| BackgroundMyocardial I/R injury is a complicated process that could damage proteins,RNA,DNA and the plasma membrane,leading to cell death and reduced cardiac output.Since mitochondria comprise at least 30% of the cardiomyocyte volume and the high energy demand of the heart,mitochondrial dysfunction has emerged as a critical factor in myocardial I/R injury.Damaged mitochondria could produce excess reactive oxygen species(ROS)and potentially activate cell death signaling.Therefore,eliminating dysfunctional mitochondria and producing healthy mitochondria are imperative for cardiomyocyte survival.Mitochondrial autophagy(termed mitophagy)is a selective form of autophagy by which damaged mitochondria are eliminated by autolysosomes,and PTEN-induced kinase 1(PINK1)and Parkin are the key proteins of mitophagy.Previous studies had demonstrated that impaired mitophagy played an important role in a number of neurodegenerative diseases.However,the role of mitophagy during myocardial I/R injury or hypoxia/reoxygenation(H/R)injury is currently unclear.Mitochondrial biogenesis is a process that produces plenty of new mitochondria.Impaired myocardial mitochondrial biogenesis can disrupt oxidative stress resistance,energy production,and diastolic function.Previous studies had revealed that impaired mitochondrial biogenesis influenced the course of heart failure and myocardial hypertrophy.Nevertheless,the role of mitochondrial biogenesis in either myocardial I/R injury or H/R injury is not yet clear.Nicotinamide riboside(NR),a newly discovered precursor of NAD+,is a functional vitamin B3 analog and has advantageous effects on metabolic regulation.Accumulating evidences have demonstrated that NR could enhance insulin sensitivity,mitochondrial biogenesis and sirtuin functions.Sirt3,a member of the sirtuin family,locates in the mitochondria and is a highly conserved NAD+-dependent deacetylase.It could deacetylate lots of proteins in mitochondria to regulate the energy and metabolic pathways.However,it is unknown whether NR could alleviate myocardial I/R injury through regulating mitophagy and mitochondrial biogenesis via Sirt3.AimThis study,from phenomenon to mec hanism,is going to make sure the role of NR promoting mitophagy and mitochondrial biogenesis in myocardial ischemia/reperfusion in vivo and in vitro and try to clarify the underlying mechanism.We hope to perfect the theoretical basis of occurrence and development of myocardial ischemia/reperfusion and supply new approaches to cure the patients suffered with myocardial ischemia/reperfusion injury.Here are the detailed aims:1.In vivo: We are going to explore whether the treatment of NR to myocardial I/R inj ury is relevant to mitophagy and mitochondrial biogenesis and try to use the pharmacological intervention and genetic technology to clarify the underlying mechanism.2.In vitro: Based on the study in vivo,we are going to use the medicine and genetic technology to intervene adult mouse cardiomyocytes to further make sure the molecular pathway of N R reducing H/R injury through mitophagy and mitochondrial biogenesis.Methods Mice were fed homemade pellets conta ining either NR(400 mg/kg/day)or N utlin-3(400 mg/kg/day,an activator of p53)for three weeks.Mice were given si RN A against Sirt3 or PGC-1? via intramyocardial injections three days before the cardiac I/R injury model.Cardiac function was measured by echocardiography,myocardial apoptosis index was determined by TUNEL,myocardial infarct size was identified by Evans Blue and TTC,autophagosomes were observed by transmission electron microscopy,and Sirt3,p53,PGC-1?,Atg5,Atg7,Beclin1,p62,LC3,Nrf1,Tfam,mito Parkin,cyto Parkin,mito PINK1,cyto PINK1,Ac-p53 and Ac-PGC-1? were assessed by western blot.Adult mouse cardiomyocytes were isolated by Langendorff and cultured for 72 h.During that time,NR(50 ? mol/L),N utlin-3(10 ? mol/L),si RNASirt3 and si RNA PGC-1? would be added to intervene cardiomyocytes.Then myocardial apoptosis index was determined by TUN EL,mitochondrial membrane potential was assessed by JC-1,autophagosomes were observed by transmission electron microscopy,and Sirt3,p53,PGC-1?,Atg5,Atg7,Beclin1,p62,LC3,Nrf1,Tfam,mito Parkin,cyto Parkin,mito PINK1,cyto PINK1,Ac-p53 and AcPGC-1? were assessed by western blot.Results 1.Compared with sham group,I/R injury reduced the cardiac function and increase the infarct size and cardiomyocytes apoptosis index.However,adding NR improved the cardiac function,reduced the infarct size and cardiomyocytes apoptosis index,and increased the level of autophagy markers,mitophagy markers and mitochondrial biogenesis markers.2.Compared with N R+I/R group,interfering Sirt3 inhibited the role o f NR.It reduced the cardiac function,increased the infarct size and cardiomyocytes apoptosis index,and reduced the level of autophagy markers,mitophagy markers and mitochondrial biogenesis markers.3.Compared with NR+I/R group,the protective role of N R and the expression of mitochondrial biogenesis markers were reduced but the levels of Sirt3,autophagy markers and mitophagy markers had no change after intervening PGC-1?,which meant that Sirt3 was the upstream of PGC-1? and NR could regulate Sirt3 to reduce I/R injury through promoting mitochondrial biogenesis.Besides,the levels of autophagy markers and mitophagy markers were increased while the expressions of Sirt3 and mitochondrial biogenesis markers had no change after activating p53,which meant that Sirt3 was the upstream of p53 and N R could regulate Sirt3 to reduce I/R injury through promoting mitophagy.4.Compared with control group,H/R injury increased the cardiomyocytes apoptosis index and reduced the mitochondrial membrane potential.Compared with H/R group,adding NR reduced the cardiomyocytes apoptosis index,improved the mitochondrial membrane potential,and increased the expression of autophagy markers,mitophagy markers and mitochondrial biogenesis markers.5.Compared with NR+H/R group,interfering Sirt3 inhibited the role of NR.It increased the cardiomyocytes apoptosis index in adult mouse,and reduced the mitochondrial membrane potential as well as the expression of autophagy markers,mitophagy markers and mitochondrial biogenesis markers.6.Compared with NR+H/R group,the protective role of N R and the expression of mitochondrial biogenesis markers were reduced but the levels of Sirt3,autophagy markers and mitophagy markers had no change after intervening PGC-1?,which meant that NR could regulate Sirt3 to reduce H/R injury through promoting mitochondrial biogenesis.Besides,the levels of autophagy markers and mitophagy markers were increased while the expressions of Sirt3 and mitochondrial biogenesis markers had no change after activating p53,which meant that NR could regulate Sirt3 to reduce H/R injury through promoting mitophagy.Conclusions1.We verified in vivo that Sirt3 was the key protein in the protective role of NR in myocardial I/R injury.Furthermore,NR could alleviate I/R injury through NR-Sirt3-p53 promoting mitophagy and NR-Sirt3-PGC-1? promoting mitochondrial biogenesis.2.The results in vitro were the same as those in vivo revealing that NR could promote Sirt3 to protect cardiomyocytes from H/R injury.And Sirt3-p53-mitophagy and Sirt3-PGC-1?-mitochondrial biogenesis were the underlying mechanisms of NR. |
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