Feng Yuanyuan, Luo Shouming, Tong Shifei, Zhong Li, Zhang Changhai, Yang Pan, Song Zhiyuan
Department of Cardiology, Southwest Hospital, Third Military Medical University, Chongqing, China.
Cardiology. 2015;132(1):49-57. doi: 10.1159/000398784. Epub 2015 Jun 4.
We aimed to investigate the role of mShox2 in generating If pacemaker current in vitro by means of electric-pulse current stimulation (EPCS) of canine mesenchymal stem cells (cMSCs).
mShox2 genetically modified cMSCs were prepared with pLentis-mShox2 red fluorescent protein. After EPCS induction, we examined the kinetic characteristics of generated inward current by means of a patch clamp. We then evaluated the expression of pacemaker-related genes, such as Nkx2.5, Tbx3, HCN4, Cx43 and Cx45, by means of qRT-PCR and Western blotting. The morphological changes and the cardiomyogenic differentiation marker cTnT were investigated at the same time.
The time- and voltage-dependent inward current recorded after mShox2 infection was confirmed to be If current. After EPCS induction, the detection rate of this If current was increased. The current amplitude and density were increased, and the channel activation curve shifted to the right. The pacemaker markers Tbx3, HCN4 and Cx45 were significantly upregulated, but the working myocardium markers Nkx2.5 and Cx43 were downregulated after mShox2 infection, and were more remarkable after EPCS induction. The cells became larger and assumed spindle and spider-like morphologies. cTnT was also detected in the experimental cells.
Our results suggest that EPCS promotes the differentiation of mShox2 genetically modified cMSCs into pacemaker-like cells, which generates more If current.
我们旨在通过对犬间充质干细胞（cMSCs）进行电脉冲电流刺激（EPCS），研究mShox2在体外产生If起搏电流中的作用。
用pLentis-mShox2红色荧光蛋白制备mShox2基因修饰的cMSCs。EPCS诱导后，我们通过膜片钳检测所产生内向电流的动力学特征。然后，我们通过qRT-PCR和蛋白质免疫印迹法评估起搏相关基因如Nkx2.5、Tbx3、HCN4、Cx43和Cx45的表达。同时研究形态学变化和心肌分化标志物cTnT。
mShox2感染后记录的时间和电压依赖性内向电流被证实为If电流。EPCS诱导后，该If电流的检出率增加。电流幅度和密度增加，通道激活曲线右移。mShox2感染后，起搏标志物Tbx3、HCN4和Cx45显著上调，但工作心肌标志物Nkx2.5和Cx43下调，EPCS诱导后更明显。细胞变大，呈现纺锤形和蜘蛛样形态。实验细胞中也检测到了cTnT。
我们的结果表明，EPCS促进mShox2基因修饰的cMSCs分化为起搏样细胞，产生更多的If电流。
