Mechanism Of UPR^mt Signaling Response Pathway Regulated By ATF6 To Reduce Myocardial Ischemia-reperfusion Injury By Improving Mitochondrial Structure And Function

ObjectiveWith the development of interventional therapy,the death rate of myocardial infarction is greatly decreased,but the death rate and the incidence of heart failure are on the rise.Therefore,myocardial ischemia reperfusion(I/R)injury should be paid attention to.Mitochondria play an important role in cardiomyocyte physiology because of their hyperdynamic characteristics.Mitochondria are selectively susceptible to myocardial I/R damage through mechanisms including oxidative stress,calcium overload,inflammation,nutrient substrate deficiency,and activation of cell death programs.In previous studies,mitochondrial quality control can protect IR damaged myocardial tissue by maintaining mitochondrial function and morphology.In addition,when myocardial I/R injury occurs,there are a large number of misfolded or unfolded proteins and peptides in mitochondria,which damage the function of myocardial cells.The buildup of too many misfolded proteins can also cause damage to heart muscle cells.Mitochondrial unfolded protein response(UPRmt)is an important part of mitochondrial quality control.Uprmt is caused by accumulation of abnormal mitochondrial proteins or interference with transcription/expression of proteins associated with mitochondrial DNA damage.Uprmt is a mitochondrial stress response that facilitates correct folding or efficient clearance of unfolded and misfolded proteins in mitochondria.Active transcription factor 6(ATF6),as one of the initiators of the endoplasmic reticulum unfolded protein response,has been identified to protect myocardial I/R injury and inhibit cellular oxidative stress response.But whether the mechanism of action is related to UPRmt remains to be investigated.In this study,we focused on whether an ATF6-regulated UPRmt signaling response pathway can improve myocardial I/R damage by improving mitochondrial structure and function,and to explore the mechanism.MethodsThis study is divided into two parts.Part Ⅰ: In vivo experiments using laboratory animals.The myocardial ischemia-reperfusion injury model was established by ligation of the anterior descending branch coronary artery(LAD)in healthy C57 BL mice for 8 weeks,which was divided into four groups: Sham operation group,Sham+(oligomycin)Omy group,I/R injury group and I/R+Omy group.UPRmt markers(mtDNAj,Clp P,Lon P1,Hsp10)were detected by qPCR.After the treatment of mice,the myocardial infarction size and cardiac function indexes(LVEF,LVSd,FS,LVDd,E/A,E/e’)were visually measured by ultrasonography,myocardial markers and heart staining.Oxidative stress,inflammation and mitochondria-related apoptotic proteins in myocardial tissue of I/R mice were verified by ELISA,Western Blot and fluorescence quantification.Mitochondrial damage in injured myocardial tissue cells was observed under fluorescence microscope,and apoptosis was observed by TUNEL method.To investigate the mechanism by which UPRmt activation improves I/R impairment.Part Ⅱ: To explore whether ATF6 could regulate UPRmt signaling pathway to reduce myocardial I/R damage by Hypoxia/Reoxygenation(H/R)injury model via H9C2 cardiomyocytes at the cellular level.The anoxic conditions of H/R cell culture were 95%N2+5%O2,p H 6.8.The experimental cells could be divided into control group,cont+Omy group,H/R group,H/R+Omy group,H/R+Omy+si-ATF6 groups.Firstly,the cell activity of each group was detected by MTT assay to determine whether the inflammatory H/R cell model was established successfully.qPCR was used to detect whether H/R treatment could up-regulate ATF6 transcription.UPRmt markers(mt DNAj,Clp P,Lon P1,Hsp10)were tested by qPCR to determine whether H/R damaged cardiomyocytes could activate UPRmt.The oxidative stress of the myocardial tissue of ATF6/UPRmt in I/R mice was verified by ELISA and fluorescence quantification.Mitochondrial damage and cell apoptosis in the injured myocardial cells were observed by fluorescence staining.ResultsIn vivo,the results showed that: 1.I/R injury could activate UPRmt in myocardium;2.2.UPRmt activation reduced I/R infarct size and markers of myocardial injury;3.Activation of UPRmt improved cardiac function in I/R mice;4.Activation of UPRmt inhibited myocardial inflammation and oxidative stress in I/R mice;5.Activation of UPRmt reduced myocardial mitochondrial damage and division in I/R mice.6.Activation of UPRmt reduced mitochondria-associated cardiomyocyte apoptosis in I/R mice.The second part of cell experiment showed that: 1.H/R treatment down-regulated the transcription of ATF6 in cardiomyocytes;2.2.si ATF6 inhibited the activation of UPRmt in H/R treated cardiomyocytes.3.H/R treated cardiomyocytes increased oxidative stress after si ATF6 transfection;4.Mitochondrial damage and division were aggravated in H/R treated cardiomyocytes after si ATF6 transfection.5.The apoptosis of H/R treated cardiomyocytes was aggravated after si ATF6 transfection.Conclusions1.UPRmt in myocardium could be activated by I/R injury,but endogenous UPRmt was not sufficient to resist myocardial I/R injury.2.Activation of UPRmt can reduce myocardial ischemia reperfusion injury by inhibiting inflammatory and oxidative stress responses in I/R damaged myocardium,thereby improving mitochondrial structure and function,and further improving myocardial apoptosis.3.ATF6 is an upstream regulator of UPRmt.4.Mechanism of UPRmt signaling response pathway regulated by ATF6 to reduce myocardial ischemia-reperfusion injury by improving mitochondrial structure and function.