Feng Yuanyuan, Luo Shouming, Yang Pan, Song Zhiyuan
Department of Cardiology, Southwest Hospital, Third Military Medical University, Chongqing 400038, P.R. China; Department of Cardiology, The 401 Hospital of PLA, Qingdao, Shandong 266071, P.R. China.
Department of Cardiology, Southwest Hospital, Third Military Medical University, Chongqing 400038, P.R. China.
Exp Ther Med. 2016 Apr;11(4):1323-1329. doi: 10.3892/etm.2016.3072. Epub 2016 Feb 11.
The 'funny' current, also known as the I current, play a crucial role in the spontaneous diastolic depolarization of sinoatrial node cells. The I current is primarily induced by the protein encoded by the hyperpolarization-activated cyclic nucleotide-gated channel 4 () gene. The functional I channel can be reconstructed in canine mesenchymal stem cells (cMSCs) transfected with mouse (m). Biomimetic studies have shown that electric pulse current stimulation (EPCS) can promote cardiogenesis in cMSCs. However, whether EPCS is able to influence the properties of the I current reconstructed in m-transfected cMSCs remains unclear. The present study aimed to investigate the effects of EPCS on the I current reconstructed in m-transfected cMSCs. The cMSCs were transfected with the lentiviral vector pLentis-m-GFP. Following transfection, these cells were divided into two groups: m-transfected cMSCs (group A), and m-transfected cMSCs induced by EPCS (group B). Using a whole cell patch-clamp technique, the I current was recorded, and group A cMSCs showed significant time and voltage dependencies and sensitivity to extracellular Cs+. The half-maximal activation (V1/2) value was -101.2±4.6 mV and the time constant of activation was 324±41 msec under -160 mV. In the group B cells the I current increased obviously and activation curve moved to right. The absolute value of V1/2 increased significantly to -92.4±4.8 mV (P<0.05), and the time constant of activation diminished under the same command voltage (251±44 vs. 324±41, P<0.05). In addition, the mRNA and protein expression levels of , connexin 43 (Cx43) and Cx45 were upregulated in group B compared with group A, as determined by reverse transcription-quantitative polymerase chain reaction and western blot analyses. Transmission electron micrographs also confirmed the increased gap junctions in group B. Collectively, these results indicated that reconstructed I channels may have a positive regulatory role in EPCS induction. The cMSCs transfected with m induced by EPCS may be a source of effective biological pacemaker cells.
“ funny”电流，也称为I电流，在窦房结细胞的自发性舒张期去极化中起关键作用。I电流主要由超极化激活的环核苷酸门控通道4（）基因编码的蛋白质诱导产生。功能性I通道可以在转染了小鼠（m）的犬间充质干细胞（cMSC）中重建。仿生学研究表明，电脉冲电流刺激（EPCS）可以促进cMSC中的心脏发生。然而，EPCS是否能够影响在转染m的cMSC中重建的I电流的特性仍不清楚。本研究旨在探讨EPCS对转染m的cMSC中重建的I电流的影响。用慢病毒载体pLentis-m-GFP转染cMSC。转染后，将这些细胞分为两组：转染m的cMSC（A组）和由EPCS诱导的转染m的cMSC（B组）。使用全细胞膜片钳技术记录I电流，A组cMSC表现出明显的时间和电压依赖性以及对细胞外Cs +的敏感性。在-160 mV下，半数最大激活（V1 / 2）值为-101.2±4.6 mV，激活时间常数为324±41毫秒。在B组细胞中，I电流明显增加，激活曲线向右移动。V1 / 2的绝对值显著增加至-92.4±4.8 mV（P <0.05），并且在相同指令电压下激活时间常数减小（251±44对324±41，P <0.05）。此外，通过逆转录定量聚合酶链反应和蛋白质印迹分析确定，与A组相比，B组中、连接蛋白43（Cx43）和Cx45的mRNA和蛋白质表达水平上调。透射电子显微镜照片也证实了B组中间隙连接增加。总体而言，这些结果表明重建的I通道可能在EPCS诱导中具有正向调节作用。由EPCS诱导的转染m的cMSC可能是有效的生物起搏细胞的来源。
