Zhou En, Zhang Tiantian, Bi Changlong, Wang Changqian, Zhang Zongqi
Department of Cardiology, Shanghai Ninth People's Hospital, School of Medicine, Shanghai Jiao Tong University, 639 Zhizaoju Road, Shanghai, 200011, China.
Shanghai Jiao Tong University School of Medicine, 227 South Chongqing Road, Shanghai, 200025, China.
Heart Vessels. 2020 Jul;35(7):1025-1035. doi: 10.1007/s00380-020-01602-3. Epub 2020 Apr 8.
Atrial fibrillation (AF) is one of the most prevalent arrhythmias. Myocardial sleeves of the pulmonary vein are critical in the occurrence of AF. Our study aims to investigate the effect of synthetic vascular smooth muscle cells (SMCs) on gap junction proteins in cardiomyocytes. (1) Extraction of vascular SMCs from the pulmonary veins of Norway rats. TGF-β was used to induce the vascular SMCs switching to the synthetic phenotype and 18-α-GA was used to inhibit gap junctions of SMCs. The contractile and synthetic phenotype vascular SMCs were cocultured with HL-1 cells; (2) Western blotting was used to detect the expression of Cx43, Cx40 and Cx45 in HL-1 cells, and RT-PCR to test microRNA 27b in vascular SMCs or in HL-1 cells; (3) Lucifer yellow dye transfer experiment was used to detect the function of gap junctions. (1) TGF- β induced the vascular SMCs switching to synthetic phenotype; (2) Cx43 was significantly increased, and Cx40 and Cx45 were decreased in HL-1 cocultured with synthetic SMCs; (3) The fluorescence intensity of Lucifer yellow was higher in HL-1 cocultured with synthetic SMCs than that in the cells cocultured with contractile SMCs, which was inhibited by18-α-GA; (4) the expression of microRNA 27b was increased in HL-1 cocultured with synthetic SMCs, which was attenuated markedly by 18-α-GA. (5) the expression of ZFHX3 was decreased in HL-1 cocultured with synthetic SMCs, which was reversed by 18-α-GA. The gap junction proteins of HL-1 were regulated by pulmonary venous SMCs undergoing phenotypic transition in this study, accompanied with the up-regulation of microRNA 27b and the down-regulation of ZFHX3 in HL-1 cells, which was associated with heterocellular gap junctions between HL-1 and pulmonary venous SMCs.
心房颤动（AF）是最常见的心律失常之一。肺静脉的心肌袖在房颤的发生中起关键作用。我们的研究旨在探讨合成型血管平滑肌细胞（SMC）对心肌细胞缝隙连接蛋白的影响。（1）从挪威大鼠肺静脉中提取血管平滑肌细胞。使用转化生长因子-β（TGF-β）诱导血管平滑肌细胞转变为合成型表型，使用18-α-甘草次酸（18-α-GA）抑制平滑肌细胞的缝隙连接。将收缩型和合成型表型的血管平滑肌细胞与HL-1细胞共培养；（2）采用蛋白质免疫印迹法检测HL-1细胞中连接蛋白43（Cx43）、连接蛋白40（Cx40）和连接蛋白45（Cx45）的表达，采用逆转录-聚合酶链反应（RT-PCR）检测血管平滑肌细胞或HL-1细胞中的微小RNA 27b（miR-27b）；（3）采用荧光黄染料转移实验检测缝隙连接的功能。（1）TGF-β诱导血管平滑肌细胞转变为合成型表型；（2）与合成型平滑肌细胞共培养的HL-1细胞中，Cx43显著增加，Cx40和Cx45减少；（3）与合成型平滑肌细胞共培养的HL-1细胞中荧光黄的荧光强度高于与收缩型平滑肌细胞共培养的细胞，18-α-GA可抑制这种现象；（4）与合成型平滑肌细胞共培养的HL-1细胞中miR-27b的表达增加，18-α-GA可显著减弱这种增加；（5）与合成型平滑肌细胞共培养的HL-1细胞中锌指蛋白X3（ZFHX3）的表达减少，18-α-GA可使其逆转。本研究中，HL-1细胞的缝隙连接蛋白受肺静脉平滑肌细胞表型转变的调控，同时伴有HL-1细胞中miR-27b的上调和ZFHX3的下调，这与HL-1细胞和肺静脉平滑肌细胞之间的异细胞缝隙连接有关。
