摘要
目的探讨Runt相关转录因子1(RUNX1)在主动脉弓缩窄(TAC)术构建小鼠心肌肥厚模型中的作用,并对其机制进行初步探讨。方法动物实验是将flox体系小鼠(RUNX1flox/flox)与RUNX1心肌特异性敲除(Cre-LoxP体系)基因工具小鼠(RUNX1^(△fl/△fl))分别进行假手术(Sham)和TAC处理,共4组。Sham小鼠仅开胸处理,TAC小鼠通过结扎主动脉弓建立小鼠心肌肥厚模型。4周后,(1)形态学观察各组小鼠心脏外观大小并比较心脏重量/体重比值(HW/BW)、心脏重量/胫骨长度比值(HW/TL);(2)比较各组小鼠心肌组织HE染色、心肌细胞横截面积(CSA)以及免疫组织化学染色(IHC),计算心肌肥厚标志物脑利尿钠肽(BNP)表达情况;(3)应用小鼠心脏超声观察并计算各组小鼠左室收缩末期容积(LVESV)、左室心肌质量(LV Mass)、左室射血分数(LVEF)以及左室短轴缩短分数(LVFS);(4)应用实时荧光定量PCR(RT-qPCR)和蛋白质印迹法(Western blotting)技术,检测各组小鼠RUNX1、BNP和肌质网/内质网钙ATP酶2a(SERCA2a)在转录水平和蛋白水平的表达情况。细胞实验是将H9c2心肌细胞通过慢病毒转染基因沉默技术分别沉默RUNX1和SERCA2a后给予血管紧张素Ⅱ(AngⅡ)干预处理,分为转染无义序列慢病毒(NS)组、血管紧张素Ⅱ(AngⅡ)+NS组、转染RUNX1沉默慢病毒(shRUNX1)/转染SERCA2a沉默慢病毒(shSERCA2a)组和AngⅡ+shRUNX1/AngⅡ+shSERCA2a组,共4组。RT-qPCR法检测各组心肌细胞RUNX1、BNP和SERCA2a表达情况,验证RUNX1与SERCA2a之间的关系。结果动物实验显示,与Sham条件相比,RUNX1flox/flox组和RUNX1^(△fl/△fl)组小鼠在TAC条件下心脏外观明显变大,心肌肥厚相关指标HW/BW、HW/TL、心肌细胞CSA、BNP表达量、LVESV以及LV Mass均显著升高,LVEF、LVFS均明显下降,这表明TAC模型成功建立且方法稳定。(1)形态学指标显示:在TAC条件下,与RUNX1flox/flox组相比,RUNX1^(△fl/△fl)组小鼠心脏外观相对较小且HW/BW、HW/TL下降具有统计学意义;而在Sham条件下,与RUNX1flox/flox组相比,RUNX1^(△fl/△fl)组小鼠心脏外观无改变且HW/BW、HW/TL无统计学差异。(2)组织学染色结果显示:在TAC条件下,HE染色显示与RUNX1flox/flox组相比,RUNX1^(△fl/△fl)组小鼠心肌细胞CSA明显变小且心肌细胞胞浆窄小、排列较整齐;IHC显示与RUNX1flox/flox组相比,RUNX1^(△fl/△fl)组小鼠BNP表达下降。而在Sham条件下,RUNX1^(△fl/△fl)组小鼠CSA、细胞形态以及BNP表达量与RUNX1flox/flox组相比无统计学差异。(3)小鼠超声心动图结果显示:在TAC条件下,与RUNX1flox/flox组相比,RUNX1^(△fl/△fl)组小鼠LVEF、LVFS均升高,LV Mass、LVESV均下降;而在Sham条件下,RUNX1^(△fl/△fl)组小鼠LVEF、LVFS、LV Mass及LVESV等指标与RUNX1flox/flox组相比无统计学差异。(4)RT-qPCR与Western blotting结果显示:与Sham条件相比,RUNX1flox/flox组小鼠在TAC条件下RUNX1及BNP表达均升高,而SERCA2a表达下降;在TAC条件下,与RUNX1flox/flox组相比,RUNX1^(△fl/△fl)组小鼠BNP表达下降,SERCA2a表达升高;而在Sham条件下,RUNX1^(△fl/△fl)组小鼠BNP和SERCA2a表达与RUNX1flox/flox组相比无统计学差异。细胞实验显示,与AngⅡ+NS组心肌细胞相比,AngⅡ+shRUNX1组细胞SERCA2a mRNA表达升高,BNP表达下降,沉默RUNX1后可上调SERCA2a mRNA的表达水平并且抑制了AngⅡ所导致的BNP mRNA表达量升高,具有统计学差异;shSERCA2a后H9c2心肌细胞与NS组细胞相比,经AngⅡ处理后SERCA2a表达下降,BNP表达明显升高,但RUNX1的表达水平未受明显影响,即SERCA2a的表达水平并不影响RUNX1的变化。结论RUNX1在TAC术构建的小鼠心肌肥厚模型中发挥着重要作用,能够促进小鼠心肌肥厚的发生发展,其作用机制可能与下调SERCA2a的表达从而导致心肌收缩及舒张功能障碍有关。
Objective To explore the role of Runt-related transcription factor 1(RUNX1)in the mouse model of myocardial hypertrophy induced by transverse aortic constriction(TAC)and to preliminarily investigate its mechanism.Methods In the animal experiment,flox system mice(RUNX1flox/flox)and RUNX1 myocardial-specific knockout(Cre-LoxP system)gene tool mice(RUNX1^(△fl/△fl))were respectively subjected to sham operation(Sham)and TAC treatment,totaling 4 groups.Sham mice only underwent thoracotomy,while TAC mice had a mouse model of myocardial hypertrophy established by ligating the aortic arch.Four weeks later,(1)morphological observation was conducted on the appearance and size of the hearts of each group of mice,and the ratios of heart weight to body weight(HW/BW)and heart weight to tibia length(HW/TL)were compared;(2)histological staining results of each group of mice were compared,including HE staining of myocardial tissue,cross-sectional area(CSA)of myocardial cells,and immunohistochemical staining(IHC)to calculate the expression of myocardial hypertrophy marker brain natriuretic peptide(BNP);(3)echocardiography was used to observe and calculate left ventricular end-systolic volume(LVESV),left ventricular myocardial mass(LV Mass),left ventricular ejection fraction(LVEF),and left ventricular fractional shortening(LVFS)in each group of mice;(4)real-time fluorescence quantitative PCR(RT-qPCR)and Western blotting techniques were used to detect the expression of RUNX1,BNP,and sarcoplasmic/endoplasmic reticulum calcium ATPase 2a(SERCA2a)at the transcriptional and protein levels in each group of mice.In the cell experiment,H9c2 cardiomyocytes were transfected with shRNA lentivirus to silence RUNX1 and SERCA2a,respectively,and then treated with angiotensinⅡ(AngⅡ).Four groups were set up:the group transfected with nonsense sequence lentivirus(NS),the AngⅡ+NS group,the group transfected with RUNX1 silencing lentivirus(shRUNX1)/transfected with SERCA2a silencing lentivirus(shSERCA2a),and the AngⅡ+shRUNX1/AngⅡ+shSERCA2a group.RT-qPCR was used to detect the expression of RUNX1,BNP,and SERCA2a in each group of cardiomyocytes to verify the relationship between RUNX1 and SERCA2a.Results In the animal experiment,compared with the Sham condition,the hearts of RUNX1flox/flox and RUNX1^(△fl/△fl)mice were significantly enlarged under TAC conditions,and the myocardial hypertrophy-related indicators HW/BW,HW/TL,CSA,BNP expression,LVESV,and LV Mass were significantly increased,while LVEF and LVFS were significantly decreased,indicating that the TAC model was successfully established and the method was stable.(1)Morphological indicators showed that under TAC conditions,compared with the RUNX1flox/flox group,the hearts of RUNX1^(△fl/△fl)mice were relatively smaller in appearance,and the HW/BW and HW/TL ratios decreased significantly;while under Sham conditions,compared with the RUNX1flox/flox group,there was no change in the appearance of the hearts of RUNX1^(△fl/△fl)mice,and there was no statistical difference in HW/BW and HW/TL.(2)Histological staining results showed that under TAC conditions,HE staining revealed that compared with the RUNX1flox/flox group,the CSA of RUNX1^(△fl/△fl)mice was significantly smaller,and the cytoplasm of myocardial cells was observed to be narrower and more orderly arranged.IHC showed that the expression of BNP in the RUNX1^(△fl/△fl)group was decreased compared with the RUNX1flox/flox group.Under Sham conditions,there was no statistically significant difference in CSA,cell morphology,and BNP expression between the RUNX1^(△fl/△fl)group and the RUNX1flox/flox group.(3)The results of echocardiography in mice showed that under TAC conditions,compared with the RUNX1flox/flox group,the LVEF and LVFS of the RUNX1^(△fl/△fl)group were increased,while the LV Mass and LVESV were decreased.Under Sham conditions,there was no statistically significant difference in LVEF,LVFS,LV Mass,and LVESV between the RUNX1^(△fl/△fl)group and the RUNX1flox/flox group.(4)The results of RT-qPCR and Western blotting showed that compared with the Sham condition,the expression of RUNX1 and BNP in the RUNX1flox/flox group was increased under TAC conditions,while the expression of SERCA2a was decreased.Under TAC conditions,compared with the RUNX1flox/flox group,the expression of BNP in the RUNX1^(△fl/△fl)group was decreased,while the expression of SERCA2a was increased.Under Sham conditions,there was no statistically significant difference in the expression of BNP and SERCA2a between the RUNX1^(△fl/△fl)group and the RUNX1flox/flox group.Cell experiments showed that compared with the AngⅡ+NS group of cardiomyocytes,the expression of SERCA2a mRNA in the AngⅡ+shRUNX1 group was increased,while the expression of BNP was decreased.Silencing RUNX1 could up-regulate the expression level of SERCA2a mRNA and inhibit the increase in BNP mRNA expression caused by AngⅡ,with statistical significance.After transfection with SERCA2a silencing lentivirus,the expression of SERCA2a in H9c2 cells was decreased and the expression of BNP was significantly increased after AngⅡtreatment compared with the NS group cells,but the expression level of RUNX1 was not significantly affected,indicating that the expression level of SERCA2a does not affect the changes in RUNX1.Conclusion RUNX1 plays an important role in the mouse model of myocardial hypertrophy constructed by TAC surgery,promoting the occurrence and development of myocardial hypertrophy in mice.The mechanism of action may be related to the down-regulation of SERCA2a expression,leading to myocardial contractile and diastolic dysfunction.
作者
邹明锐
丁立群
王玉玖
杨丽娟
韩曰信
刘宝辉
ZOU Mingrui;DING Liqun;WANG Yujiu;YANG Lijuan;HAN Yuexin;LIU Baohui(Department of Cardiovascular Surgery,Affiliated Hospital of Binzhou Medical University,Binzhou 256603,Shandong,China;Medical Research Center,Affiliated Hospital of Binzhou Medical University,Binzhou 256603,Shandong,China)
出处
《心血管病学进展》
2025年第8期760-768,共9页
Advances in Cardiovascular Diseases
基金
山东省自然科学基金(ZR2020QH017)。
关键词
Runt相关转录因子1
心肌肥厚
主动脉弓缩窄
肌质网/内质网钙ATP酶2a
Runt-related transcription factor 1
Cardiac hypertrophy
Transverse aortic constriction
Sarcoplasmic/endoplasmic reticulum calcium ATPase 2a
