The Effect Of Deacetylase Sirt1 On Cardiac Protection Via Regulation Of Mitochondrial Fusion And Fission In Response To Hypoxia

BackgroundHypoxia is the pathophysiological basis of multiple cardiovascular diseases.With the increase of the extent and duration of hypoxia,metabolic disorders and dysfuction occurs in cell.As a double membrane-bound organelle,mitochondrion produces energy through oxidative phosphorylation and plays an important role in cellular metabolism.As the result of hypoxic injury,the cellular powerhouse,mitochondria experience a series of change,including mitochondrial morphology alteration.Studies have shown that the morphology of mitochondria is linked to cellular functions,which include calcium signaling,the generation of ROS,neuron plasticity,muscle atrophy and lymphocyte migration.On the one hand,mitochondrial fusion defection can cause mitochondrial dysfunction,may be caused by impaired mitochondrial matrix exchange capability.On the other hand,blocking mitochondrial fission may result in impairment of autophagy mediated damaged mitochondrial clearance,or influence mitochondrial function through uncovered mechanism.Mitochondrial fusion and fission are mediated by dynamin-related large GTPases.Mitochondrial fission are mediated by Drp1 and Fis1.Fis1 RNAi or Drp1 dominant negative mutation inhibits the mitochondrial fusion and fission cycle,resulting accumulation of damaged mitochondria.In many experimental models,researchers have found that inhibiting mitochondrial fission or enhancing fusion benifits tissue IR injury,metabolic disorders as well as neurodegenerative diseases.Previous studies have found that cardiac Sirt1 expression inhibition or inactivation increase apoptosis,and Sirt1 overexpression plays a protective role in rat heart ischemia-reperfusion injury.Nevertheless,the roles of Sirt1 in hypoxic heart are largely unknown.Sirt1 regulates mitochondrial biogenesis and mitophagy,which are closely related to mitochondrial fission and fusion.We speculate that Sirt1 may participate the regulation of mitochondrial fusion and fission in response to hypoxia,exerting a cardiac protection.ObjectivesTo explore the changes of mitochondrial fission and fusion under hypoxia and the potential role and mechanism of Sirt1 in this condition.Methods1.29 children diagnosed with congenital heart disease were enrolled from the Institute of Cardiovascular Surgery,Xinqiao Hospital in 2014,including 15 cyanotic cases and14 acyanotic cases.Western blot was used to assess the expression of Drp1 and Fis1 in myocardial tissues of right ventricular outflow tract.2.Rat H9c2 cells were cultured under hypoxic condition(94% N2,5% CO2,1% O2)for 0,2,4,8,12 h.Whole cell protein was harvested at the indicated time to detect the protein expression level of Drp1 and Fis1.H9c2 cells were transfected with mitochondria-targeted ds RED(mtRFP)to assess the mitochondrial morphology alteration under hypoxia condition.3.Under hypoxia condition,HA-K38A-DRP1 plasmid(a dominant negative mutant form of Drp1)and hFis1 plasmid were transfected into H9c2 cells(empty vector was used as a control),LDH test was employed to assess cytotoxcity.4.MtRFP was co-transfected with empty vector,sh-Sirt1 or Sirt1 plasmid into H9c2 cells.After 12 h normoxic or hypoxic culture,cells were assessed by confocal microscopy to observe the changes of mitochondrial fission and fusion.5.Ad-Sirt1 was used to over-express and Lv-sh-Sirt1 was employed to inhibit Sirt1,Ad-GFP and Lv-Sh-scramble were used as control,and then cells were cultured in hypoxic environment for 12 h.Western blot was used to evaluate the expression of Drp1 and Fis1.Results1.Compared with the acyanotic group,the expression of Drp1 and Fis1 increased significantly in the heart samples of patients with cyanotic heart disease(p<0.05).H9c2 cells were cultured in a hypoxic incubator,western blot analysis demonstrated that Drp1 and Fis1 increased gradually after hypoxia exposure.Mitochondrial morphology analysis showed that the percentage of cells containing fragmented mitochondria increased significantly [(64±5.29%)vs(12.33±3.51),P<0.05 ] Transfection of H9c2 cells with DRP1K38 A plasmid increased while h Fis1 plasmid transfection decreased cell activity.2.Overexpression of Sirt1 decreased the percentage of cells containing fragmented mitochondria significantly in response to hypoxia(p<0.05),and Sirt1 inhibition increased the percentage of cells containing fragmented mitochondria(p<0.05).Sirt1 overexpression reduced cell death after hypoxic incubation.Under hypoxic condition,compared with the control group,Sirt1 overexpression decreased Drp1 and Fis1 expression levels(P <0.05),while Drp1 and Fis1 expression levels increase after Sirt1 inhibition(p<0.05).Conclusions:Under hypoxic environment,Sirt1 inhibits mitochondrial fission through decreasing Drp1 and Fis1 expression to protect the heart.