细胞骨架及极性在心肌致密化不全患者来源心肌样细胞中的改变

Changes of cytoskeleton and polarity in cardiomyocyte-like cells derived from left ventricular noncompaction patients

目的探讨心肌致密化不全(left ventricular noncompaction,LVNC)患者来源的心肌样细胞(cardiomyocytes,CMs)骨架与极性的改变及机制。方法以LVNC患者及正常人来源的多潜能干细胞(induced pluripotent stem cells,iPSCs)诱导分化的心肌样细胞为模型,进行转录组测序,透射电镜观察两组细胞骨架改变,免疫荧光观察细胞骨架相关蛋白纤维状肌动蛋白(F-actin)、微管蛋白二聚体(ɑ/β-tublin)、极性蛋白par6和黏着斑蛋白(vinculin)在两组心肌细胞中的表达和分布,Western blot检测细胞骨架及极性Rho/Rock通路关键蛋白Ras同源基因家族成员A(ras homolog gene family member A,RhoA)的表达。结果两组细胞均阳性表达胚胎干细胞表面分子标志物Oct4、Sox2、SSEA4、TRA1-81,且可诱导产生具有自主搏动节律的心肌细胞团,免疫荧光检测示心肌细胞表面标志cTnT、ɑ-actinin表达阳性,转录组测序结果显示有多个差异基因与微管联系紧密;与正常组相比,透射电镜观察到疾病组心肌细胞骨架排列紊乱,免疫荧光发现骨架相关蛋白ɑ/β-tublin表达下调[n=62,(226.4±65.1),Z=-3.451,P<0.01],F-actin表达下调[n=62,(236.4±33.2),P<0.001],vinculin表达下调且分布不均匀,于细胞膜处明显浓聚[n=57,(137.2±35.9),Z=-3.852,P<0.001],极性蛋白par6表达上调[n=64,(-132.7±43.5),Z=-3.148,P<0.01];Western blot结果提示患者组RhoA蛋白表达水平明显上调(P<0.01)。结论LVNC患者来源的心肌细胞骨架和极性存在异常。

Objective To investigate the changes and mechanism of the cytoskeleton and polarity in cardiomyocyte-like cells derived from patients with left ventricular noncompaction(LVNC).Methods After induced pluripotent stem cells(iPSCs)were isolated from LVNC patients and normal individuals,respectively,the obtained cells were induced to differentiate into cardiomyocyte-like cells.Then transcriptome sequencing was performed.Transmission electron microscopy(TEM)was employed to observe the changes of the myocardial cytoskeleton.Immunofluorescence assay was used to detect the location and expression of cytoskeleton-associated proteins,fibrous actin(F-actin),tubulin dimer(ɑ/β-tublin),and polar protein Par6 and vinculin.Western blotting was used to detect the expression of Rho/Rock pathway key protein,Ras homolog gene family member A(RhoA).Results Both the obtained iPSCs derived from LVNC patients and normal persons positively expressed the surface molecular markers of embryonic stem cells,Oct4,Sox2,SSEA4,and TRA1-81,and could be induced into cardiomyocyte-like clusters with autonomous pulsating rhythm.Immunofluorescence assay indicated that the cardiomyocyte-like cells expressed the surface markers of cardiomyocytes,cTnT andɑ-actinin.The results of transcriptome sequencing showed that there were multiple differential genes related closely to microtubules between the 2 kinds of cardiomyocyte-like cells.Compared with the cells derived from the normal individuals,there was a disordered arrangement of the cytoskeleton with TEM,significantly decreased expression levels of ɑ/β-tublin(n=62,226.4±65.1,P<0.01,Z=-3.451),F-actin(n=62,(236.4±33.2),P<0.001),and vinculin(n=57,137.2±35.9,P<0.001,Z=-3.852).and it was unevenly distributed and obviously concentrated in the cell membrane,and obviously up-regulation of Par6(n=64,-132.7±43.5,P<0.01,Z=-3.148)with the aid of immunofluorescence assay.Western blotting demonstrated that the expression of RhoA was significantly increased in the cells derived from the patient group(P<0.01).Conclusion The cytoskeleton and polarity of cardiomyocyte-like cells derived from LVNC patients are indeed abnormal.