Sirt1在缺氧条件下对心肌细胞线粒体融合与分裂中的作用

Role of Sirt1 in mitochondrial fusion and fission of hypoxic cardiac myocytes

目的探讨缺氧条件下线粒体动态学变化及Sirt1在其中的作用。方法 1收集2014年于新桥医院心血管外科行手术治疗的先心病患儿29例,其中非紫绀型先心病14例,紫绀型先心病15例,取术中所切除的右室流出道心肌组织为心肌标本,Western blot检测促线粒体分裂蛋白Drp1与Fis1表达情况。2将H9c2心肌细胞置入缺氧培养箱(94%N2,5%CO2,1%O2)中进行培养,检测缺氧0、2、4、8、12 h后促线粒体分裂蛋白Drp1与Fis1的表达水平。3将线粒体靶向质粒分别同空载质粒、Sirt1过表达质粒和Sirt1干扰质粒共转染H9c2细胞,常氧或缺氧培养箱中培养12 h后,激光共聚焦显微镜下观察线粒体融合、分裂情况;4运用腺病毒Ad-Sirt1和慢病毒Lv-Sh-Sirt1分别在H9c2细胞中过表达或干扰Sirt1后,将细胞置于缺氧培养箱中培养12 h,检测Drp1与Fis1表达水平;5分别转染空载质粒、Sirt1过表达质粒和Sirt1干扰质粒至H9c2细胞后,缺氧培养12 h,LDH试剂盒检测细胞毒性。结果 1同非紫绀组相比,紫绀组心肌中Drp1与Fis1表达显著增高,Drp1相对灰度值:[(0.47±0.11)vs(0.76±0.12)、Fis1相对灰度值(0.53±0.02)vs(0.83±0.03),P<0.05];2细胞水平研究结果显示Drp1与Fis1的蛋白表达随缺氧的时间的延长呈递增趋势;3激光共聚焦结果表明,Sirt1过表达后能够一定程度地缓解缺氧诱导的线粒体分裂(P<0.05),而当Sirt1受到干扰后,缺氧所诱导的线粒体分裂水平明显提高(P<0.05);4在缺氧条件下,过表达Sirt1能够抑制Drp1与Fis1蛋白表达水平(P<0.05),而Sirt1被抑制后,Drp1与Fis1蛋白表达水平增加(对照组,过表达组,低表达组Drp1相对灰度值依次为[(0.47±0.02)vs(0.36±0.02)vs(0.78±0.04);Fis1相对灰度值依次为(0.64±0.02)vs(0.42±0.02)vs(0.80±0.04),P<0.05];5Sirt1过表达能够降低缺氧刺激后H9c2心肌细胞的死亡率。结论 Sirt1可能通过调控促线粒体分裂蛋白Drp1与Fis1表达水平促进心肌细胞在缺氧条件下的适应。

Objective To determine the changes of mitochondrial dynamics under hypoxia and the potential role of Sirt1 in this condition. Methods Twenty-nine children diagnosed with congenital heart diseases were recruited from the institute of cardiovascular surgery of Xinqiao hospital since 2014,and included 14 acyanotic cases and 15 cyanotic cases. The myocardial tissues of right ventricular outflow tract were taken for further analysis. Western blot analysis was employed to detect the expression levels of mitochondrial pro-fission proteins Drp1 and Fis1 in the myocardial tissues. H9c2 cells were cultured in a hypoxic incubator（ 94% N2,5% CO2,1% O2） for 0,2,4,8 and 12 h respectively,and Drp1 and Fis1 expression levels were detected by Western blotting. Mitochondria-targeted red fluorescent protein（ mt-RFP）-expressing plasmid together with blank vector,Sirt1 overexpression plasmid or Sh-Sirt1 interference plasmid co-transfected H9c2 cells,and then the cells were cultured in a normoxic or hypoxic incubator. Confocal microscopy was used to visualize mitochondrial morphology. Adenovirus-Sirt1 or lentivirus-Sh-Sirt1 was added to H9c2 cells to activate or inhibit Sirt1 expression,and Drp1 and Fis1 expressions were measured by Western blotting after 12 h hypoxia treatment. LDH assay was employed to assess cytotoxicity after the H9c2 cells were transfected with the empty vector,Sirt1 overexpression plasmid or Sh-Sirt1 interference plasmid. Results Compared with the acyanotic group,Drp1 and Fis1 expression levels were significantly increased in the cyanotic group（ Drp1： 0. 47 ± 0. 11 vs 0. 76 ± 0. 12,Fis1： 0. 53 ± 0. 02 vs 0. 83 ± 0. 03,P 〈0. 05）. In H9c2 cells,Drp1 and Fis1 were activated after hypoxia exposure in a time-dependent manner. Confocal images of H9c2 cells expressing mt-RFP showed that Sirt1 overexpression significantly suppressed hypoxia-induced mitochondrial fission whereas Sirt1 interference promoted mitochondrial fission（ P 0. 05）. Compared with the control group,the expression levels of Drp1 and Fis1 were decreased after Sirt1 overexpression,and prominently increased after Sirt1 interference（ Drp1： 0. 47 ± 0. 02 vs 0. 36 ± 0. 02 vs 0. 78 ± 0. 04,Fis1： 0. 64± 0. 02 vs 0. 42 ± 0. 02 vs 0. 80 ± 0. 04,P 0. 05）. Sirt1 overexpression decreased the mortality rates of H9c2 cells challenged by hypoxia（ P 〈0. 05）. Conclusion Sirt1 may be involved in hypoxic adaptation through regulating the expression of Drp1 and Fis1 in the cardiac myocytes.