Role And Mechanism Of PXDN In Regulating Cardiomyocyte Ferroptosis And Mediating Myocardial Injury After Myocardial Infarction

ObjectiveHeart failure（HF）is the leading cause of death worldwide.After myocardial infarction（MI）,ischemic and hypoxic stimulation leads to non-necrotizing death of cardiomyocytes at the infarct edge and distal end and further aggravates myocardial injury,which is a key factor leading to the development of fatal heart failure.Thus,elucidating the mechanism of non-necrotizing death of cardiomyocytes after myocardial infarction is of great significance for the prevention and treatment of heart failure after MI.Ferroptosis is a newly discovered form of iron-dependent programmed cell death characterized by oxidative stress and accumulation of lipid peroxidation.In recent years,studies have found that ferroptosis plays an important role in various cardiovascular diseases such as atherosclerosis,but its role and regulatory mechanism in myocardial injury after MI are still unclear.Previous studies have confirmed that peroxidasin（PXDN）activates oxidative stress and is involved in the occurrence and development of various cardiovascular diseases,but its role in myocardial injury and post-MI heart failure remains unknown.This study aims to elucidate the role and molecular mechanism of PXDN in regulating cardiomyocyte ferroptosis to mediate myocardial injury after MI,and to provide experimental basis and theoretical support for finding new intervention targets in the clinical treatment of post-MI heart failure.Methods1.Western blot and quantitative real time polymerase chain reaction（q RT-PCR）were used to detect PXDN expression in infarcted border zone myocardial tissue of ischemic cardiomyopathy patients and MI mice,and hypoxia-stimulated cardiomyocytes.2.MI model was established by ligation of left anterior descending in PXDN knockout and wild-type mice of the same genetic background and the survival of mice was observed.Western blot and q RT-PCR were used to detect protein and gene expression of myocardial tissue,and colorimetric assay was used to determine heme levels.Cardiac structure and function assessment:echocardiography was used to detect cardiac function,TTC staining to observe the size of infarction,masson staining to evaluate the degree of myocardial fibrosis,and ELISA to detect the concentration of plasma biomarkers of myocardial injury.Detection of ferroptosis-related indicators:prussian blue staining was used to observe myocardial iron deposition,spectrophotometry to detect myocardial MDA and ATP content,transmission electron microscopy to evaluate mitochondrial damage,C11 BODIPY fluorescent probe to detect myocardial lipid peroxidation levels,and dihydroethidium（DHE）fluorescent probe to detect reactive oxygen species（ROS）generation.3.Myocardial H9C2 cells were cultured in vitro and treated with hypoxia,ferric citrate and ferroptosis agonists（erastin and RSL3）,and PXDN was silenced by si RNA.Western blot,q RT-PCR,colorimetric assay,spectrophotometry,transmission electron microscopy,C11BODIPY and DHE fluorescent probes were used to detect heme,protein and gene levels,as well as changes of ferroptosis-related indicators.4.Myocardial H9C2 cells were treated with ferric citrate and ferroptosis agonists,and were infected with lentivirus to overexpress PXDN and sh RNA to silence p53.Various methods were used to detect changes in protein,gene levels and ferroptosis-related indicators.5.Myocardial H9C2 cells were treated with ferroptosis agonist,and PXDN was silenced by si RNA,and then the proteasome inhibitor MG132and autophagy inhibitor DC661 were added.The p53 protein level was detected by western blot.6.Myocardial H9C2 cells were treated with ferroptosis agonists,and PXDN was silenced by si RNA,followed by hypochlorous acid（HOCl）treatment.Multiple methods were used to detect the changes of heme,protein and gene levels,and ferroptosis-related indicators.7.Myocardial H9C2 cells were treated with ferroptosis agonists,and PXDN was silenced by si RNA,followed by the addition of nuclear export inhibitor leptomycin B（LMB）and proteasome inhibitor MG132.Various methods were used to detect the changes of heme,protein and gene levels,and ferroptosis-related indicators.The nuclear and cytoplasmic proteins were isolated and extracted,and the protein expression of p53 was evaluated by western blot.8.PXDN gene knockout and wild-type mice with the same genetic background were continuously given 5%ferric citrate（20 mg/day）in drinking water,intraperitoneal injection of erastin（20 mg/kg）and RSL3（10 mg/kg）,and the survival of mice was observed.Plasma and myocardial tissue were collected on day 14 after MI.Echocardiography,masson staining,and ELISA were used to evaluate cardiac structure and cardiac function.Various methods were used to detect the changes of heme,protein and gene levels,and ferroptosis-related indicators.Results1.The expression of PXDN in myocardial tissue of patients with ischemic cardiomyopathy was increased significantly;Protein levels of PXDN was gradually increased in myocardial tissue of mice after MI,and the highest expression was observed on the 14th day.The expression of PXDN in myocardial H9C2 cells was significantly increased after hypoxia stimulation,and was the highest at 1%O₂for 24 h.2.Myocardial systolic function of MI mice was significantly decreased,myocardial fibrosis was severe,and plasma levels of myocardial injury biomarkers were increased.After PXDN knockout,the cardiac systolic function of MI mice was improved,infarction area was reduced,fibrosis was alleviated,plasma levels of myocardial injury biomarkers were decreased,and the survival rate of mice was increased.3.After MI,mice myocardial tissue iron deposition was increased,protein expression of cell death signals was elevated,myocardial mitochondrial damage,lipid peroxidation and ROS production were increased,and p53 expression was elevated.After knockout of PXDN,myocardial tissue iron deposition was reduced,the ferroptosis marker levels were significantly decreased,myocardial mitochondrial damage,lipid peroxidation and ROS production were reduced,and p53 expression was decreased in MI mice.4.After hypoxia,exogenous iron and ferroptosis agonist treatment,myocardial H9C2 cells showed increased levels of ferroptosis markers,increased mitochondrial damage,increased lipid peroxidation and ROS production,and increased p53 expression.After silencing PXDN,the levels of ferroptosis markers,mitochondrial damage,lipid peroxidation and ROS production were reduced,and the expression of p53 was decreased in H9C2 cells.5.After PXDN overexpression,the levels of p53 and its downstream molecules,ALOX15 and SAT1,were significantly upregulated in iron citrate and ferroptosis agonist-stimulated myocardial H9C2 cells,with increased levels of ferroptosis markers,increased mitochondrial damage,and increased lipid peroxidation and ROS production.After further silencing of p53,the expression of p53 downstream molecules was downregulated,the levels of ferroptosis markers were decreased,mitochondrial damage was reduced,and cellular lipid peroxidation and ROS production were decreased.6.After stimulation with ferric citrate and ferroptosis agonists,nuclear p53 expression was significantly upregulated in myocardial H9C2cells and the level of p53 target genes was increased;after silencing PXDN,nuclear p53 protein expression was reduced while its m RNA level did not change significantly;after further addition of the proteasome inhibitor MG132,p53 level was significantly increased.7.Heme level was decreased in myocardial tissue of MI mice and increased after knockout of PXDN.In addition,heme level was reduced in myocardial H9C2 cells after stimulation with iron citrate and ferroptosis agonists and increased after silencing of PXDN.Further treatment with HOCl resulted in decreased cellular heme level,increased levels of p53 and its downstream molecules,free ubiquitin and p53 target genes,increased levels of cellular ferroptosis markers,increased mitochondrial damage and increased cellular lipid peroxidation and ROS production.8.After nuclear export inhibitor LMB and proteasome inhibitor MG132 treatment,p53 and its downstream molecules and free ubiquitin levels decreased by PXDN silencing were reversed,but there was no significant change in heme level.In addition,the levels of p53 target genes,ferroptosis markers,mitochondrial damage,lipid peroxidation and ROS production were increased.9.After administration of ferric citrate,erastin and RSL3 and MI modeling,the survival rate of mice was decreased,the cardiac systolic function was decreased,myocardial fibrosis and plasma levels of myocardial injury biomarkers increased.The levels of p53 and its downstream molecules,free ubiquitin and p53 target genes in mice myocardial tissue were increased,while the levels of heme decreased.In addition,the levels of ferroptosis markers,mitochondrial damage,lipid peroxidation and ROS production were increased.Ferric citrate,erastin and RSL3 treatment aggravated myocardial injury and ferroptosis in MI mice.After PXDN knockout,the survival rate of mice in ferric citrate,erastin,RSL3 and MI treatment groups was increased,the cardiac function was improved,the PXDN/HOCl/heme/p53 pathway was inhibited,and the degree of myocardial ferroptosis and myocardial injury was alleviated.Conclusion1.The expression of PXDN was upregulated in myocardial tissues of patients with heart failure after ischemic cardiomyopathy.2.Knockout of PXDN alleviates cardiomyocytes mitochondrial damage,reduces myocardial lipid peroxidation,reduces cardiomyocytes ferroptosis,improves cardiac function and survival rate of MI mice;3.PXDN regulates cardiomyocyte ferroptosis through the HOCl/heme/p53 pathway and promotes exogenous iron and different ferroptosis agonists（erastin and RSL3）induced myocardial and post-MI myocardial injury.