The Mechanism Of FSP1 In Ferroptosis Induced By Myocardial Ischemia-Reperfusion Injury

Background:Acute myocardial infarction（AMI）is characterized by a high incidence,disability rate,and mortality rate.Percutaneous coronary intervention（PCI）has been increasingly performed in recent years;however,ischemia-reperfusion injury（IRI）during the operation worsens myocardial cell damage,leading to malignant arrhythmia and heart failure.Therefore,reducing IRI is an urgent clinical problem.Despite many studies on the mechanisms of IRI,therapeutic targeting of these pathological and physiological processes has been unsatisfactory.This indicates that the current research has not fully elucidated all the mechanisms of myocardial IRI,making IRI research a challenging and ongoing topic in the medical field.Recent studies have shown that ferroptosis is involved in myocardial IRI,but the specific mechanism is not yet clear.The classical ferroptosis signaling pathway is mediated by glutathione peroxidase 4（GPX4）.However,in 2019,Ferroptosis Suppressor Protein 1（FSP1）was discovered to significantly reduce cell ferroptosis when overexpressed in cells,making it the first enzymatic system found to compensate for the loss of GPX4.Based on the above research background,we propose the scientific hypothesis that during acute myocardial infarction,dangerous factors such as ischemia and hypoxia act on myocardial cells,inhibiting the expression of FSP1,an important regulatory factor in the ferroptosis signaling pathway,inducing ferroptosis of myocardial cells and promoting myocardial cell ischemia-reperfusion injury.Objective:This study aims to investigate the role of FSP1 in ferroptosis-induced myocardial ischemia-reperfusion injury.Methods:1.H9C2 myocardial cells were subjected to hypoxia/reoxygenation（H/R）to simulate in vivo IRI.Cell proliferation was detected using the CCK8 assay,while cell membrane integrity was measured using the LDH assay.2.H9C2 myocardial cells were divided into three groups:Control group,H/R medium group,and H/R medium+Ferrostatin-1（Fer-1）group（H/R cells with Fer-1,an iron-mediated cell death inhibitor）.Western blotting was used to detect the expression of classical iron-mediated cell death-related proteins in H9C2 myocardial cells,including GPX4,ACSL4,NOX1,and COX2.3.H9C2 myocardial cells were divided into three groups:Control group,H/R group,and H/R medium+Fer-1 group（H/R cells with Fer-1）.The levels of total iron and Fe²⁺in cells were measured using an iron assay kit,while mitochondrial Mito SOX levels and lipid ROS levels were observed using immunofluorescence and flow cytometry,respectively.4.FSP1 overexpression（OE）and knockdown（KD）plasmids were transferred into H9C2 myocardial cells to establish FSP1 overexpression or knockdown cell models.Western blotting was used to detect FSP1 protein expression in cells after hypoxia/reoxygenation and plasmid transfection.5.H9C2 myocardial cells were divided into four groups:NC group,H/R medium group,H/R medium+FSP1 OE group（H/R cells with FSP1 OE plasmids）,and H/R medium+FSP1 KD group（H/R cells with FSP1 KD plasmids）.Cell proliferation was measured using the CCK8 assay,while cell toxicity was detected using the LDH assay.The levels of total iron and Fe²⁺in cells were measured using an iron assay kit,while lipid ROS levels were measured using flow cytometry.Results:1.H9C2 cardiomyocytes exhibited decreased cell viability and increased cytotoxicity after hypoxia/reoxygenation（H/R）,confirming the successful construction of the H/R model.2.Classic ferroptosis-associated protein GPX-4 expression decreased,while ACSL4,NOX1,and COX2 expression increased in H9C2 cardiomyocytes after H/R.Fer-1 inhibited the changes in these iron-dependent death-associated proteins induced by H/R,indicating the occurrence of ferroptosis.3.H/R led to a significant increase in Fe²⁺,total iron,Mito SOX,and lipid ROS levels in H9C2 cardiomyocytes,which were significantly inhibited by Fer-1 treatment,further confirming the occurrence of ferroptosis.4.FSP1 expression decreased in H9C2 cardiomyocytes after H/R.Transfection with FSP1 OE plasmid significantly increased FSP1 protein expression compared to the hypoxia/reoxygenation group,while transfection with FSP1 KD plasmid decreased FSP1 protein expression,further validating the successful construction of the FSP1overexpression or knockdown cell model.5.Overexpression of FSP1 reduced H/R-induced cytotoxicity and increased cell viability,while knockdown of FSP1 increased H/R-induced cytotoxicity and decreased cell viability.6.Overexpression of FSP1 inhibited the levels of Fe²⁺,total iron,and lipid ROS induced by H/R,thereby inhibiting ferroptosis.In contrast,knockdown of FSP1promoted H/R-induced increases in Fe²⁺,total iron,and lipid ROS levels,thereby promoting ferroptosis.Conclusion:The research findings indicate that hypoxia/reoxygenation can promote ferroptosis by reducing FSP1 expression in H9C2 myocardial cells.