心肌细胞FoxO1特异性敲除对心肌梗死的影响及机制

Effects of cardiomyocyte-specific FoxO1 knockout on ventricular remodeling post myocardial infarction in mice

目的探讨心肌细胞FoxO1特异性敲除对小鼠急性心肌梗死后纤维瘢痕和心功能的影响。方法使用他莫昔芬(0.1mg/g/d)对Myh6-ER-Cre^(+)FoxO1^(loxP/loxP)小鼠连续腹腔注射5 d的方法诱导心肌细胞内FoxO1的敲除。H9C2心肌细胞转染FoxO1的siRNA诱导心肌细胞内FoxO1的表达下调。使用Western Blot法检测FoxO1的蛋白表达水平,以明确敲减效率。选取10周左右的心肌细胞FoxO1特异性敲除(CKO组)和注射玉米油的对照小鼠(对照组)通过前降支结扎的方法制备心肌梗死模型。心脏超声检测各组心功能改变,Masson染色检测各组心脏梗死纤维瘢痕面积。TUNEL和心肌细胞标志蛋白α-Actinin免疫荧光双染法检测心肌细胞凋亡情况。realtime-PCR法检测细胞凋亡相关基因的表达。Caspase3/7活性检测心肌细胞活性。结果Western Blot显示CKO组小鼠心脏组织中FoxO1的表达显著下降,表明敲减成功。术后28 d的生存曲线显示CKO组小鼠的生存率高于对照组。与对照组相比,CKO组的梗死纤维瘢痕的面积显著减小,心功能显著提高(P<0.05)。CKO组心脏组织中促凋亡相关基因Bax和Bim的表达显著降低(P<0.05),梗死交界区心肌细胞凋亡数量也少于对照组(P<0.05)。H9C2心肌细胞使用siRNA抑制FoxO1的表达后,缺氧6 h后的心肌细胞中Caspase3/7的活性显著下降(P<0.05),下调细胞内促凋亡基因(Bim、Bax)的表达和上调抗凋亡基因(Bcl2、Bcl-xL)的表达(P<0.05)。结论心肌细胞特异性敲除FoxO1可减轻心肌细胞缺氧诱导的损伤,从而在小鼠急性心肌梗死时起到心脏保护作用。

Objective To investigate the effect of cardiomyocyte-specific deletion of FoxO1 on myocardial fibrosis and cardiac function after acute myocardial infarction in mice.Methods Tamoxifen(0.1mg/g/d)intraperitoneal injection to Myh6-ER-Cre^(+)FoxO1^(loxP/loxP)mice for 5 days was used to induce FoxO1 knockout,and Foxo1-siRNA transfection was used to induce FoxO1 inhibition in H9C2 cell line.The FoxO1 knockout efficiency were identified by Western Blot.10 weeks,male cardiomyocyte-specific FoxO1 deletion mice(CKO group)and corn oil injected control mice(control group)were used for myocardial infarction operation by ligating the anterior descending branch.After myocardial infarction operation,cardiac function was detected by echocardiography,cardiac fibrosis was evaluated by Masson staining.The apoptosis of cardiomyocytes was measured by TUNEL andα-Actinin immunofluorescence double staining and realtime-PCR with apoptosis associated genes.Caspase3/7 activity was used to examine the cell viability of cardiomyocytes.Results The expression of FoxO1 in heart tissue and H9C2 cell was decreased significantly.Compared with the control group,cardiomyocyte-specific FoxO1 deletion could improve the cardiac function and mitigate the infarction scar size(P<0.05).FoxO1 deletion could decrease the cardiomyocyte apoptosis and the mRNA levels of pro-apoptosis genes Bax and Bim(P<0.05).In H9C2 cardiomyocytes,FoxO1 knockdown by siRNA could attenuate the increased activity of Caspase 3/7(P<0.05),reduce the expression of pro-apoptosis genes(Bax,Bim)and increase the expression of anti-apoptosis genes(Bcl2,Bcl-xL)under hypoxia condition(P<0.05).Conclusions Cardiomyocyte-specific FoxO1 deletion has obviously protective effects on myocardial fibrosis and cardiac function after acute myocardial infarction in mice.The mechanism may be associated with the decreased cardiomyocyte apoptosis.