Ischemia/reperfusion-induced Myocardial Injury In Mice With Diabetes Mellitus: Role Of Sirt1

Background and Aim: Ischemia/reperfusion(I/R)myocardial injury refers to reperfusion itself induces further myocardial injury.The reperfusion of myocardium in patients with diabetes mellitus(DM)leads to exaggerated I/R injury and results in worse clinical outcomes than those of patients without DM.However,the underlying mechanism(s)by which DM sensitizes the myocardium to I/R injury remains unclear.According to pathological studies,the major form of cardiomyocyte death in the peri-infarct region is thought to be apoptosis,especially during reperfusion,and mitochondria have been found to play a major role in apoptotic cell death.Mitochondrial morphological dynamics is known as mitochondria sustain fission and fusion cycle,which is important to maintain the healthy function of mitochondria and their host cells.Excessive mitochondrial fission promotes cell apoptosis.Mitochondrial fission is controlled by Dynamin-related protein 1(Drp1),and the Drp1 activity is regulated by phosphorylation of its serine residues.However,it remains unknown whether the excessive of activation of Drp1 causes the increased vulnerability of the myocardium to I/R induced injury in diabetes.Silent information regulator factor 2 related enzyme 1(Sirt1)is a member of the class III group of histone deacetylases,which can regulate the protein expression by deacetylatetranscription factors,and modulate protein function by post-translational modification pathway.Recently studies indicate that Sirt1 has beneficial actions on various cardiovascular pathologies.The objective of the present study is to explore the alterations of myocardial Sirt1 expression and activity in mice with DM,and their impact on the I/R-induced myocardial injury in mice with DM.Most importantly,current study aims to elucidate the intracellular signaling pathway involved in the myocardial protection of Sirt1 in diabetic mice challenged with I/R injury.Methods: 1)In vivo study: DM Mouse model was induced in 6 weeks old male C57BL/6mice with intraperitoneal injection of streptozotocin(STZ,50 mg/kg,daily for 3 consecutive days).Mouse model of myocardial I/R injury was induced by ligation of left anterior descending coronary artery and followed by releasing.To increase the Sirt1 activity,Sirt1 activator was intraperitoneal injected to the DM mice.Myocardial Sirt1 expression was assessed with Western blot,and Sirt1 direct fluorescent screening assay kit was used to access Sirt1 activity.Mitochondrial morphology was accessed by transmission electron microscope.Myocardial apoptosis was determined with TUNEL staining.Evans Blue and 2,3,5-triphenyltetrazoliumchloride(TTC)wereused to stain myocardial tissueto determine the infarct area.Echocardiography(preclinical ultrasound equipped with 40-MHz linear array transducer with nominal in-plane spatial resolution of 40 ?m)was used to evaluate the mice cardiac function.2)In vitro study: Cardiomyocytes(CM)were isolated from neonatal mouse hearts and cultured,and high concentration of glucose(HG,30 m M)was choosed to treat the CM in order to mimic the in vivo conditions of DM.CM were exposed to anoxia/reoxygenation(A/R,an in vitro countpart to I/R in vivo)process.Sirt1 activator or transfection with Sirt1 CRISPR activation plasmid were used to increase Sirt1 activity.Wortmannin was used to inhibit the phosphorylation of Protein kinase B(Akt).Akt si RNA was used to inhibit CM Akt expression.Mitochondria/Cytosol fractionation kit was used to isolate mitochondria.CM protein expression was assessed with Western blot.Sirt1 activity was accessed by Sirt1 direct fluorescent screening assay kit.CM apoptosis was assessed by determination of caspase 3 activity,fragmented DNA(Cell death ELISA)and TUNEL staining.To determine the mitochondrial reactive oxygen species,live myocytes staining with Mito SOX and imaged immediately on a fluorescent microscope was used.The mitochondrial reactive oxygen species production was quantified by measuring red fluorescence.To determine the morphology mitochondrial,live myocytes were infected with a modified baculovirus vector and imaged with laser-scanning microscope.Results: 1)Eight weeks after induction of DM,mouse myocardial levels of Sirt1 expression were decreased,and myocardial Sirt1 activity was reduced as compared to those of control mice.The decrease myocardial Sirt1 protein and activity in diabetic myocardial were associated with increase in I/R-induced myocardial injury.I/R challenge to hearts of diabetic mice resulted in increased myocardial apoptosis,myocardial infarct size,and decreased myocardial function as compared with those of I/R challenged hearts of mice without DM.Pre-treatment of diabetic hearts with Sirt1 activator attenuated the increased myocardial apoptosis,decreased myocardial infarct size,and improved myocardial function in mice with DM.2)Condition of CM with HG down-regulated Sirt1 protein expression.Sirt1 activity was decreased in CM with HG.A/R challenge to CM preconditioned with HG incurred increased CM apoptosis as compared with A/R challenged naive CM.Pretreatment of the HG preconditioned CM with Sirt1 activator or transfection of the CM with Sirt1 CRISPR activation plasmid prevented the exaggerated A/R-induced CM apoptosis.3)A/R challenge to CM preconditioned with HG incurred exaggerated mitochondrial fragmentation and oxidant stress.Mitochondrial fragmentation is controlled by dynamin related protein 1(Drp1).The Drp1 activity is modulated by inhibitory phosphorylation of Drp1 Ser637.A/R challenge to CM resulted in decrease in inhibitory Drp1 Ser637 phosphorylation and promoted Drp1 mitochondrial translocation,those effects were exaggerated when the CM were preconditioned with HG.Pretreatment of the HG-conditioned CM with Sirt1 activator attenuated the exaggeration of A/R-induced decrease in Drp1 Ser637 phosphorylation and increase in Drp1 mitochondrial translocation.The effects of Sirt1 activator were further confirmed by transfection of the CM with Sirt1 CRISPR activation plasmid.4)Cardiomyocytes preconditioned with HG shown decreased Sirt1 expression,this was associated with decreased Akt activation as indicated by decreased Akt Ser473 phosphorylation.Inhibition of Akt with Wortmannin decreased Drp1 Ser637 phosphorylation and promoted Drp1 activation.Up-regulation of CM Sirt1 by transfection of the cells with Sirt1 CRISPR activation plasmid increased Akt activation(Akt Ser473 phosphorylation)and decreased Drp1 activation(Drp1 Ser637 phosphorylation)and inhibited Drp1 mitochondria translocation in HG conditioned CM challenged with I/R.Conclusion: 1)DM decreases myocardial levels of Sirt1 expression and activity which is associated with exaggerated I/R injury in DM.2)Increasing mitochondrial fission contributes to exaggerated myocardial ischemia/reperfusion injury in DM.3)The resultant of decreased Sirt1 is down-regulation of Akt phosphorylation at Ser473 and subsequently decreased phosphorylation of Drp1 at Ser637 is enhanced activation of Drp1 mediated mitochondrial fission after I/R in the diabetic myocardium and subsequent exaggeration of myocardial injury.4)The Sirt1/Akt/Drp1 axis plays an essential role in increased vulnerability of diabetic myocardium to I/R injury.