The pathogenesis of myocardial ischemia/reperfusion injury (MIRI) is complicated and involves various factors, including iron overload, oxidative stress, mitochondrial damage, calcium overload, and inflammation. Hence, the aim of this study was to explore the molecular mechanisms of MIRI and verify the myocardial protection mechanism of carnosic acid (CA) in MIRI. In vivo and in vitro MIRI models were established. The results of this study verified that pretreatment with CA effectively increased cell viability, reduced the apoptosis rate and lactate dehydrogenase activity, maintained mitochondrial dynamic balance, and inhibited iron overload and abnormal lipid metabolism in cardiomyocytes with anoxia/reoxygenation treatment. Pretreatment with CA not only improved cardiac function and reduced infarct size in rats treated with ischemia/reperfusion (I/R) but also decreased reactive oxygen species levels and the rate of apoptotic cardiomyocytes in myocardium induced by I/R. These cardioprotective effects of CA could be inhibited by treatment with adenoviral vector expressing Mfn2 short hairpin RNA. In general, CA pretreatment protected cardiomyocytes against MIRI injury by inhibiting ferroptosis and maintaining mitochondrial dynamic homeostasis through an Mfn2-dependent mechanism.

Abdominal aortic aneurysm (AAA) progression is closely linked to inflammation and endothelial dysfunction. Our previous study has demonstrated that increased CD95 ligand (CD95L) and its downstream effector Caspase-8 in the aortic tissue, contributed to AAA by modulating inflammation. However, how the CD95L/Caspase-8 modulated aneurysmal inflammation remains poorly understood. This study investigates how CD95L/Caspase-8 signaling drives endothelial pyroptosis to exacerbate AAA. Using a CaCl2-induced AAA murine model and primary mouse aortic endothelial cells (MAECs), we demonstrate that CD95L triggers endothelial pyroptosis, characterized by NLRP3 inflammasome activation, Gasdermin D N-terminal (GSDMD-N) cleavage, and Caspase-8/Caspase 1 activation. Electron microscopy confirmed pyroptotic morphology, while flow cytometry excluded apoptosis or necrosis. CD95L elevated IL-1β/IL-18 secretion, which was abolished by Caspase-8 siRNA or inhibitor Z-IETD-FMK. Mechanistically, CD95L suppressed Caspase-8 phosphorylation at Tyr380, enabling its activation of GSDMD-dependent pyroptosis. In vivo, CaCl2-induced AAA mice exhibited aortic dilation, elastin degradation, and endothelial-specific pyroptosis, all attenuated by endothelial-targeted Caspase-8 knockdown via AAV9-shRNA. This intervention reduced NLRP3 and GSDMD-N expression while preserving vascular integrity. Similarly, SRC kinase activation mitigated pyroptosis markers and aortic damage. These findings establish CD95L as a key mediator of endothelial pyroptosis in AAA via Caspase-8 dephosphorylation and NLRP3/GSDMD-N activation. Targeting Caspase-8 or enhancing SRC activity represents a promising therapeutic strategy to curb AAA progression by preserving endothelial homeostasis.

Myocardial ischemia-reperfusion (I/R) injury disrupts intracellular ion homeostasis, triggering intracellular sodium overload and ultimately leading to cell death. However, the specific molecular mechanisms driving this pathological process remain incompletely understood. Weighted gene co-expression network analysis (WGCNA) was applied to myocardial I/R injury transcriptomic datasets from the GEO database to identify genes associated with sodium accumulation. Subsequently, three machine learning algorithms (LASSO, Random Forest, and Support Vector Machine) were integrated to further screen for core hub genes. Single-cell RNA sequencing (scRNA-seq) data were utilized to determine the cellular localization of these key genes, followed by in vivo validation using a murine myocardial I/R injury model. WGCNA identified 11 candidate genes highly correlated with sodium accumulation. Further screening via machine learning algorithms pinpointed MYD88 and GPR35 as core genes. Single-cell analysis revealed that MYD88 was specifically upregulated in macrophages, suggesting that sodium overload might exacerbate myocardial I/R injury by mediating inflammatory responses. In vivo experiments confirmed a significant elevation of sodium ion concentration in myocardial tissues following I/R, accompanied by a marked upregulation of MYD88 expression. This study confirms that myocardial I/R injury triggers sodium overload and identifies MYD88 as a candidate molecule closely associated with sodium overload-induced cell death (SOICD). These findings provide a novel perspective for further exploring the pathological mechanisms underlying myocardial I/R injury.

In the original publication [...].

PurposeDiabetic cardiomyopathy (DCM), which is diabetes mellitus-induced cardiomyopathy, significantly elevates the risk of heart failure and sudden cardiac death. No specific treatments for DCM are currently available. Gallic acid (GA) is a polyhydroxyphenolic compound that has been shown to inhibit ferroptosis and maintain mitochondrial homeostasis, with potential therapeutic effects in various cardiac diseases. However, its specific role and underlying mechanisms in DCM remain unexplored.MethodsAn in vitro model was established using H9C2 cells pretreated with high glucose plus palmitate, and an in vivo type 2 diabetes mellitus model generated by treating rats with streptozotocin-induced and feeding a high-fat diet. The protective effects of GA and its mechanism of action were evaluated using various methods, including flow cytometry, Western blotting (WB), and transmission electron microscopy. Bioinformatics analysis identified potential target genes for GA’s cardioprotection, which were subsequently validated using pAD/TSPO (for overexpression) and pAD/FTMT-shRNA (for silencing) constructs.ResultsGA treatment decreased PTGS2, lactate dehydrogenase, malondialdehyde, ferrous iron, ROS, and oxidized glutathione disulfide (GSSG) levels and increased cell viability, glutathione (GSH) levels, the GSH/GSSG ratio, and GPX4 protein levels in the injury models. GA markedly attenuated mitochondrial ultrastructural damage and promoted mitochondrial homeostasis. These protective effects were abrogated by TSPO overexpression and FTMT silencing.ConclusionGA was shown to attenuate diabetic cardiomyopathy by inhibiting ferroptosis and protecting mitochondria via the TSPO/FTMT signaling pathway.

Catheter ablation (CA) is a recognized treatment for people experiencing symptomatic paroxysmal or persistent atrial fibrillation (AF). Pulmonary vein isolation (PVI) is the predominant therapeutic intervention for AF globally. According to PVI, some individuals may require supplementary ablation interventions. Literature has shown that the closeness of the esophagus to the posterior wall of the left atrium might result in injury to the vagal nerve branches during CA, causing conditions such as "gastroparesis" or "pyloric spasms." This report details a case of superior mesenteric artery compression syndrome following AF ablation, which did not improve with conservative management and was ultimately addressed by surgical procedure.

PURPOSE:Ischemia-reperfusion injury remains a major problem following myocardial infarction. Alpinetin (ALPT) has been reported to exhibit cardioprotective effects as well as resistance to ischemia-reperfusion injury. However, its role and mechanism during myocardial ischemia-reperfusion injury are unknown. METHODS:The anoxia/reoxygenation (A/R) injury model of H9c2 cells and the ischemia reperfusion (I/R) injury model of Sprague-Dawley rats were used in this study. Multiple indicator evaluations, flow cytometry, western blot, and transmission electron microscopy were performed to assess the protective effect of alpinetin pretreatment and its mechanism of action. In addition, the role of mitochondrial ferritin (FTMT) in alpinetin-based protection was investigated using pAD/FTMT-shRNA. The experimental findings were ultimately validated in rat I/R injury models. RESULTS:Similar to ferrostatin-1, alpinetin decreased prostaglandin-endoperoxide synthase 2 (PTGS2), lactate dehydrogenase, malondialdehyde, ferrous iron, reactive oxygen species, and oxidized glutathione disulfide (GSSG) levels and increased cell viability, glutathione (GSH) levels, the GSH/GSSG ratio, and glutathione peroxidase 4 protein levels in the injury models. Alpinetin also reversed A/R injury-induced increased caspase-3 activity and apoptosis rate and decreased Bcl-2/Bax ratio and mitochondrial membrane potential level. Of note, alpinetin attenuated mitochondrial damage induced by A/R injury. These protective effects were blocked via FTMT silencing. CONCLUSION:Alpinetin protects against myocardial ischemia-reperfusion injury by inhibiting ferroptosis and apoptosis via FTMT.

Ischaemic cardiomyopathy is becoming one of the most prevalent cardiovascular diseases among the global elderly population. However, the underlying molecular mechanisms remain incompletely understood. Our previous study demonstrated that VDAC1 plays a significant role in MI/RI. Furthermore, FTMT plays a pivotal role in iron metabolism. However, the precise molecular functions of VDAC1 and FTMT in MI/RI remain to be elucidated. In vitro H9c2 cells A/R and in vivo SD rat MI/RI models were constructed. The present study reports that VDAC1 levels were increased and FTMT levels were decreased in A/R. The overexpression of VDAC1 resulted in an exacerbation of the A/R-induced injury, characterised by an increase in oxidative stress, a reduction in the GSH/GSSG ratio, the formation of reactive oxygen species, elevated levels of lipid peroxidation, and the deposition of iron. In contrast, FTMT overexpression reversed these alterations and mitigated mitochondrial dysfunction by downregulating VDAC1, PTGS2 levels, upregulating GPX4 levels, inhibiting MPTP over-opening and stabilising MMP. Additionally, knockdown of VDAC1 alleviated A/R-induced ferroptosis. In vivo experiments showed that overexpression of FTMT improved cardiac function in rats, as evidenced by the reduction of MI/RI-induced serum CK-MB, LDH and Fe2+ content and the shrinkage of myocardial infarction area. Moreover, HE, DHE staining and TEM observations showed that the overexpression of FTMT ameliorated MI/RI-induced myocardial tissue and mitochondrial damage. Furthermore, the overexpression of FTMT was found to inhibit MI/RI-induced ferroptosis. In general, our study is the first to demonstrate that FTMT overexpression alleviates ferroptosis and mitochondrial dysfunction by regulating VDAC1, thereby reducing MI/RI injury.

The study aimed to elucidate the underlying pharmacological mechanism of the traditional Chinese medicine Pue in ameliorating myocardial ischemia-reperfusion injury (MIRI), a critical clinical challenge exacerbated by reperfusion therapy. In vivo MIRI and in vitro anoxia/reoxygenation (A/R) models were constructed. The results demonstrated that Pue pretreatment effectively alleviated MIRI, as manifested by diminishing the levels of serum CK-MB and LDH, mitigating the extent of myocardial infarction and enhancing cardiac functionality. Additionally, Pue significantly alleviated histopathological damage in MIRI-treated myocardium, as evidenced by HE staining and TUNEL assay. In vitro, Pue pretreatment significantly alleviated A/R-induced damage by decreasing LDH levels, increasing cellular activity, inhibiting autophagic lysosomal overactivation, inhibiting oxidative stress (ROS, LIP ROS, MDA), increasing antioxidant defense (SOD, GSH-Px), and increasing P62 protein expression while decreasing LC3II/I ratio. Furthermore, Pue inhibited apoptosis and maintained mitochondrial homeostasis by up-regulating the expression of Hairy and Enhancer of Split-1 (HES1) protein, which was crucial for its cardioprotective effects. Nevertheless, the cardioprotective efficacy of Pue pretreatment was negated via the knockdown of HES1 protein expression via pAD/HES1-shRNA transfection. In conclusion, Pue effectively ameliorated HES1-mediated MIRI-induced autophagy, apoptosis, and mitochondrial dysfunction.

Pulmonary artery hypertension (PAH) is characterized by a cancer-like metabolic shift towards aerobic glycolysis. Nuclear Receptor Binding SET Domain Protein 2 (NSD2), a histone methyltransferase, has been implicated in PAH, yet its precise role remains unclear. In this study, we induced PAH in C57BL/6 mice using monocrotaline (MCT) and observed increased FOLR1 expression in PAH tissues, which was suppressed by NSD2 knockdown. Silencing NSD2 or FOLR1 inhibited the proliferation and migration of pulmonary artery endothelial cells (PAECs) and alleviated PAH phenotypes, right ventricular dysfunction, and pulmonary artery remodeling. Mechanistically, NSD2 knockdown prevented nuclear translocation of FOLR1 and its interaction with H3K36me2. Metabolic analysis revealed that NSD2 or FOLR1 knockdown reversed the increased oxygen consumption rate, extracellular acidification rate, glucose consumption, lactate production, and G6PD activity in MCT-treated PAECs. Furthermore, NSD2 or FOLR1 silencing decreased the expression of key glycolytic genes (HK2, TIGAR, and G6PD) by suppressing their promoter activity and weakening the interaction between FOLR1/H3K36me2 and these gene promoters. Our findings suggest that NSD2-mediated H3K36me2 recruits FOLR1 to promote PAH, and FOLR1 acts as a transcriptional factor to upregulate glycolytic gene expression in PAECs.

BACKGROUND AND AIM:Dilated cardiomyopathy is a major cause of heart failure, and hypertrophic cardiomyopathy is a common cause of sudden cardiac death in young adults. Epidemiological studies reporting the association between these cardiomyopathies and common cardiovascular risk factors, including smoking, alcohol, and obesity, are limited, and the published studies are mostly observational, making them vulnerable to bias. METHODS AND RESULTS:We performed a two-sample Mendelian randomization analysis to assess whether cardiovascular risk factors were causally associated with dilated and hypertrophic cardiomyopathies. Independent genetic variants associated with body mass index, smoking, and alcohol were selected as instrumental variables, with two sets of instrumental variables utilized for alcohol. Dilated cardiomyopathy data on 355,318 samples and hypertrophic cardiomyopathy data on 489,727 samples were obtained from a European population-based genome-wide association study (GWAS) meta-analysis. The large GWAS data sample size improved the statistical power. Our results showed significant associations between a genetic predisposition for smoking and the risk of dilated cardiomyopathy (odds ratio (OR) = 1.33; 95 % confidence level (CI): 1.07-1.67; p = 0.012) and between a genetic predisposition for obesity and the risk of dilated cardiomyopathy (OR = 1.62; 95 % CI, 1.30-2.02; p = 1.51 × 10-5). The results of the other associations were not significant. CONCLUSIONS:This study suggests that smoking and obesity are causally associated with an increased risk of dilated cardiomyopathy.

Despite improvements in interventional techniques leading to faster myocardial reperfusion postmyocardial infarction, there has been a significant rise in the occurrence of myocardial ischaemia/reperfusion injury (MI/RI). A deeper understanding of the underlying mechanisms of MI/RI could offer a crucial approach to reducing myocardial damage and enhancing patient outcomes. This study examined the myocardial protective properties of puerarin (PUE) in the context of MI/RI using hypoxia/reoxygenation (H/R) or ischaemia/reperfusion (I/R) injury models were employed in H9c2 cells and C57BL/6 mice. Our findings demonstrate that pretreatment with PUE effectively mitigated cardiomyocyte ferroptosis, restored redox balance, preserved mitochondrial energy production and maintained mitochondrial function following MI/RI. Furthermore, these cardioprotective effects of PUE were found to be mediated by the downregulation of voltage-dependent anion channel 1 (VDAC1) protein. These data reveal a novel mechanism by which PUE inhibits MI/RI and reveal that this protective effect of PUE is dependent on the downregulation of VDAC1.

The present study aimed to explore how resveratrol (Res) confers myocardial protection by attenuating ferroptosis. In vivo and in vitro myocardial ischemia/reperfusion injury (MIRI) models were established, with or without Res pretreatment. The results showed that Res pretreatment effectively attenuated MIRI, as evidenced by increased cell viability, reduced lactate dehydrogenase activity, decreased infarct size, and maintained cardiac function. Moreover, Res pretreatment inhibited MIRI-induced ferroptosis, as shown by improved mitochondrial integrity, increased glutathione level, decreased prostaglandin-endoperoxide synthase 2 level, inhibited iron overload, and abnormal lipid peroxidation. Of note, Res pretreatment decreased or increased voltage-dependent anion channel 1/glutathione peroxidase 4 (VDAC1/GPX4) expression, which was increased or decreased via anoxia/reoxygenation (A/R) treatment, respectively. However, the overexpression of VDAC1 via pAd/VDAC1 and knockdown of GPX4 through Si-GPX4 reversed the protective effect of Res in A/R-induced H9c2 cells, whereas the inhibition of GPX4 with RSL3 abolished the protective effect of Res on mice treated with ischemia/reperfusion.Interestingly, knockdown of VDAC1 by Si-VDAC1 promoted the protective effect of Res on A/R-induced H9c2 cells and the regulation of GPX4. Finally, the direct interaction between VDAC1 and GPX4 was determined using co-immunoprecipitation. In conclusion, Res pretreatment could protect the myocardium against MIRI-induced ferroptosis via the VDAC1/GPX4 signaling pathway.

Several studies have shown that berberine (BBR) is effective in protecting against myocardial ischemia‑reperfusion injury (MI/RI). However, the precise molecular mechanism remains elusive. The present study observed the mechanism and the safeguarding effect of BBR against hypoxia/reoxygenation (H/R) myocardial injury in H9c2 cells. BBR pretreatment significantly improved the decrease of cell viability, P62 protein, Rho Family GTPase 3 (RhoE) protein, ubiquinone subunit B8 protein, ubiquinol‑cytochrome c reductase core protein U, the Bcl‑2‑associated X protein/B‑cell lymphoma 2 ratio, glutathione (GSH) and the GSH/glutathione disulphide (GSSG) ratio induced by H/R, while reducing the increase in lactate dehydrogenase, microtubule‑associated protein 1 light 3 protein, caspase‑3 activity, reactive oxygen species, GSSG and malonaldehyde caused by H/R. Transmission electron microscopy and LysoTracker Red DND‑99 staining results showed that BBR pretreatment inhibited H/R‑induced excessive autophagy by mediating RhoE. BBR also inhibited mitochondrial permeability transition, maintained the stability of the mitochondrial membrane potential, reduced the apoptotic rate, and increased the level of caspase‑3. However, the protective effects of BBR were attenuated by pAD/RhoE‑small hairpin RNA, rapamycin (an autophagy activator) and compound C (an AMP‑activated protein kinase inhibitor). These new findings suggested that BBR protects the myocardium from MI/RI by inhibiting excessive autophagy, maintaining mitochondrial function, improving the energy supply and redox homeostasis, and attenuating apoptosis through the RhoE/AMP‑activated protein kinase pathway.

The present study aimed to explore how resveratrol (Res) confers myocardial protection by attenuating ferroptosis. In vivo and in vitro myocardial ischemia/reperfusion injury (MIRI) models were established, with or without Res pretreatment. The results showed that Res pretreatment effectively attenuated MIRI, as evidenced by increased cell viability, reduced lactate dehydrogenase activity, decreased infarct size, and maintained cardiac function. Moreover, Res pretreatment inhibited MIRI-induced ferroptosis, as shown by improved mitochondrial integrity, increased glutathione level, decreased prostaglandin-endoperoxide synthase 2 level, inhibited iron overload, and abnormal lipid peroxidation. Of note, Res pretreatment decreased or increased voltage-dependent anion channel 1/glutathione peroxidase 4 (VDAC1/GPX4) expression, which was increased or decreased via anoxia/reoxygenation (A/R) treatment, respectively. However, the overexpression of VDAC1 via pAd/VDAC1 and knockdown of GPX4 through Si-GPX4 reversed the protective effect of Res in A/R-induced H9c2 cells, whereas the inhibition of GPX4 with RSL3 abolished the protective effect of Res on mice treated with ischemia/reperfusion.Interestingly, knockdown of VDAC1 by Si-VDAC1 promoted the protective effect of Res on A/R-induced H9c2 cells and the regulation of GPX4. Finally, the direct interaction between VDAC1 and GPX4 was determined using co-immunoprecipitation. In conclusion, Res pretreatment could protect the myocardium against MIRI-induced ferroptosis via the VDAC1/GPX4 signaling pathway.

Introduction:Excessive generation of reactive oxygen species (ROS) following myocardial ischemia-reperfusion (I/R) can result in additional death of myocardial cells. The rapid clearance of ROS after reperfusion injury and intervention during subsequent cardiac repair stages are crucial for the ultimate recovery of cardiac function.Methods:Magnesium-doped mesoporous bioactive glasses were prepared and loaded with the antioxidant drug gallic acid into MgNPs by sol-gel method. The antioxidant effects of MgNPs/GA were tested for their pro-angiogenic and anti-inflammatory effects based on the release characteristics of GA and Mg2+ from MgNPs/GA. Later, we confirmed in our in vivo tests through immunofluorescence staining of tissue sections at various time points that MgNPs/GA exhibited initial antioxidant effects and had both pro-angiogenic and anti-inflammatory effects during the cardiac repair phase. Finally, we evaluated the cardiac function in mice treated with MgNPs/GA.Results:We provide evidence that GA released by MgNPs/GA can effectively eliminate ROS in the early stage, decreasing myocardial cell apoptosis. During the subsequent cardiac repair phase, the gradual release of Mg2+ from MgNPs/GA stimulated angiogenesis and promoted M2 macrophage polarization, thereby reducing the release of inflammatory factors.Conclusion:MgNPs/GA acting on multiple cell types is an integrated solution for comprehensive attenuation of myocardial ischaemia-reperfusion injury and cardiac function protection.

Background: Pathological cardiac hypertrophy poses a significant threat to human health by leading to ventricular remodeling. CircRNAs play a potential role in the dysregulation of cardiac hypertrophy and recent evidence highlights their translational ability in various diseases. However, it remains unclear if circRNAs have a protein-coding role in myocardial hypertrophy and ventricular remodeling. This study aims to investigate the role of translatable circRNA in the pathogenesis of myocardial hypertrophy. Methods: The Transverse Aortic Constriction (TAC) induced hypertrophy mouse model was constructed. The heart function was evaluated by C57BL/6 mice. The myocardial structure injury and fibrosis were analyzed by HE staining and Masson staining. CircRNAs Microarray assay was used to screen the dysregulated circRNAs. The recombinant adenovirus-associated virus was constructed to over-expression or knockdown FTO or circFTO. Mass spectrometry analysis, Dual-luciferase reporter assay, and Polysome profiling analysis were performed to detect the circFTO encoded protein circFTO-36aa. Results: The study screened for dysregulated circRNAs in sham and transverse aortic constriction (TAC) and found that an up-regulated circular RNA, circFTO is generated from the back-splicing of FTO exon 5 and exon 7. Silencing circFTO by AAV significantly weakened the TAC-induced hypertrophy phenotype. Moreover, the study identified a novel protein, FTO-36aa, coded by circFTO that caused the pro-hypertrophy effect of circFTO. FTO-36aa promoted the ubiquitination-mediated protein degradation of FTO, which suppressed the demethylation of RNA, elevating the global m6A methylation. It was verified that the m6A reader, IGF2BP2, recognized the circFTO/FTO-36aa elevated m6A methylation and increased mRNA stabilities of m6A methylated hypertrophic genes. Conclusion: Overall, this study sheds light on the functional importance of alternative splicing-generated circFTO and its coded FTO-36aa during myocardial hypertrophy. The findings provide fundamental insights into the mechanisms of m6A methylation regulation in hypertrophic cardiomyocytes.

The present study aimed to elucidate the role of autophagy-related genes (ARGs) in calcific aortic valve disease (CAVD) and their potential interactions with immune infiltration via experimental verification and bioinformatics analysis. A total of three microarray datasets (GSE12644, GSE51472 and GSE77287) were obtained from the Gene Expression Omnibus database, and gene set enrichment analysis was performed to identify the relationship between autophagy and CAVD. After differentially expressed genes and differentially expressed ARGs (DEARGs) were identified using CAVD samples and normal aortic valve samples, a functional analysis was performed, including Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses, protein-protein interaction network construction, hub gene identification and validation, immune infiltration and drug prediction. The results of the present study indicated a significant relationship between autophagy and CAVD. A total of 46 DEARGs were identified. GO and pathway enrichment analyses revealed the complex roles of DEARGs in regulating CAVD, including multiple gene functions and pathways. A total of 10 hub genes were identified, with three (SPP1, CXCL12 and CXCR4) consistently upregulated in CAVD samples compared with normal aortic valve samples in multiple datasets and experimental validation. Immune infiltration analyses demonstrated significant differences in immune cell proportions between CAVD samples and normal aortic valve samples, thus showing the crucial role of immune infiltration in CAVD development. Furthermore, therapeutic drugs were predicted that could target the identified hub genes, including bisphenol A, resveratrol, progesterone and estradiol. In summary, the present study illuminated the crucial role of autophagy in CAVD development and identified key ARGs as potential therapeutic targets. In addition, the observed immune cell infiltration and predicted autophagy-related drugs suggest promising avenues for future research and novel CAVD treatments.

This study aimed to explore the effects of peroxisome proliferator-activated receptor α (PPAR-α), a known inhibitor of ferroptosis, in Myocardial ischemia/reperfusion injury (MIRI) and its related mechanisms. In vivo and in vitro MIRI models were established. Our results showed that activation of PPAR-α decreased the size of the myocardial infarct, maintained cardiac function, and decreased the serum contents of creatine kinase-MB (CK-MB), lactate dehydrogenase (LDH), and Fe2+ in ischemia/reperfusion (I/R)-treated mice. Additionally, the results of H&E staining, DHE staining, TUNEL staining, and transmission electron microscopy demonstrated that activation of PPAR-α inhibited MIRI-induced heart tissue and mitochondrial damage. It was also found that activation of PPAR-α attenuated MIRI-induced ferroptosis as shown by a reduction in malondialdehyde, total iron, and reactive oxygen species (ROS). In vitro experiments showed that intracellular contents of malondialdehyde, total iron, LDH, reactive oxygen species (ROS), lipid ROS, oxidized glutathione disulphide (GSSG), and Fe2+ were reduced by the activation of PPAR-α in H9c2 cells treated with anoxia/reoxygenation (A/R), while the cell viability and GSH were increased after PPAR-α activation. Additionally, changes in protein levels of the ferroptosis marker further confirmed the beneficial effects of PPAR-α activation on MIRI-induced ferroptosis. Moreover, the results of immunofluorescence and dual-luciferase reporter assay revealed that PPAR-α achieved its activity via binding to the 14-3-3η promoter, promoting its expression level. Moreover, the cardioprotective effects of PPAR-α could be canceled by pAd/14-3-3η-shRNA or Compound C11 (14-3-3η inhibitor). In conclusion, our results indicated that ferroptosis plays a key role in aggravating MIRI, and PPAR-α/14-3-3η pathway-mediated ferroptosis and mitochondrial injury might be an effective therapeutic target against MIRI.

The early restoration of hemodynamics/reperfusion in acute myocardial infarction (AMI) is an effective therapeutic strategy to reduce sudden death and improve patient prognosis. However, reperfusion induces additional cardiomyocyte damage and cardiac tissue dysfunction. In this context, turmeric‑derived curcumin (Cur) has been shown to exhibit a protective effect against myocardial ischemia/reperfusion injury (I/RI). The molecular mechanism of its activity, however, remains unclear. The current study investigated the protective effect of Cur and its molecular mechanism via in vitro experiments. The Cell Counting Kit‑8 and lactate dehydrogenase (LDH) assay kit were used to assess the cell viability and cytotoxicity. The contents of malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase, glutathione (GSH)/glutathione disulfide (GSSG), total iron, ferrous iron, caspase‑3 and reactive oxygen species (ROS) were measured using an appropriate kit. Western blotting was used to detect the expression of relevant proteins. The levels of apoptosis, mitochondrial permeability transition pore (MPTP) opening, and mitochondrial membrane potential (MMP) were detected by flow cytometry. The study findings indicated that anoxia/reoxygenation (A/R) injury significantly decreased cell viability, increased in LDH and caspase‑3 activities, induced ferroptosis, increased apoptosis and overactivated autophagy. However, pretreatment with Cur or ferrostatin‑1 (Fer‑1, a ferroptosis inhibitor) significantly increased A/R‑reduced cell viability, SOD, glutathione peroxidase activity, GSH/GSSH ratio and HES1 and glutathione peroxidase 4 protein expression; attenuated A/R‑induced LDH, MDA, total iron, ferrous iron, prostaglandin‑endoperoxide synthase 2 protein expression and prevented ROS overproduction and MMP loss. In addition, Cur inhibited caspase‑3 activity, upregulated the Bcl‑2/Bax ratio, reduced apoptotic cell number and inhibited MPTP over‑opening. Furthermore, Cur increased P62, LC3II/I, NDUFB8 and UQCRC2 expression and upregulated the p‑AMPK/AMPK ratio. However, erastin (a ferroptosis activator), pAD/HES1‑short hairpin RNA, rapamycin (an autophagy activator) and Compound C (an AMPK inhibitor) blocked the protective effect of Cur. In conclusion, Cur pretreatment inhibited ferroptosis, autophagy overactivation and oxidative stress; improved mitochondrial dysfunction; maintained energy homeostasis; attenuated apoptosis; and ultimately protected the myocardium from A/R injury via increased HES1 expression.