The SIRT1-SIRT3 Axis Protects Cardiomyocytes Against Myocardial Ischemia-reperfusion Injury By Regulating Mitophagy-related Ferroptosis Via The PINK1/Parkin Signaling Pathway

With the incidence rate of ischemic cardiomyopathy（ICM）increasing year by year,the problem of ischemia-reperfusion injury has aroused great concern of clinicians.Mitochondrial injury and subsequent ferroptosis are the main causes of myocardial ischemia-reperfusion injury（MIRI）.MIRI has become an important cause of poor prognosis in patients with ICM.As yet,the mechanism of MIRI remains unknown and there are no effective treatments.Therefore,it is important to elucidate its pathogenesis and develop potential therapeutic targets.Sirtuin1（SIRT1）and Sirtuin3（SIRT3）are two well-characterized members of the silent information regulator 2（SIR2）protein family.Both SIRT1 and SIRT3 have been proved to play important roles in protecting cardiomyocytes against MIRI,but almost all studies focused on the single gene level of either,and there were few reports on the collaborative relationship between the two.The purpose of this study was to explore the internal relationship between SIRT1 and SIRT3,as well as their relationship with mitophagy-related ferroptosis during MIRI.It was found that SIRT1 and SIRT3 were abnormally expressed during MIRI through bioinformatics analysis and cell experiments,and the expressions of SIRT1 and SIRT3 were inhibited by each other,and they formed a regulatory axis with internal relationship,which can regulate the balance of mitophagy in myocardial cells through PINK1/Parkin signaling pathway during MIRI,so as to regulate the process of cell ferroptosis and alleviate the injury of myocardial cells caused by ischemia-reperfusion.Therefore,this study has made some new discoveries and insights on the role of SIRT1 and SIRT3 in MIRI,which can provide potential therapeutic targets for the prevention and treatment of MIRI in patients with ICM.Part I:Expression and correlation analysis of SIRT1/SIRT3 in ICMBackgrounds:In the current studies,the expressions of SIRT1/SIRT3 in patients with ICM are unclear and there are many contradictions.In addition,it is not clear whether SIRT1 and SIRT3 have cooperation during MIRI.Objective:To explore the expression and correlation of SIRT1/SIRT3 in myocardial tissue and peripheral blood of patients with ICM at the gene level,and to validate the results of the analysis at the molecular level through cell experiments,by which to preliminarily analyze its role of SIRT1/SIRT3 in the occurrence and development of MIRI in patients with ICM.Methods:The"merge"function in R software was used to merge the myocardial tissue sample datasets GSE5406,GSE1869 and GSE974 from patients with ICM in the GEO database.The peripheral blood samples datasets GSE48060 and GSE97320from patients with ICM in the GEO database were also merged.After the"Combat"function being used to remove their batch effect,the SIRT1 and SIRT3 expression data matrices were extracted,and the"ggboxplot"in R software was used to visualize the expression difference results;"ggscatter"was used to visualize the expression correlation results.Then,we constructed a cell model of MIRI in vitro,by which the expressions of SIRT1/SIRT3 and their correlations were verified using small interference technology,so as to clarify the internal relationship between SIRT1 and SIRT3,and confirm the existence of SIRT1-SIRT3 axis.Results:The expression of SIRT1 in the myocardial tissue and peripheral blood of patients with ICM was lower than that in the control group（P<0.05）,while there was no significant difference in SIRT3 between the two groups.The expressions of SIRT1 and SIRT3 were negatively correlated in myocardial tissue and peripheral blood of patients with ICM（P<0.01）.The above results were verified by the H/R cell model of MIRI in vitro.It was found that the expression of SIRT1 was significantly lower in the H/R group than in the control group（P<0.05）,while the expression of SIRT3 remained unchanged in both groups.Interestingly,the expression of SIRT1was significantly increased in the sh SIRT3 group,and the expression of SIRT1 in the sh SIRT1+sh SIRT3 group was also significantly higher than that in the sh SIRT1 group（P<0.05）;More interestingly,the expression of SIRT3 in the sh SIRT1 group was also significantly increased,and the expression of SIRT3 in the sh SIRT1+sh SIRT3 group was also significantly higher than that in the sh SIRT3 group（P<0.05）.Conclusions:SIRT1 and SIRT3 were abnormally expressed in patients with ICM,and they formed a regulatory axis with intrinsic mutual inhibition,which participated in the occurrence and development of MIRI.Part II:Abnormal SIRT1-SIRT3 axis promotes the occurrence and development of MIRI through ferroptosisBackgrounds:The process of MIRI is accompanied by ferroptosis.Mitochondrial injury and subsequent ferroptosis are the main causes of MIRI.In the previous process of bioinformatics analysis,we also found that changes in the expressions of SIRT1-SIRT3 axis will cause changes in the expression of genes related to ferroptosis.Therefore,we speculated that SIRT1-SIRT3 axis had some relationships with ferroptosis,and they may be involved in the regulation of ferroptosis,which in turn contributes to the occurrence and development of MIRI.Objective:In this section,we intended to explore the relationship between SIRT1-SIRT3 axis and ferroptosis of cardiomyocytes during MIRI,as well as its specific effects on ferroptosis.Methods:The gene expression matrix related to ferroptosis from the public dataset GEO116250 was first extracted.After differentially expressed analysis,we extracted the core differentially expressed genes（DEGs）using LASSO algorithm.Finally,we analyzed the correlation between SIRT1/SIRT3 and these core ferroptosis-related DEGs to preliminarily evaluate the correlation between SIRT1/SIRT3 and ferroptosis.Subsequently,we further explored the specific effects of SIRT1/SIRT3 on ferroptosis in myocardial cells through cell model.The expressions of SIRT1 and SIRT3 protein in H9c2 cells were down-regulated by small interference technique.After hypoxia/reoxygenation（H/R）treatment,the change of Fe²⁺concentration was detected by Fe²⁺detection kit;MDA kit detects the change of MDA content;JC-1 membrane potential detection kit was used to detect the degree of mitochondrial damage.Results:We extracted 218 genes related to ferroptosis from GEO116250 dataset,and extracted 15 DEGs through differentially expressed analysis,including 8up-regulated genes and 7 down-regulated genes.Then,3 core DEGs related to ferroptosis were further extracted using LASSO algorithm.The result of correlation analysis showed that SIRT1/SIRT3 was closely related to these 3 core DEGs（P<0.05）.Subsequently,we used small interference technology to knock down the expressions of SIRT1 and SIRT3 proteins in H9c2 cell line.After H/R treatment,it was found that the concentration of Fe²⁺,the content of MDA and the proportion of mitochondrial damage in SIRT1 and SIRT3 interference groups were all increased（P<0.05）.Conclusions:SIRT1-SIRT3 axis was closely related to ferroptosis during MIRI.Silencing of SIRT1-SIRT3 axis will increase the incidence of ferroptosis in myocardial cells.So,the abnormality of SIRT1-SIRT3 axis promotes the occurrence and development of MIRI through ferroptosis.In addition,the phenotypes further confirmed that SIRT1 and SIRT3 formed a regulatory axis that is both mutually inhibitory and indispensable.Part III:SIRT1-SIRT3 axis modulates ferroptosis via the PINK1/Parkin signaling pathway in cardiomyocytesBackgrounds:In the previous process of bioinformatics analysis,we found that the changes of SIRT1-SIRT3 axis gene expressions would cause the changes of mitophagy-related gene expressions,and the disruption of mitophagy balance would lead to increased cell ferroptosis.Therefore,we inferred that SIRT1-SIRT3 axis regulated the incidence of myocardial cell ferroptosis by regulating the dynamic balance of mitophagy.Objective:In this section,we intended to explore the specific mechanism and signaling pathway by which SIRT1-SIRT3 axis regulated ferroptosis in myocardial cells during MIRI.Methods:First,the genes related to mitophagy were extracted from the public dataset GEO116250.After correlation analysis,the genes closely related to SIRT1/SIRT3 were identified,which were verified by cell model and in-depth researches were carried out on this basis.In the part of experimental verifications,we used Western blotting to detect the expression changes of mitophagy-related proteins to verify the results of bioinformatics analysis after down-regulating the expressions of SIRT1 and SIRT3 proteins in H9c2 cells by gene knockout technology.Finally,key molecules from these autophagy-related proteins were extracted,and the changes of cell phenotypes were observed after knocking out their expressions so as to verify the signaling pathway.Meanwhile,we observed whether the SIRT1 agonist resveratrol（Res）and/or SIRT3 agonist honokiol（HKL）could reverse the effect caused by these key molecule expression silencing.Results:We found that the changes of SIRT1-SIRT3 axis gene expressions were accompanied by the changes of PINK1,Parkin,LC3 and P62 expressions,which were all genes in the PINK1/Parkin signaling pathway.This result was verified by cell model experiment in vitro.After the expression of PINK1 being knocked down in H9c2 cells,who showed ferroptotic phenotypes consistent with down-regulation of SIRT1-SIRT3 axis,such as increased concentration of Fe²⁺,increased content of MDA,increased proportion of mitochondrial damage and increased apoptosis（P<0.05）,while Res and HKL could effectively reverse the above changes（P<0.05）.Conclusions:SIRT1-SIRT3 axis regulates the dynamic balance of mitophagy via the PINK1/Parkin signal pathway,so as to regulates the ferroptosis process in myocardial cells.The abnormality of SIRT1-SIRT3 axis promotes the occurrence and development of MIRI through mitophagy-dependent ferroptosis.