Disheveled binding antagonist of β-catenin 1 interacted with β-catenin and connexin 43 in human-induced pluripotent stem cells-derived cardiomyocytes.
Disheveled binding antagonist of β-catenin 1 interacted with β-catenin and connexin 43 in human-induced pluripotent stem cells-derived cardiomyocytes.
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β-连环蛋白 1 的蓬乱结合拮抗剂与人诱导多能干细胞衍生的心肌细胞中的 β-连环蛋白和连接蛋白 43 相互作用
DOI：
10.1080/21655979.2022.2070448
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发表时间：
2022-05
期刊：
影响因子：
4.9
通讯作者：
Wu Z
中科院分区：
文献类型：
--
作者：
Hou J;Huang S;Long Y;Feng K;Shang L;Zhou Z;Yue Y;Huang X;Chen G;Wu Z
以前，我们证明了β-catenin 1（DACT 1）的结合拮抗剂通过调节心肌细胞中连接蛋白43和β-catenin的重组参与心房颤动。然而，对DACT 1在人类正常心肌细胞中的作用知之甚少。因此，我们使用来自人胚胎干细胞（ESC）和诱导多能干细胞（iPSC）的心肌细胞（CM）来研究DACT 1的作用及其与β-连环蛋白和连接蛋白43的联系。虽然ESC-CM和iPSC-CM使用商业分化试剂盒进行分化，但通过免疫荧光检测心脏特异性标志物。免疫印迹法检测DACT 1的表达水平，免疫荧光法和免疫共沉淀法检测DACT 1与连接蛋白43（connexin 43）和β-连环蛋白（β-catenin）的相互作用。对H1-CM和SF-CM进行心脏特异性标志物免疫染色，包括肌钙蛋白I、肌钙蛋白T、α-辅肌动蛋白、NKX2.5和GATA 6。虽然DACT 1在H1 ESC和SF-iPSC中均不表达，但它在分化的CM中高度表达，并且也定位于分化的CM的细胞质和细胞核中。有趣的是，在同一多核细胞中，不同核中DACT 1的表达是不同的。此外，DACT 1与β-catenin共定位于分化型CM的胞质和胞核中，与Cx43共定位于分化型CM的核周区和差距连接处。Co-IP结果显示DACT 1可直接与β-catenin和connexin 43结合。总之，DACT 1与人诱导多能干细胞衍生的心肌细胞中的β-catenin和connexin 43相互作用。
Previously, we demonstrated that the disheveled binding antagonist of β-catenin 1 (DACT1) was involved in atrial fibrillation by regulating the reorganization of connexin 43 and β-catenin in cardiomyocytes. Little is known, however, about DACT1 in human normal myocardial cells. Therefore, we used cardiomyocytes (CMs) derived from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) to investigate the role of DACT1 and its connection with β-catenin and connexin 43. While the ESC-CMs and iPSC-CMs were differentiated using commercial differentiation kits, the cardiac-specific markers were detected by immunofluorescence. The expression level of DACT1 was detected using western blotting, whereas the interaction of DACT1 and connexin 43 or β-catenin was detected by immunofluorescence and co-immunoprecipitation (co-IP) assays. Both H1-CMs and SF-CMs were immunostained for cardiac-specific markers, including Troponin I, Troponin T, α-actinin, NKX2.5, and GATA6. While DACT1 was not expressed in both H1 ESCs and SF-iPSCs, it was, however, highly expressed in differentiated CMs, being also localized in the cytoplasm and the nucleus of differentiated CMs. Interestingly, the DACT1 expression in different nuclei was different in the same multinucleated cell. Moreover, DACT1 colocalized with β-catenin in both the cytoplasm and nucleus of differentiated CMs, and it also colocalized with connexin 43 in the perinuclear region and the gap junctions of differentiated CMs. Co-IP results showed that DACT1 could directly bind to β-catenin and connexin 43. Taken together, DACT1 interacted with β-catenin and connexin 43 in human-induced pluripotent stem cells-derived cardiomyocytes.
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