagen volume fraction of the left ventricle of rats from Aging Control group and Aging RES group (n=6 per group).\u003c/p\u003e\n\u003cp\u003eThe data are given as mean ± SEM and compared by Student’s t test.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7140279/v1/85abc30defc1f1a727452a5a.png"},{"id":88011188,"identity":"78d46dc8-6e1f-45de-8c75-44363c2a83f0","added_by":"auto","created_at":"2025-07-31 12:06:02","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":146086,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSirt1 deficiency exacerbates age-related heart failure through enhancing ferroptosis via GATA4-HADHA-GPX4 signaling pathway.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7140279/v1/4afebecabee3b66ff4f32f89.png"},{"id":105888553,"identity":"fc29238c-ff29-43f3-a767-a778976589d8","added_by":"auto","created_at":"2026-04-01 07:44:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":13275563,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7140279/v1/5aca8f13-02ee-4c80-a67b-1081783f3845.pdf"},{"id":88011182,"identity":"18e415dd-c6a9-4009-9e56-fc48a7f0702e","added_by":"auto","created_at":"2025-07-31 12:06:02","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2007389,"visible":true,"origin":"","legend":"supplemental figure","description":"","filename":"supplementfigure.docx","url":"https://assets-eu.researchsquare.com/files/rs-7140279/v1/12213862ad654418d223b92a.docx"},{"id":88011194,"identity":"e08a3c1a-d425-4185-a70e-97a9320fca65","added_by":"auto","created_at":"2025-07-31 12:06:02","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":9309123,"visible":true,"origin":"","legend":"uncropped blots used for the study","description":"","filename":"Uncroppedblotsusedfrothestudy.docx","url":"https://assets-eu.researchsquare.com/files/rs-7140279/v1/955eabd932c07dce141b2eb0.docx"}],"financialInterests":"(Not answered)","formattedTitle":"Sirt1 deficiency promotes age-related heart failure through enhancing ferroptosis via GATA4-HADHA-GPX4 axis","fulltext":[{"header":"1. Introdcution","content":"\u003cp\u003eHeart failure (HF) is a leading cause of morbidity and mortality worldwide, affecting an estimated 56\u0026nbsp;million individuals globally\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. Advanced age has been identified as a major risk factor for HF. By 2050, the global population of individuals aged 65 and over is projected to reach approximately 2\u0026nbsp;billion, and the number of people 80 years or older is projected to reach 425 million\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e, further exacerbating the burden of HF. Over the past few decades, the prevalence of HF has steadily increased due to the advancing age of the population and extended life\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. Among the general adult population, HF prevalence ranges from 1\u0026ndash;3%. This rate dramatically rises to 8% in individuals aged between 65 to 74 years, and it further surges to a staggering 16.1% in those aged over 74 years\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. Given the significant growth of the aging population, age-related HF has emerged as a formidable challenge in the realm of global healthcare. Despite the urgency for effective therapeutic interventions, the precise mechanisms that driving age-related HF continue to be enigmatic. This presents a critical knowledge gap in our current understanding, underscoring the necessity for further research in this field.\u003c/p\u003e\u003cp\u003eThe pathophysiology of age-related HF is a complex interplay of various factors, many of which are not yet fully understood. Extensive research has explored the role of free radicals in the aging process. As the body ages, its ability to produce free radicals escalates, while its capacity to clear these potentially harmful molecules diminishes. This imbalance can lead to an accumulation of reactive oxygen species (ROS), disrupting cellular redox homeostasis and potentially triggering a cascade of age-related diseases\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. It was reported that excessive ROS are likely to disrupt cellular redox homeostasis and induce ferroptosis\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. Ferroptosis, a novel form of regulated cell death, is characterized by the lethal accumulation of iron-dependent lipid peroxides\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. The role of ferroptosis in age-related diseases has been the subject of increasing scientific interest. Mounting studies have implicated ferroptosis in a range of age-related conditions, including Alzheimer's disease\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e, Parkinson's disease\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e, osteoporosis, and osteoarthritis\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. More recently, the spotlight has turned to the potential role of ferroptosis in cardiovascular diseases\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e, including diabetic cardiomyopathy\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e, doxorubicin-induced cardiomyopathy\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e, abdominal aortic aneurysm\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e, myocardial ischemia-reperfusion injury\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e, and myocardial infarction\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. Despite these advances, the role of ferroptosis in age-related HF and the underlying mechanisms remains a mystery, and warrant further exploration.\u003c/p\u003e\u003cp\u003eIn our present study, we elucidated the role of ferroptosis in age-related HF, and deciphered underlying mechanisms. Our findings found that aging rats displayed aggravated ferroptosis in the left ventricle, which was exacerbated by a high-iron diet.\u003c/p\u003e\u003cp\u003eInterestingly, ferroptosis inhibitor (ferrostatin-1) improved cardiac function of aging rats. Mechanically, HADHA deficiency in the heart of aging rats leads to mitochondrial dysfunction, which in turn, results in an accumulation of reactive oxygen species (ROS), triggering a depletion of glutathione (GSH) and a downregulation of GPX4 expression. These changes increased the level of ferroptosis in cardiomyocytes, ultimately causing cardiac structural remodeling and the onset of HF. Furthermore, we found that the downregulation of Sirt1 reduced the expression of HADHA through restraint of GATA4 expression. A Sirt1 agonist, resveratrol, effectively reduced the level of ferroptosis in the hearts of aging rats and improved cardiac function. In summary, these findings illuminate the fundamental mechanisms of HF within the context of aging, particularly from the perspective of ferroptosis. It opens up a new avenue of research and intervention strategies, suggesting that modulation of ferroptosis could be a viable approach in the prevention and treatment of HF in the aging population.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Experimental animals\u003c/h2\u003e\u003cp\u003eThe animal experiments in this study were performed in accordance with the Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee at the Harbin Medical University (\u003cb\u003eEthical approval number:2020123\u003c/b\u003e). Male SD adult rats (200-250g) were purchased from Beijing Vital River Laboratory Animal Technology Co, Ltd (Beijing, China) and raised at the Experimental Animal Center of Harbin Medical University (Harbin, China).Cardiomyocyte-specific knockout GPX4 mice (Myh6-cre\u003csup\u003e+\u003c/sup\u003eGPX4\u003csup\u003efl/fl\u003c/sup\u003e) were generated by Cyagen Biosciences, Inc (Suzhou,China). Briefly, Myh6-cre\u003csup\u003e+\u003c/sup\u003eGPX4\u003csup\u003efl/fl\u003c/sup\u003e were generated by crossing Myh6-Cre\u003csup\u003e+\u003c/sup\u003e mice with GPX4\u003csup\u003eflox/flox\u003c/sup\u003e mice. Myh6-cre\u003csup\u003e+\u003c/sup\u003eGPX4\u003csup\u003efl/fl\u003c/sup\u003e offspring were born at the expected Mendelian ratio and were viable. Control mice for this group are sex- and age-matched wild type control. The animals were kept in cages with light/dark cycles of 12 h and fed with food and water available ad libitum in SPF conditions.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Treatment with high-iron diet\u003c/h2\u003e\u003cp\u003eTo evaluate the effect of iron supplementation on age-related HF, 18-month old rats were randomly divided into two groups, one group received standard diet, another group received high-iron diet (1.5g/kg) for 4 months. Furthermore, 2-month old rats were randomly divided into two groups, one group received standard diet, another group received high-iron diet (1.5g/kg) for 4 months.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e\u003cb\u003e2.3 Treatment with ferrostatin-1\u003c/b\u003e\u003c/h2\u003e\u003cp\u003eTo testify the role of ferroptosis in age-related HF, 18-month old rats were randomly divided into two groups, one group were intraperitoneal injected with ferroptosis inhibitor ferrostatin-1 (0.8mg/kg), once a week for 4 months, the Vehicle control group were treated with saline of equal volume.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Treatment with acetylcysteine\u003c/h2\u003e\u003cp\u003eFor acetylcysteine intervention experiment, 18-month old rats were randomly divided into two groups, one group received acetylcysteine dissolved in water (free drinking: 600mg/L), another group received water (free drinking) for 4 months. Furthermore, we used D-galactose to establish aging rat model via subcutaneous injection at a dose of 150mg/kg/day. Then, rats were randomly divided into two groups, one group received acetylcysteine dissolved in water (free drinkin: 600mg/L), another group received water (free drinking) for 12 weeks.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Treatment with resveratrol\u003c/h2\u003e\u003cp\u003eTo assess the potential of resveratrol in age-related HF, 18-month old rats were randomly divided into two groups, one group received resveratrol (10mg/kg/d) through gavage for 4 months, another group received control solvent of equal volume.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Echocardiography\u003c/h2\u003e\u003cp\u003eTransthoracic echocardiography was performed on anesthetized rats using a Vivid 7 echo machine (GE Healthcare, Milwaukee, WI, USA) with two-dimensional M-mode analysis. The rats were anesthetized by 1% sodium pentobarbital (30mg/kg) intraperitoneal injection, then the left ventricular ejection fraction (LVEF), and left ventricular fractional shortening (LVFS) were tested for at least five nonstop cardiac cycles.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7 Histopathology\u003c/h2\u003e\u003cp\u003eThe ventricular tissue of rats were collected, fixed overnight in 4% paraformaldehyde, embedded in paraffin, and serially sectioned at 4\u0026micro;m thickness. The tissues were then stained with hematoxylin and eosin (H\u0026amp;E) for routine histological examination. To measure collagen deposits, select sections were stained with Masson\u0026rsquo;s trichrome staining. Fibrotic area was quantified using ImageJ software and collagen volume fraction was calculated as collagen area/total area\u0026times;100%.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8 Analysis of oxylipins\u003c/h2\u003e\u003cp\u003eThe ventricular tissue samples were collected for oxylipidomics. Through targeted metabolomics using UPHLC-MS/MS with a EXIONLC System (SCIEX) connected to a SCIEX 6500 QTRAP\u0026thinsp;+\u0026thinsp;MS/MS system equipped equipped with an IonDrive Turbo V electrospray ionization (ESI) interface. Lipids were separated using a 1.7 \u0026micro;m C18 column (150*2.1mm). SCIEX Analyst Work Station Software (Version 1.6.3) and Multiquant 3.03 software were employed for MRM data acquisition and processing.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.9 Culture of primary rat cardiomyocytes and fibroblasts\u003c/h2\u003e\u003cp\u003ePrimary cardiomyocytes were isolated from the hearts of neonatal Sprague-Daw rats (1\u0026ndash;3 days old). Briefly, the hearts was aseptically dissected and cut to small pieces. Then digested in 0.25% tryspin with gently shaking, and digestive fluid was collected in DMEM supplemented with 10% fetal bovine serum, centrifuged at 1200 rpm for 5 minutes. The cell pellet was then resuspended in DMEM containing 10% FBS and 1% penicillin-streptomycin. The cells were transferred to a culture dish and allowed to adhere for 90 minutes. Non-adherent cells, which primarily contain cardiomyocytes, were then transferred to new six-well plates and incubated at 37\u0026deg;C in a 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere. Upon reaching a fusion rate of 70%-80% and exhibiting good condition, the treatment was initiated. We used H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (50\u0026micro;mol/L) to establish a senescent cell model.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.10 MTT assay\u003c/h2\u003e\u003cp\u003eCell viability was assessed utilizing the MTT Cell Proliferation Assay (Beyotime, Shanghai, China), following the manufacturer's instructions. Briefly, cells were cultured in 96-well plates. Subsequent to the designated treatment, 10\u0026micro;L of MTT reagent was incorporated into each well containing phenol red-free culture medium, followed by an incubation period of 4 hours at 37\u0026deg;C. Then, DMSO was added to the cells, and the absorbance was measured by a microplate reader (Thermo, Massachusetts, USA).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e2.11 JC-1 staining\u003c/h2\u003e\u003cp\u003eThe mitochondrial membrane potential (MMP) was detected by JC-1 staining kit (Beyotime) in accordance to the manufacturer\u0026rsquo;s instructions. Briefly, primary rat cardiomyocytes were subjected to incubation with JC-1 staining solution (5pg/ml) at 37\u0026deg;C for 20 min. Subsequently, the cells were rinsed meticulously with JC-1 staining buffer to remove excess dye. Mitochondrial depolarization is indicated by an increase in the green/red fuorescence intensity ratio.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e2.12 Intracellular reactive oxygen species (ROS) detection\u003c/h2\u003e\u003cp\u003eIntracellular levels of ROS were determined using Dihydroethidium (S0063, Beyotime). In brief, primary rat cardiomyocytes cultured were incubated with 5\u0026micro;M Dihydroethidium in serum-free medium at 37℃ for 30min away from light. Post incubation, cells were washed thoroughly to remove unincorporated dye, followed by examination using an immunofluorescence microscope (Zeiss, Jena, Germany).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e2.13 Western blot\u003c/h2\u003e\u003cp\u003eThe total protein samples were extracted from tissues of rats or primary cultured cardiomyocytes and cardiac fibroblasts. Briefly, approximately 30\u0026thinsp;~\u0026thinsp;50 \u0026micro;g of proteins were fractionated by 8\u0026thinsp;~\u0026thinsp;12% SDS-PAGE. Proteins were transferred to PVDF membranes (Millipore, Billerica, MA, USA). The samples were then incubated with primary antibodies for Tf (1:500, 17435-1-AP, Proteintech, Wuhan, China), FTH1 (1:500, bs-5907R, Bioss, Beijing, China), GPX4 (1:500, ab125066, Abcam, Cambridge, UK), HADHA (1:2000, 10758-1-AP, Proteintech, Wuhan, China) and Sirt1 (IP-1:500, 60303-1-Ig, Proteintech, Wuhan, China; IB-1:500, ab189494, Abcam, Cambridge, UK), GATA4 (1:500, 19530-1-AP, Proteintech, Wuhan, China), and GAPDH (1:10000, ab 128915, Abcam, Cambridge, UK) at 4℃ overnight. After washing, the membrane was incubated with anti-IgG horseradish peroxidase-conjugated secondary antibody (Jackson Immuno Research, West Grove, PA, USA). The membranes were exposed to ECL buffer and detected by ChemiDoc XRS gel documentation system (Bio-Rad, Hercules, CA, USA). Protein bands were analyzed by Bio-rad software and standardized with internal reference.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e2.14 Co-immunoprecipitation\u003c/h2\u003e\u003cp\u003eThe cardiomyocytes were transfected with negative control or si-Sirt1 plasmid. Total of 200\u0026micro;L 1x IP lysis buffer (containing protease inhibitor) was added to the collected cardiomyocytes, and the cells were lysed on ice for 15 min, and then centrifuged at 13500 g for 15 min to obtain supernatant. After that, the precleaned lysates were mixed with primary anti-Sirt1. The mixture was shaken gently at 4℃ and incubated overnight. The protein A/G beads were washed one time with lysis buffer and collected by magnetic separation. Then, 20 \u0026micro;L protein A/G beads were added to the mixture and gently shaken at 4℃ for 2 h. The samples were washed 3 times with lysis buffer and the supernatant was carefully removed by magnetic separation. Total of 24 \u0026micro;L of lysis buffer and 6 ul of 5x SDS sample buffer were added, and the samples were boiled for 10 min. The protein A/G beads were discarded by magnetic separation and supernatant was collected. Finally, 15 \u0026micro;L of each sample was separated by SDS-PAGE for Western blot analysis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e2.15 Statistical analysis\u003c/h2\u003e\u003cp\u003eStatistical analysis was performed using GraphPad Prism 8.0 software (GraphPad Software, Inc, La Jolla, CA). Shapiro-Wilk test was used for normality test. Continuous variables were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of mean (SEM) or median and interquartile range. Categorical variables were represented as numbers and percentages. Two-group comparisons were performed using non-paired Student\u0026rsquo;s t-test or Mann-Whitney U test for continuous variables. Variables with more than two groups were analyzed by one-way ANOVA, followed by Tukey tests. Two-tailed and \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered statistically significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e3.1 GPX4 Deficiency Predisposes Mice to Age-Related HF\u003c/h2\u003e\u003cp\u003eAging is an important risk factor for heart failure. We observed cardiac function of 6-month-old and 22-month-old rats through echocardiography. Compared to the young rats, the aging rats demonstrated a significant reduction in cardiac ejection fraction (EF) and fractional shortening (FS) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-C), accompanied by an increase in plasma NT-proBNP levels \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). These findings substantiate a decline in cardiac function in aging rats, while the underlying mechanisms remain elusive. Mounting research suggests that oxidative stress has been implicated in various age-related diseases\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. We found that the levels of reactive oxygen species (ROS) was also significantly increased in aging rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). To explore the role of oxidative stress in age-related HF, we examined the expression of a wide range of genes involved in the ROS detoxification system in the left ventricles of young and aging rats. We observed a significant reduction in the levels of antioxidant genes glutathione peroxidase 4 (GPX4) and superoxide dismutase (Sod1) in the left ventricles of aging rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF).\u003c/p\u003e\u003cp\u003eGPX4 is a pivotal enzyme that perform the essential function of mitigating lipid peroxidation\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. A decrease in GPX4 can heighten the susceptibility to ferroptosis\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e, increase the vulnerability to age-related diseases\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. To further validate the pivotal role of GPX4 in age-related HF, we generated cardiomyocyte-specific GPX4 knockout mice (Myh6-cre\u003csup\u003e+\u003c/sup\u003eGPX4\u003csup\u003efl/fl\u003c/sup\u003e) and established an aging model using D-galactose on both wild type mice and Myh6-cre\u003csup\u003e+\u003c/sup\u003eGPX4\u003csup\u003efl/fl\u003c/sup\u003e mice(Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). As expected, GPX4 conditional knockout significantly downregulated EF and FS \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH-J\u003cb\u003e)\u003c/b\u003e, increased NT-proBNP\u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eK\u003cb\u003e)\u003c/b\u003e, and aggravated cardiac structural disorder, and increased cardiac fibrosis levels in aging model(Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eL-N). Taken together, those findings confirmed the crucial role of GPX4 in age-related HF.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003col\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eRepresentative M-mode images of left ventricular wall motion in the hearts of young rats and aging rats.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eThe statistical data of cardiac ejection fraction (EF) of young rats and aging rats (n\u0026thinsp;=\u0026thinsp;8 per group).\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eThe statistical data of cardiac fraction shortening (FS) of young and aging rats (n\u0026thinsp;=\u0026thinsp;8 per group).\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eQuantitative analysis of plasma NT-proBNP levels of young and aging rats (n\u0026thinsp;=\u0026thinsp;8 per group).\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eRepresentative images of ROS staining in the left ventricle of young and aging rats.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eThe relative expression of the indicated antioxidation genes was measured using real-time PCR in the left ventricle of young rats and aging rats (n\u0026thinsp;=\u0026thinsp;5 per group).\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eSchematic illustration of the experimental design. We established an aging mouse model with D-galactose (200mg/kg/day) via subcutaneous injection for 6 weeks.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eRepresentative M-mode images of heart in from WT group and Myh6-cre\u003csup\u003e+\u003c/sup\u003eGPX4\u003csup\u003efl/fl\u003c/sup\u003e group.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eThe statistical data of left ventricular ejection fraction (EF) of mice from WT group and Myh6-cre\u003csup\u003e+\u003c/sup\u003eGPX4\u003csup\u003efl/fl\u003c/sup\u003e group (n\u0026thinsp;=\u0026thinsp;6 per group).\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eThe statistical data of left ventricular fraction shortening (FS) of mice from WT group and Myh6-cre\u003csup\u003e+\u003c/sup\u003eGPX4\u003csup\u003efl/fl\u003c/sup\u003e group (n\u0026thinsp;=\u0026thinsp;6 per group).\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eThe levels of plasma NT-ProBNP in mice from WT group and Myh6-cre\u003csup\u003e+\u003c/sup\u003eGPX4\u003csup\u003efl/fl\u003c/sup\u003e group (n\u0026thinsp;=\u0026thinsp;6 per group).\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eRepresentative images of HE staining of the left ventricle of mice from WT group and Myh6-cre\u003csup\u003e+\u003c/sup\u003eGPX4\u003csup\u003efl/fl\u003c/sup\u003e group.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eRepresentative images of Masson staining of the left ventricle of mice from WT group and Myh6-cre\u003csup\u003e+\u003c/sup\u003eGPX4\u003csup\u003efl/fl\u003c/sup\u003e group.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eThe collagen volume fraction of the left ventricle of mice from WT group and Myh6-cre\u003csup\u003e+\u003c/sup\u003eGPX4\u003csup\u003efl/fl\u003c/sup\u003e group (n\u0026thinsp;=\u0026thinsp;6 per group).\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003c/ol\u003e\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003eThe data are given as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM and compared by Student\u0026rsquo;s t test.\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Aged rats displayed aggravated ferroptosis in the heart\u003c/h2\u003e\u003cp\u003eThe induction of ferroptosis depends on the oxidation of polyunsaturated fatty acids (PUFAs), which serve as precursors for bioactive oxylipins. Intriguingly, the oxidative lipidomics revealed a remarkable increase in the levels of multiple arachidonic acid and linoleic acid derived oxidized fatty acid metabolites in the ventricular tissues of aging rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-D). Consistently, we observed an increase in iron deposition (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE), and elevated expression of the ferroptosis-related protein TF, along with a decrease in FTH1 and GPX4 expression levels in the aging rats compared to the young rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). More interestingly, we established a senescent cardiomyocyte model using H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA-B\u003c/b\u003e), and found that both the levels of ROS and iron deposition were higher in senescent cardiomyocytes than that in the control group (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC\u003c/b\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG\u003cb\u003e)\u003c/b\u003e, along with a elevated expression of TF, and reduced FTH1 and GPX4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH). These results suggest that ferroptosis may be a crucial mechanism contributing to the decline in cardiac function observed in aging rats.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003col\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003ePrincipal component analysis (PCA) of oxidized fatty acid metabolites in the left ventricle from young and aging rats (n\u0026thinsp;=\u0026thinsp;8 per group).\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003c/ol\u003e\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003e\u003cb\u003e(B-D)\u003c/b\u003e Quantitative analysis of arachidonic acid (AA) metabolites, linoleic acid (LA) metabolites and docosahexaenoic acid (DHA) metabolites in young and aging rats (n\u0026thinsp;=\u0026thinsp;8 per group).\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003e\u003cb\u003e(E)\u003c/b\u003e Representative image of Perls\u0026rsquo; Blue staining in the left ventricle of young and aging rats.\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003e\u003cb\u003e(F)\u003c/b\u003e Representative bands and quantification of expressions of TF, FTH1 and GPX4 in heart of young and aging rats (n\u0026thinsp;=\u0026thinsp;6 per group).\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003e\u003cb\u003e(G)\u003c/b\u003e Representative images of FerroOrange detection in cardiomyocytes of Control group and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e group.\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003e\u003cb\u003e(H)\u003c/b\u003e Representative bands and quantification of the expression of Tf, FTH1 and GPX4 in cardiomyocytes of Control group and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e group (n\u0026thinsp;=\u0026thinsp;6 per group).\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eThe data are given as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM and compared by Student\u0026rsquo;s t test.\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003e3.3 Ferroptosis is responsible for the occurance of age-related HF.\u003c/h2\u003e\u003cp\u003eTo ascertain the role of ferroptosis in age-related HF, we administered a high-iron diet to aging rats for 4 months (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Iron supplementation significantly upregulated the expression of TF, and decreased FTH1 and GPX4 expression in the ventricular tissues of aging rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).Compared to the control group, the EF and FS were significantly reduced, and the level of plasma NT-proBNP was increased in the high-iron diet group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC-F). Concurrently, the high-iron diet led to an aggravated cardiac structural disorder, and exacerbated fibrosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG-I). Interestingly, when administered a high-iron diet to young rats, we found that iron supplementation did not affect their cardiac function or ferroptosis levels (\u003cb\u003eFig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e\u003c/b\u003e). To further demonstrate that ferroptosis is a critical pathological mechanism mediating age-related HF, we administered a ferroptosis inhibitor (Ferrostatin-1, Fer-1) to aging rats (\u003cb\u003eFig. S3A)\u003c/b\u003e. Notably, Fer-1 significantly reduced the expression of TF and increased the expression levels of FTH1 and GPX4 in the cardiac tissue of aging rats (\u003cb\u003eFig. S3B\u003c/b\u003e). Compared to the control group, the aging rats received Fer-1 exhibited improved cardiac function (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eJ-M). These findings suggest that ferroptosis is a critical pathological mechanism for age-related HF.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003col\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eSchematic illustration of the experimental design for HID. 18 months old rats were randomly divided into two groups, receiving a standard diet (ND) or a high-iron diet (HID) for 4 months.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eRepresentative bands and quantification of the expression of Tf, FTH1 and GPX4 in the heart of rats from Aging ND group and Aging HID group (n\u0026thinsp;=\u0026thinsp;6 per group).\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eRepresentative M-mode images of heart in ND-fed and HID-fed aging rats.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eThe statistical data of left ventricular ejection fraction (EF) of rats in Aging ND group and Aging HID group (n\u0026thinsp;=\u0026thinsp;6 per group).\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eThe statistical data of left ventricular fraction shortening(FS) of rats in Aging ND group and Aging HID group (n\u0026thinsp;=\u0026thinsp;6 per group).\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eThe levels of plasma NT-proBNP in rats from Aging ND group and Aging HID group (n\u0026thinsp;=\u0026thinsp;6 per group).\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eRepresentative images of HE staining in ND-fed and HID-fed aging rats.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eRepresentative images of Masson staining in ND-fed and HID-fed aging rats.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eThe collagen volume fraction of the left ventricle of rats from Aging ND and Aging HID group (n\u0026thinsp;=\u0026thinsp;6 per group).\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eRepresentative M-mode images of heart in Aging Control group and Aging Fer-1 group.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eThe statistical data of cardiac ejection fraction (EF) of rats in Aging Control group and Aging Fer-1 group (n\u0026thinsp;=\u0026thinsp;6 per group).\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eThe statistical data of cardiac fraction shortening (FS) of rats in Aging Control group and Aging Fer-1 group (n\u0026thinsp;=\u0026thinsp;6 per group).\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eThe levels of plasma NT-proBNP in rats from Aging Control group and Aging Fer-1 group (n\u0026thinsp;=\u0026thinsp;6 per group).\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003c/ol\u003e\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003eThe data are given as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM and compared by Student\u0026rsquo;s t test.\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003e3.4 HADHA deficiency in aging rats contributes to ferroptosis.\u003c/h2\u003e\u003cp\u003eTo elucidate the mechanism underlying ferroptosis in aging rats, we conducted a proteomics on the hearts of aging rats received normal diet and high-iron diet, revealing a significant differences in the protein expression profiles between the two groups \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of these differentially expressed proteins indicated that the fatty acid metabolism pathway was the most enriched term \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB\u003cb\u003e)\u003c/b\u003e. Notably, the expression level of HADHA was significantly downregulated in the cardiac tissues of aging rats on a high-iron diet (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). HADHA, the alpha subunit of the mitochondrial trifunctional protein, catalyzes the beta-oxidation of fatty acids, playing a crucial role in maintaining fatty acid oxidation in cardiomyocytes\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. We corroborated the downregulation of HADHA expression in the hearts of aging rats received a high-iron diet as well as aging rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD-F).\u003c/p\u003e\u003cp\u003eIt has been reported that HADHA deficiency leads to mitochondrial dysfunction, which in turn triggers the accumulation of ROS\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. Interestingly, we discovered that silencing HADHA led to an increase in ROS levels and a significant decrease in mitochondrial membrane potential (MMP) in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced senescent cardiomyocytes (\u003cb\u003eFig. S4A-C\u003c/b\u003e). The cell viability and ATP levels were also reduced in senescent cardiomyocytes with HADHA deficiency (\u003cb\u003eFig. S4D-E\u003c/b\u003e).Cellular experiments revealed that silencing HADHA increased iron deposition (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG-H), upregulated Tf expression, and decreased the expression levels of FTH1 and GPX4 in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced senescent cardiomyocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ). GSH, a vital cellular antioxidant, which depletion can lead to the downregulation of GPX4, eventually heightening the cell's vulnerability to ferroptosis\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. We observed a remarkable depletion of GSH in cardiomyocytes following the silencing of HADHA(Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI).However, silencing HADHA has no effects on the level of ferroptosis in senescent cardiac fibroblasts (\u003cb\u003eFig. S5A-B\u003c/b\u003e). Additionally, overexpression of HADHA abrogated the increase in ROS levels, the decrease in cell viability, GSH and ATP induced by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (\u003cb\u003eFig. S6A-E\u003c/b\u003e). Taken together, HADHA deficiency causes ferroptosis in cardiomyocytes.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003col\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eThe volcano plot of proteomics data showed diferentially expressed genes between ND-fed and HID-fed aging rats. n\u0026thinsp;=\u0026thinsp;3 biological replicates/group. Downregulation and up-regulation are shown in blue and red, respectively.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eKEGG analyses of proteomics data showing the top 8 enriched pathways in the heart between ND-fed and HID-fed aging rats.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eThe expression of HADHA in ND-fed and HID-fed aging rats determined by proteomics.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eRepresentative bands showing the expression of HADHA in the heart of rats from Aging ND and Aing HID groups, and rats from Young and Aging groups.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eQuantification of expressions of HADHA in the heart of rats from Aging ND and Aing HID groups (n\u0026thinsp;=\u0026thinsp;6 per group).\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eQuantification of expressions of HADHA in the heart of rats from Young and Aging groups (n\u0026thinsp;=\u0026thinsp;6 per group).\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003c/ol\u003e\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003e\u003cb\u003e(G-J)\u003c/b\u003e Cardiomyocytes were first transfected with plasmids to silence HADHA. After 24 hours, following a change of culture media,they were treated with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 24 hours before conducting assays for relevant markers.\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003cp\u003e\u003col\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eRepresentative image of FerroOrange detection in cardiomyocytes.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eFluorescence intensity of FerroOrange detection in cardiomyocytes (n\u0026thinsp;=\u0026thinsp;4 per group).\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eThe levels of GSH in cardiomyocytes (n\u0026thinsp;=\u0026thinsp;5 per group).\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eRepresentative bands and quantification of the expression of Tf, FTH1 and GPX4 in cardiomyocytes (n\u0026thinsp;=\u0026thinsp;6 per group).\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003c/ol\u003e\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003eThe data are given as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM and compared by Student\u0026rsquo;s t test or one way ANOVA.\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e\u003ch2\u003e3.5 HADHA deficiency induces ferroptosis through regulation of GSH-GPX4.\u003c/h2\u003e\u003cp\u003eIn order to further substantiate that HADHA deficiency promotes ferroptosis via GSH depletion, we administered N-acetylcysteine (NAC), a precursor of GSH, to the HADHA-silenced cardiomyocytes. Notably, NAC supplementation reduced the accumulation of ROS, led to an increased in MMP levels, accompanied by decreased expression of Tf, and increased expression of FTH1 and GPX4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-D). Those results indicated that HADHA deficiency induces ferroptosis through regulation of GSH-GPX4.\u003c/p\u003e\u003cp\u003eTo substantiate the potential of NAC in age-related HF, 18-month old rats were treated with NAC for 4 months (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). We found that supplementation of NAC reduced the expression of TF, while increased FTH1 and GPX4 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). Notably, NAC ameliorated cardiac function of aging rats, supported by the elevated EF and FS, and reduced NT-proBNP (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG-J). Furthermore, NAC significantly alleviated cardiac structural disorder and fibrosis levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eK-M). To further evalulate the therapeutic potential of NAC for age-related HF, we established an aging rat model using D-galactose, followed by intervention with either a control solvent or NAC \u003cb\u003e(Fig. S7A)\u003c/b\u003e. As expected, NAC significantly downregulated TF expression, and upregulated FTH1 and GPX4 expression in the hearts of aging rats induced by D-galactose (\u003cb\u003eFig. S7B\u003c/b\u003e). NAC treatment significantly upregulated EF and FS \u003cb\u003e(Fig. S7C-E)\u003c/b\u003e, reduced NT-proBNP\u003cb\u003e(Fig. S7F)\u003c/b\u003e, and rectified cardiac structural disorder, and reduced cardiac fibrosis levels (\u003cb\u003eFig. S7G-I\u003c/b\u003e). Taken together, these findings provide evidence that NAC prevents the occurrence of age-related HF by reducing cardiac ferroptosis levels.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003e\u003cb\u003e(A-D)\u003c/b\u003e Cardiomyocytes were first transfected with plasmids to silence HADHA. After 24 hours, they were treated with H2O2 for 24 hours. Following a change of culture media, the cells were treated with control solvent or NAC for 24 hours before conducting assays for relevant markers.\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003cp\u003e\u003col\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eRepresentative images of ROS staining and JC-1 staining in cardiomyocytes.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eThe fluorescence intensity of ROS staining of cardiomyocytes (n\u0026thinsp;=\u0026thinsp;5 per group).\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eThe JC-1 polymer/monomer fluorescence ratio of cardiomyocytes (n\u0026thinsp;=\u0026thinsp;5 per group).\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eRepresentative bands and quantification of expressions of Tf, FTH1 and GPX4 in cardiomyocytes (n\u0026thinsp;=\u0026thinsp;6 per group).\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eSchematic illustration of the experimental design for NAC intervention. 18 months old rats were randomly divided into two groups, receiving control solvent (Aging Control group) or NAC (Aging NAC group) through drinking for 4 months.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eRepresentative bands and quantification of the expression of Tf, FTH1 and GPX4 in the heart of rats from Aging Control group and Aging NAC group (n\u0026thinsp;=\u0026thinsp;4 per group).\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eRepresentative M-mode images of heart in Aging Control group and Aging NAC group.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eThe statistical data of left ventricular ejection fraction (EF) of rats from Aging Control group and Aging NAC group (n\u0026thinsp;=\u0026thinsp;4 per group).\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eThe statistical data of left ventricular fraction shortening (FS) of rats from Aging Control group and Aging NAC group (n\u0026thinsp;=\u0026thinsp;4 per group).\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eThe levels of plasma NT-ProBNP in rats from Aging Control group and Aging NAC group (n\u0026thinsp;=\u0026thinsp;4 per group).\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eRepresentative images of HE staining of the left ventricle of rats from Aging Control group and Aging NAC group.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eRepresentative images of Masson staining of the left ventricle of rats from Aging Control group and Aging NAC group.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eThe collagen volume fraction of the left ventricle of rats from Aging Control group and Aging NAC group (n\u0026thinsp;=\u0026thinsp;4 per group).\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003c/ol\u003e\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003eThe data are given as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM and compared by Student\u0026rsquo;s t test.\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003e3.6 Sirt1 deficiency downregulates HADHA expression by inhibiting GATA4 expression.\u003c/h2\u003e\u003cp\u003eSirtuins have been recognized as crucial regulators in anti-aging processes. Among them, Sirt1 has garnered significant attention as a potential therapeutic target for the mitigation of age-related disorders, including Alzheimer's disease and Parkinson's disease\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e. In our previous studies, we found a correlation between the reduction of Sirt1 in the atria of aging rats and the incidence of age-related atrial fibrillation. However, whether Sirt1 is involved in age-related HF remians unclear. Intriguingly, we found that silencing Sirt1 in cardiomyocytes led to a downregulation of HADHA, accompanied by an increase in Tf expression and a decrease in GPX4 and FTH1 levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). However, the result of protein interaction prediction analysis showed no binding sites between Sirt1 and HADHA, while the PCR data confirmed that the mRNA levels of HADHA were significantly decreased in cardiomyocytes with Sirt1 silencing, indicating that Sirt1 may affect HAHDA expression by regulating the transcription factor of HADHA. Database analysis revealed that HADHA expression can be regulated by GATA4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB-C), a transcription factor of HADHA. To confirm that Sirt1 can bind to GATA4 and regulate GATA4 expression, we performed co-immunoprecipitation experiments. As expected, the binding between Sirt1 and GATA4 was observed. Furthermore, silencing Sirt1 led to a downregulation of GATA4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). These findings suggest that Sirt1 deficiency downregulates GATA4, thereby inhibiting HADHA expression and promoting the occurrence of ferroptosis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec25\" class=\"Section2\"\u003e\u003ch2\u003e3.7 Supplementation of resveratrol restrains ferroptosis and protects aging rats from heart failure.\u003c/h2\u003e\u003cp\u003eResveratrol is a known activator of Sirt1. To confirm the role of Sirt1 deficiency is the key mechanism for age-related HF, we administrated resveratrol to 18-month-old aged rats for 4 months(Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). Compared to the control group, the resveratrol-treated group exhibited significantly upregulated expression levels of Sirt1, HADHA, and GATA4 in cardiac tissues(Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF). Simultaneously, resveratrol notably reduced TF expression levels, while it upregulated FTH1 and GPX4 expression levels in the heart tissues of aging rats(Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG-H), suggesting a significant alleviation of cardiac ferroptosis. Additionally, resveratrol markedly improved cardiac function\u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eI-L\u003cb\u003e)\u003c/b\u003e, and alleviated cardiac structural disorder and cardiac fibrosis in aging rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eM-O). These findings suggest that Sirt1 is a potential therapeutic target in aging-related HF. The use of resveratrol, as a Sirt1 activator, may represent a promising therapeutic strategy for the treatment and management of this age-related cardiac condition.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003col\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eCardiomyocytes were transfected with plasmids to silence Sirt1.Representative bands and quantification of expressions of HADHA, Tf, FTH1 and GPX4 in cardiomyocytes (n\u0026thinsp;=\u0026thinsp;5 per group).\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eMolecular docking of Sirt1 and HADHA.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eThe relative mRNA expression of HADHA in cardiomyocytes (n\u0026thinsp;=\u0026thinsp;6 per group).\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eRepresentative Western blots and quantification of protein expressions of Sirt1 and GATA4 (n\u0026thinsp;=\u0026thinsp;6 per group). Fractions of cardiomyocytes were immunoprecipitated with anti-Sirt1 antibody, and immunoblotting was performed with Sirt1 antibody and GATA4 antibody.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eSchematic illustration of the experimental design for resveratrol intervention. 18 months old rats were randomly divided into two groups, receiving control solvent (Aging Control group) or resveratrol (Aging RES group) through gavage for 4 months.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eRepresentative bands of expressions of Sirt1, HADHA and GATA4 in the heart of rats from Aging Control group and Aging RES group.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eRepresentative bands of expressions of Tf, FTH1 and GPX4 in the heart of rats from Aging Control group and Aging RES group.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eQuantification of protein expressions of Sirt1, HADHA, GATA4, Tf, FTH1 and GPX4 in the heart of rats from Aging Control group and Aging RES group (n\u0026thinsp;=\u0026thinsp;6 per group).\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eRepresentative M-mode images of heart in from Aging Control group and Aging RES group.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eThe statistical data of left ventricular ejection fraction (EF) of rats from Aging Control group and Aging RES group (n\u0026thinsp;=\u0026thinsp;6 per group).\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eThe statistical data of left ventricular fraction shortening (FS) of rats from Aging Control group and Aging RES group (n\u0026thinsp;=\u0026thinsp;6 per group).\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eThe levels of plasma NT-ProBNP in rats from Aging Control group and Aging RES group (n\u0026thinsp;=\u0026thinsp;6 per group).\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eRepresentative images of HE staining of the left ventricle of rats from Aging Control group and Aging RES group.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eRepresentative images of Masson staining of the left ventricle of rats from Aging Control group and Aging RES group.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eThe collagen volume fraction of the left ventricle of rats from Aging Control group and Aging RES group (n\u0026thinsp;=\u0026thinsp;6 per group).\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003c/ol\u003e\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003eThe data are given as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM and compared by Student\u0026rsquo;s t test.\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis research presents groundbreaking pathophysiological perspectives on the intricate link between ferroptosis and age-related HF, thereby paving the way for a novel therapeutic approach in managing heart failure associated with aging. Herein, we revealed that ferroptosis is the predominant mechanism for age-related HF. In the heart of aging rats, the expression of HADHA was significantly decreased, which led to mitochondrial dysfunction, causing the accumulation of reactive oxygen species, following the exhaustion of GSH and remarkable downregulation of GPX4, inducing ferroptosis. Furthermore, we found that Sirt1 deficiency contributed to the downregulation of HADHA in aging rats through binding GATA4. Supplementation of resveratrol, an agonist of Sirt1, effectively mitigated ferroptosis and protected aging rats from HF. Taken together, targeting ferroptosis may be a potential therapeutic target for age-related HF.\u003c/p\u003e\u003cp\u003eHeart failure is a significant health threat, characterized by high morbidity and mortality rates. Its incidence notably escalates with age, and the pathophysiology of heart failure in the elderly remains complex and not fully understood. In the present study, we observed a remarkable increase in ferroptosis levels in the cardiac tissues of aging rats. Ferroptosis is an iron-dependent form of cell death characterized by lipid peroxidation. Mounting studies have linked ferroptosis with various age-related disorders\u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e. Recently, an increasing body of research has demonstrated the involvement of ferroptosis in the development and progression of various cardiovascular diseases, such as myocardial ischemia-reperfusion injury and cardiomyopathy\u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e. Targeting ferroptosis has emerged as a potential therapeutic strategy for various diseases, such as age-related diseases\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e, cancer\u003csup\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e, and cardiovascular diseases\u003csup\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e. Our findings revealed that a high-iron exacerbated ferroptosis in the cardiac tissues of aging rats, leading to a decline in cardiac function. Conversely, ferroptosis inhibitors can effectively prevent this decline in cardiac function. These results underscore ferroptosis as a critical mechanism mediating aging-related HF.\u003c/p\u003e\u003cp\u003eHADHA is one of the subunits of hydroxyacyl CoA dehydrogenase trifunctional multienzyme complex, which catalyzes the last three reactions of the mitochondrial fatty acid β-oxidation\u003csup\u003e[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/sup\u003e. HADHA plays a essential role in mitochondrial fatty acid oxidation and as an acyltransferase in cardiolipin remodeling for cardiac homeostasis\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. Impairment of lipid oxidation can cause accumulation of lipid peroxides, which is an important mechanism for ferroptosis. We found that the expression of HADHA was significantly decreased in the heart of aging rats, and further decreased in high-Iron diet fed aging rats. HADHA deficiency led to mitochondrial dysfunction and generation of large amounts of reactive oxygen species in cardiomyocytes. The accumulation of reactive oxygen species caused GSH depletion and downregulation of GPX4, which eventually triggering ferroptosis. We used NAC, a precursor of GSH, comfirmed that HADHA deficiency induced accumulation of reactive oxygen species and GSH depletion is the key mechanism for ferroptosis. We firstly revealed the causal relationship between HADHA deficiency and ferroptosis in age-related HF.\u003c/p\u003e\u003cp\u003eSirt1, is a highly conserved NAD\u003csup\u003e+\u003c/sup\u003e-dependent class III histone/protein deacetylase of sirtuins family, has been identified as important anti-aging regulatory factors\u003csup\u003e[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/sup\u003e. Targeting Sirt1 has emerged as a novel measures for age-related disorders\u003csup\u003e[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]\u003c/sup\u003e. Our previous study demonstrated that Sirt1 deficiency contributed to age-related atrial fibrillation through regulation of necroptosis. Berger also revealed the downregulation of Sirt1 in senescence and ageing\u003csup\u003e[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]\u003c/sup\u003e. In our present study, silencing Sirt1 led to a decrease in the expression of HADHA through binding and downregulating the levels of GATA4, a transcription factor of HADHA. Furthermore, Sirt1 deficiency aggravated ferroptosis of cardiomyocytes. Administration of resveratrol, an agonist of Sirt1, exhibited a potential to alleviate ferroptosis and prevented aging rats from cardiac dysfunction. It was reported that melatonin MT1 receptors prevents α-syn-induced ferroptosis in Parkinson's disease through enhancement of the Sirt1/Nrf2/Ho1/Gpx4 pathway\u003csup\u003e[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]\u003c/sup\u003e, consistent with our study that indicating the potential of Sirt1 activation in suppression of ferroptosis.\u003c/p\u003e\u003cp\u003eIn summary, our study provides novel insights into the crucial role of ferroptosis in the progression of age-related HF. We demonstrated that aging rats displayed accumulated lipid peroxides and aggravated ferroptosis in the heart, while administration of ferroptosis inhibitor effectively prevented the decline of cardiac function in aging rats. Specifically, we found that the decline of Sirt1 in aging rats causing the downregulation of HADHA through binding its transcription factor GATA4. HADHA deficiency caused mitochondrial dysfunction and accumulation of ROS, which inducing exhaustion of GSH and downregulation of GPX4, leading to ferroptosis and age-related HF. These findings highlight that targeting HADHA or Sirt1 may be a novel measures for the prevention and treatment of age-related HF.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAged-related Sirt1 deficiency leads to the downregulation of HADHA through inhibiting GATA4. The decreased HADHA induces mitochondrial dysfunction, resulting in an accumulation of reactive oxygen species, which in turn depletes GSH, causing a downregulation of GPX4, ultimately triggers cardiac ferroptosis and aged-related cardiac dysfunction.\u003c/p\u003e\u003cp\u003e\u003cb\u003eLimitations\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSeveral limitations are acknowledged in our study. Firstly, due to the inherent challenges in obtaining human cardiac tissue, our findings related to the expression levels of HADHA and related mechanism lack validation in human samples. Secondly, our assertion of the protective effects of resveratrol against age-related HF is based solely on animal models. Further studies, particularly clinical trials in human subjects, are needed to validate the therapeutic potential of resveratrol in the context of age-related HF. Finaly, our study primarily focused on the role of HADHA deficiency-induced ferroptosis and did not comprehensively explore the contributions of other proteins or other forms of cell death.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCRediT authorship contribution statement\u003c/b\u003e\u003c/p\u003e\u003cp\u003eYun Zhang and Yu Duan conducted the statistical analyses, interpreted the results, and prepared the manuscript with input from all authors. Yingchun Luo analyzed the proteomics databases. Xuejie Han drafted the manuscript. Yue Li and Yong Zhang designed the experiments. Yun Zhou, Yunlong Gao and Hui Yu contributed to the animal experiments, while Qian Xu and Ying Wei performed the cell experiments and qRT-PCR experiments. Ruoxin Min, Yong Hong, and Xuanrui Ji supervised and performed sample data collection. Yun Zhang and Yu Duan have reviewed and verified the underlying data. All authors have read and approved the final version of the manuscript.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eDeclaration of competing interest\u003c/h2\u003e\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e\u003cp\u003eThis work was supported by grants from the National Key R\u0026amp;D Program of China (2024YFA1307001) to Y. L, the National Natural Science Foundation of China (82200413) to Y.Z, the State\u0026ensp;Key\u0026ensp;Program\u0026ensp;of\u0026ensp;National\u0026ensp;Natural\u0026ensp;Science\u0026ensp;Foundation\u0026ensp;of\u0026ensp;China (No.82330014) to Y. L, the National Natural Science Foundation of China (No.82070336) to Y. L, and the National Natural Science Foundation of China (No. 82400375) to XJ. H.\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e\u003cp\u003eData will be made available on request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHaddad F, Saraste A, Santalahti KM, et al. Smartphone-Based Recognition of Heart Failure by Means of Microelectromechanical Sensors. JACC Heart Fail. 2024: S2213-1779(24)00165-3 [pii].\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhang ZD, Milman S, Lin JR, et al. Genetics of extreme human longevity to guide drug discovery for healthy ageing. 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Exp Mol Med. 2023. 55(6): 1232\u0026ndash;1246.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang Y, Xie F, He Z, et al. Senescence-Targeted and NAD(+)-Dependent SIRT1-Activated Nanoplatform to Counteract Stem Cell Senescence for Promoting Aged Bone Regeneration. Small. 2024. 20(12): e2304433.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMengozzi A, Costantino S, Paneni F, et al. Targeting SIRT1 Rescues Age- and Obesity-Induced Microvascular Dysfunction in Ex Vivo Human Vessels. Circ Res. 2022. 131(6): 476\u0026ndash;491.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eXu C, Wang L, Fozouni P, et al. SIRT1 is downregulated by autophagy in senescence and ageing. Nat Cell Biol. 2020. 22(10): 1170\u0026ndash;1179.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLv QK, Tao KX, Yao XY, et al. Melatonin MT1 receptors regulate the Sirt1/Nrf2/Ho-1/Gpx4 pathway to prevent α-synuclein-induced ferroptosis in Parkinson's disease. J Pineal Res. 2024. 76(2): e12948.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Unsectioned Paragraphs","content":"\u003cp\u003e*Corresponding auth: [email protected] (Yun Zhang), [email protected] (Yue Li), [email protected](Haibo Jia).\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cell-death-and-disease","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddis","sideBox":"Learn more about [Cell Death \u0026 Disease](http://www.nature.com/cddis/)","snPcode":"41419","submissionUrl":"https://mts-cddis.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Heart failure, Aging, Ferroptosis, HADHA, Sirt1","lastPublishedDoi":"10.21203/rs.3.rs-7140279/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7140279/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAging is a major contributor to the escalating prevalence of heart failure (HF). Ferroptosis has been implicated in age-related disorders and cardiovascular diseases.\u003c/p\u003e\u003cp\u003eThe role of ferroptosis in age-related HF and the underlying mechanisms remain ambiguous. Herein, we found that aging rats displayed compromised cardiac function, with molecular characteristics indicative of ferroptosis, including diminished glutathione peroxidase 4 (GPX4) levels and heightened lipid peroxidation. Notably, a high-iron diet exacerbated ferroptosis and promoted cardiac dysfunction, while ferrostatin-1, a specific ferroptosis inhibitor, rescued this phenotype. Proteomic data analysis uncovered that the expression of hydroxyacyl-CoA dehydrogenase subunit A (HADHA) was significantly reduced in aging rats fed a high-iron diet. HADHA deficiency resulted in mitochondrial dysfunction and an accumulation of reactive oxygen species, leading to glutathione (GSH) exhaustion, causing the downregulation of GPX4 and subsequent ferroptosis. Furthermore, it was confirmed that the reduction of Sirt1 in the hearts of aging rats caused the downregulation of HADHA by binding and inhibited the expression of GATA4, a transcription factor of HADHA, as evidenced by co-immunoprecipitation experiments. Furthermore, resveratrol, a Sirt1 agonist, effectively shielded aging rats from HF by upregulating HADHA and mitigating ferroptosis. In conclusion, this study underscores the significance of ferroptosis in age-related HF, and suggests that targeting HADHA or Sirt1 may present potential strategies for the prevention and treatment of age-related HF.\u003c/p\u003e","manuscriptTitle":"Sirt1 deficiency promotes age-related heart failure through enhancing ferroptosis via GATA4-HADHA-GPX4 axis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-31 12:05:56","doi":"10.21203/rs.3.rs-7140279/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2025-08-18T10:39:40+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-08-11T13:05:13+00:00","index":1,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-08-10T12:06:59+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-08-07T11:56:44+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-07-28T00:30:16+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2025-07-27T19:05:31+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-17T13:44:58+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-16T13:02:08+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cell Death \u0026 Disease","date":"2025-07-16T13:02:07+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-death-and-disease","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddis","sideBox":"Learn more about [Cell Death \u0026 Disease](http://www.nature.com/cddis/)","snPcode":"41419","submissionUrl":"https://mts-cddis.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"81ce67f8-9fc3-4508-bfc3-02e943c7aba4","owner":[],"postedDate":"July 31st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":52188071,"name":"Biological sciences/Cell biology/Senescence"},{"id":52188072,"name":"Biological sciences/Cell biology/Cell death"}],"tags":[],"updatedAt":"2026-04-01T07:43:32+00:00","versionOfRecord":{"articleIdentity":"rs-7140279","link":"https://doi.org/10.1038/s41419-026-08634-z","journal":{"identity":"cell-death-and-disease","isVorOnly":false,"title":"Cell Death \u0026 Disease"},"publishedOn":"2026-03-23 04:00:00","publishedOnDateReadable":"March 23rd, 2026"},"versionCreatedAt":"2025-07-31 12:05:56","video":"","vorDoi":"10.1038/s41419-026-08634-z","vorDoiUrl":"https://doi.org/10.1038/s41419-026-08634-z","workflowStages":[]},"version":"v1","identity":"rs-7140279","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7140279","identity":"rs-7140279","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}