The Role And Mechanism Of Sirt1 Modulating Autophagy And Apoptosis In Cardioprotection Under Chronic Hypoxia

BackgroundCyanotic Congenital heart disease（CHD）belongs to the complex CHDS,and the majority of the complex CHDs are cyanotic.Cyanotic CHDs are caused by right-to-left shunt in the heart,and chronic hypoxia is their vital patho-physiological feature.Patients suffer from cyanotic CHD always have low SaO₂,but they can survive for a long time until receive surgery,suggesting that there may be some compensatory adaptation mechanism generated in cardiomyocytes to help itself cope with hypoxic stress.Therefore,it is of great importance to find the underlying mechanisms of hypoxia adaptation in cardiomyocytes,which may help to provide improved strategies for cardioprotection and improve the outcome of clinical therapies.Autophagy is a cellular degradation process that cytoplasmic proteins and organelles are sequestered into vesicles called autophagosomes,and then delivered for lysosomal degradation.Under stress free condition,basic autophagy is beneficial for the clearance of damaged proteins and organelles.Autophagy contributes to survival in harsh environment by degrading proteins,lipids,and organelles to provide energy and nutrients.AMPK is a highly conservative Ser/Thr kinase that senses energy status.AMPK was activated when the ATP content is low in cell to provide ATP by promoting metabolic catabolism and inhibiting metabolic synthesis.Moreover,AMPK participates in autophagy,it inhibits mTORC1 and activates ULK1 to promote autophagy.Although the exact mechanism of cyanotic CHD pathogenesis is still unclear,cell death caused by apoptosis has been shown to play a vital role in the development of cyanotic CHD.Apoptosis is a precise and complicate process.Recently,ER-stress dependent apoptosis has been demonstrated to participate in apoptosis of myocardiocyte in hypoxic rats.Endoplasmic reticulum is the central organelle for protein folding,calcium store,and lipid synthesis.Perturbations in ER function caused by hypoxia and other forms of cellular stress cause ER stress,then trigger the unfolded protein response（UPR）.The UPR is tightly controlled to relieve the cellular stress and re-gain protein homeostasis.There are three branches of UPR,including IRE1α（inositol-requiring protein-1α）,PERK（protein kinase RNA（PKR）-like ER kinase）,and ATF6（activating transcription factor 6）.Unrelieved ER stress often leads to apoptotic cell death via a yet unclear mechanism.IRE1αis the most conservative sensor of ER stress,it activates the Apoptotic-Signaling Kinase-1（ASK1）,which stimulates activation of JNK and p38 MAPK to promote apoptosis.It has been reported that cytokine functions on IRE1αand promotes apoptosis via JNK activation.The role of IRE1αsignaling in chronic hypoxic cardiomyocytes is unclear yet.Silent mating type information regulation 2 homolog 1（Sirt1）,a nicotinamide adenine dinucleotide（NAD+）dependent deacetylase,is widely expressed in eukaryotic cells.It has been reported that Sirt1 is involved in a lot of cellular processes,including autophagy,apoptosis,caloric restriction,cell differentiation,anti-aging and DNA damage repair.Of note,Sirt1 exerts beneficial effects in cardiovascular disease,such as ischemia/reperfusion injury,hypertrophy,and atherosclerosis.In addition,Sirt1 has been related to hypoxia by deacetylating HIF-1αand HIF-2α.These results suggest that Sirt1 may play a role in hypoxia adaptation of cardiomyocytes,but the mechanism remain elusive.Based on previous literature,we hypothesize that Sirt1 may participate in hypoxia adaptation of cardiomyocytes by regulating autophagy and apoptosis.ObjectivesThe study intends to exam the expression of autophagy related,apoptosis related proteins as well as Sirt1 in myocardium of clinical samples.A chronic hypoxia cell model was established.Silence and overexpression of Sirt1 is achieved by adenoviruses Ad-Sirt1 and Ad-Sh-Sirt1 respectively,and then autophagy and apoptosis were detected.The regulatory of Sirt1 on AMPK and IRE1αis explored in H9C2 cell line.A Chronic hypoxia mice model was established to study the role of pharmacological modulation of Sirt1 in autophagy and apoptosis in the myocardium of mice.Methods1.Collection of human myocardial samples:The myocardial tissues were obtained from the right ventricular outflow tract form patients with cyanotic CHD（diagnosed Tetralogy of Fallot）and acyanotic CHD（diagnosed ventricular septal defect combined with right ventricular outflow tract obstruction）.Expressions of LC3-II,p62,cleaved Caspase 3,Bcl2and Sirt1 was detected by Western blot.2.Research on the role of Sirt1 on autophagy in H9C2 cells subjected to chronic hypoxia.2.1 Establishment of chronic hypoxia model:After serum-starved overnight,H9C2 cells were cultured in hypoxic incubator containing a gaseous mixture of O₂ 1%,CO₂ 5%,N₂ 94%for 24 hours.The normoxia group was cultured in an environment with O₂ 21%.2.2 Adenoviruses Ad-Sirt1 and Ad-Sh-Sirt1 was used to overexpress or inhibit the expression of Sirt1,Ad-LacZ was used as a control.Autophagy related proteins were detected as well.2.3 Cells were divided into Normoxia+Ad-LacZ group,Hypoxia+Ad-LacZ group,Normoxia+Ad-Sh-Sirt1 group,Hypoxia+Ad-Sh-Sirt1 group.Western blot was used to detect the expressions of LC3-II and p62.2.4 Cells were divided into Normoxia+Ad-LacZ group,Hypoxia+Ad-LacZ group,Hypoxia+Ad-Sirt1 group,Hypoxia+Ad-Sh-Sirt1 group.Western blot was employed to detect the expressions of AMPK and p-AMPK.2.5 Cells were divided into Normoxia+Ad-LacZ group,Hypoxia+Ad-LacZ group,Hypoxia+Ad-Sirt1 group,Hypoxia+Ad-Sirt1+Compound C group.Expressions of LC3-II and p62 was detected by Western blot.3.Research on the role of Sirt1 on apoptosis in H9C2 cells subjected to chronic hypoxia.3.1 Cells were divided into Normoxia+Ad-LacZ group,Hypoxia+Ad-LacZ group,Hypoxia+Ad-Sirt1 group,Hypoxia+Ad-Sh-Sirt1 group.The expressions of cleaved Caspase 3 and Bcl2 were detected by Western blot.Apoptosis are detected by flow cytometry.Western blot was used to detect the expressions of IRE1αand p-IRE1α.3.2 Lentivirus Lv-Sh-IRE1αwas used to interfere IRE1α,and Ad-IRE1αwas used to overexpress IRE1α.Their efficiency was confirmed by Western blot.Cells were divided into Lv-Sh-Scramble+Normoxia group,Lv-Sh-Scramble+Hypoxia group,Lv-Sh-IRE1α+Normoxia group,Lv-Sh-IRE1α+Hypoxia group,Ad-LacZ+Normoxia group,Ad-LacZ+Hypoxia group,Ad-IRE1α+Normoxia group,Ad-IRE1α+Hypoxia group,8 groups in total.Expressions of cleaved Caspase 3,Bcl2,p-JNK,p-JNK,p-c-jun,c-jun,NF-κB p65 and NF-κB p-p65 was examed by Western blot.3.3 Cells are divided into Ad-LacZ+Hypoxia group,Ad-IRE1α+Hypoxia group,Ad-Sirt1+Hypoxia group,Ad-IRE1α+Ad-Sirt1+Hypoxia group.Expressions of cleaved Caspase 3,Bcl2,p-JNK,p-JNK,p-c-jun,c-jun,NF-κB p65,and NF-κB p-p65 was examed by Western blot.4.Effects of pharmacological activator and inhibitor of Sirt1 on autophagy and apoptosis in the myocardium of hypoxic mice.4.1 8-10 weeks old male C57BL/6J mice were divided into Normoxia+DMSO group,Hypoxia+DMSO group,Hypoxia+EX-527 group,Hypoxia+SRT1720 group（n=10）.The Ruskinn hypoxia workstation was used to establish a hypoxia mice model,the O₂ content is10%.The O₂ content is 21%in the normoxia group.The hypoxia time was set for 2 weeks.4.2 At the endpoint of hypoxia treatment,hemoglobin and hematocrit values are detected to value the effects of hypoxia,and the hearts of mice were harvest.The expression of HIF-1αwas detected by immunofluorescence technique.4.3 Western blot was used to detect the expressions of Sirt1,Acet-p53,and p53 in heart tissues.4.4 Western blot was used to detect the expressions of LC3-II,p62,p-AMPK and AMPK in each group.Immunofluorescence technique was used to exam LC3 expression.4.5 Western blot was used to detect the expressions of cleaved Caspase 3,Bcl2,p-IRE1αand IRE1αin each group.TUNEL was used to detect the apoptotic cell rate in each group.Results1.Western blot results suggest that expression of Sirt1 was significantly increased in patients with cyanotic CHDs than in acyanotic CHDS.In addition,LC3-II and cleaved caspase 3 were increased,while p62 and Bcl2 were decreased in cyanotic group.2.Compared with Ad-LacZ,Ad-Sirt1 significantly increased the expression of Sirt1 while Ad-Sh-Sirt1 decreased the expression of Sirt1.Ad-Sirt1 treatment result in an increase in LC3-II and a decrease in p62 expression.Ad-Sh-Sirt1 results in an opposite effect.Ad-Sh-Sirt1 treatment decreased LC3-II expression and increased p62 expression when compared with the hypoxia control group.The p-AMPK/AMPK ratio was increased in Ad-Sirt1+Hypoxia group and decreased in Ad-Sh-Sirt1+Hypoxia group when compared with Hypoxia group.Ad-Sirt1 treatment increased the expression of LC3-II and decreased the expression of p62 compared to the hypoxia group,but the pro-autophagy effect of Ad-Sirt1was abolished when Compound C was added.3.Flow cytometry was used to detect cellular apoptosis.Ad-Sirt1 treatment decreased the apoptotic cell rate,while Ad-Sh-Sirt1 increased the apoptotic cell rate,compared to the hypoxia control group.Western blot results showed that Ad-Sirt1 decreased the expression of cleaved Caspase 3and enhanced the expression of Bcl2.Ad-Sh-Sirt1 resulted an opposite trend.The p-IRE1α/IRE1αratio was decreased in Ad-Sirt1+Hypoxia treatment group,and increased in Ad-Sh-Sirt1+Hypoxia treatment group compared with the hypoxia control group.Ad-IRE1αtreatment increased cleaved Caspase 3 and decreased Bcl2 expression compared to the hypoxia control group,while Lv-Sh-IRE1αtreatment resulted in an opposite trend.Further study showed that the ratios of p-JNK/JNK,p-cjun/cjun,and p-p65/p65 NF-κB were upregulated in Ad-IRE1αtreatment group,but downregulated in Lv-Sh-IRE1αtreatment group.Moreover,the upregulated ratios of p-JNK/JNK,p-cjun/cjun,and p-p65/p65 NF-κB by Ad-Ad-IRE1αwere alleviated by Sirt1 overexpression.4.The blood hemoglobin levels and hematocrit values were significantly increased in the hypoxia group.The immunofluorescence intensity of HIF-1αwas significantly increased in the myocardium of hypoxic mice.Compared to the hypoxia control group,the expression of Sirt1 was decreased and the ratio of acetyl-p53/p53 was significantly upregulated in the inhibitor,EX-527,group.An opposite trend was observed in the Sirt1 activator group（SRT1720）.Compared to the hypoxia control group,the p-AMPK/AMPK ratio and LC3-II expression was decreased and p62 was increased significantly in the Sirt1 inhibitor treated group.However,the Sirt1 activator resulted in increased p-AMPK/AMPK ratio and LC3-II expression,and lowered p62 expression.Immunofluorescence results showed that SRT1720increased while EX-527 treatment decreased LC3 intensity.Western blot results showed that the p-IRE1α/IRE1αratio and cleaved Caspase 3 expression were increased,and Bcl2 was decreased in the EX-527 group,compared to the hypoxia control group.However,SRT1720led to an opposite trend.In addition,TUNEL staining showed that SRT1720 decreased while EX-527 treatment increased the apoptotic cell rates,compared to the hypoxia group.Conclusions1.The expression level of Sirt1 is significantly increased in cyanotic group.Autophagy and apoptosis are triggered in the myocardium of cyanotic patients.2.Up-regulation of Sirt1 could enhance autophagy in hypoxic H9C2 cardiomyocytes,and the pro-autophagy is medicated by AMPK.3.Up-regulation of Sirt1 could decrease cellular apoptosis induced by hypoxia in H9C2cells.IRE1αis a target protein of Sirt1.IRE1α/JNK/c-jun and NF-κB p65 signaling pathway could be obviously influenced when Sirt1 was activated in hypoxic cardiomyocytes.4.Pharmacological activation of Sirt1 by SRT1720 could enhance autophagy and alleviate apoptosis in myocardium of hypoxic mice.Inhibiting Sirt1 by Ex-527 resulted opposite trend.