Department of Cardiology, Renmin Hospital of Wuhan University, Wuhan, 430060, PR China; Cardiovascular Research Institute, Wuhan University, Wuhan, 430060, PR China; Hubei Key Laboratory of Cardiology, Wuhan, 430060, PR China.
Department of Cardiology, Renmin Hospital of Wuhan University, Wuhan, 430060, PR China; Cardiovascular Research Institute, Wuhan University, Wuhan, 430060, PR China; Hubei Key Laboratory of Cardiology, Wuhan, 430060, PR China.
Biomed Pharmacother. 2020 Oct;130:110612. doi: 10.1016/j.biopha.2020.110612. Epub 2020 Aug 6.
The TBX3(T-box 3)transcription factor is considered as an essential factor in sinoatrial node formation. While the effect of TBX3 in the differentiation of sinoatrial node cells from embryonic stem cells(ESCs) has been recognized, its role in human induced pluripotent stem cell derived cardiomyocytes(hiPSCMs) has not been addressed. Therefore, the purpose of the present study was to investigate whether overexpression of TBX3 in hiPSCs could increase their differentiation into pacemaker-like cells.
The hiPSCs were transfected with TBX3 gene during differentiation into cardiomyocytes(CMs). The hiPSCMs were analyzed using immunofluorescence, RT-qPCR, flow cytometry, whole-cell patch clamp recording to identify the differentiation effect exerted by TBX3. We discovered that hiPSCs transfected with TBX3 showed more proportions of NKX2.5-cTNT + sinoatrial node cells and faster contracting rates.
The results showed increment in transcription factor TBX18, SHOX2; hyperpolarization-activated cyclic nucleotide (HCN) channel: HCN1, HCN2, HCN4, connexin 45(CX45), Na + Ca2+ exchanger(NCX) in TBX3 transfected hiPSCMs. Sinoatrial node cell specific If current and action potential were also confirmed by patch clamp in TBX3 transfected hiPSCMs and the pacemaker-like cells were able to pace hiPSCMs ex vivo.
In conclusion, the present study demonstrated that overexpression of TBX3 could increase the differentiation of hiPSCs into pacemaker-like cells. Our study provide new strategy to construct a biological pacemaker, however, further study is still needed to identify the efficacy and safety of using the pacemaker-like cells to produce biological pacemaker in vivo.
TBX3（T 盒转录因子 3）转录因子被认为是窦房结形成的必要因素。虽然 TBX3 对胚胎干细胞（ESCs）来源的窦房结细胞分化的作用已得到认可，但它在人诱导多能干细胞（hiPSCs）来源的心肌细胞（hiPSCMs）中的作用尚未得到解决。因此，本研究旨在探讨 TBX3 在 hiPSCs 中的过表达是否能增加其分化为起搏样细胞的能力。
在 hiPSCs 向心肌细胞（CMs）分化过程中，用 TBX3 基因转染 hiPSCs。采用免疫荧光、RT-qPCR、流式细胞术、全细胞膜片钳记录等方法分析 hiPSCMs，以鉴定 TBX3 发挥的分化作用。我们发现，转染 TBX3 的 hiPSCs 显示出更多比例的 NKX2.5-cTNT+窦房结细胞和更快的收缩率。
结果显示，TBX3 转染的 hiPSCMs 中转录因子 TBX18、SHOX2 增加；超极化激活环核苷酸（HCN）通道：HCN1、HCN2、HCN4、连接蛋白 45（CX45）、Na+Ca2+交换体（NCX）增加。在 TBX3 转染的 hiPSCMs 中，也通过膜片钳证实了窦房结细胞特异性 If 电流和动作电位，并且起搏样细胞能够在体外起搏 hiPSCMs。
总之，本研究表明，TBX3 的过表达可以增加 hiPSCs 向起搏样细胞的分化。我们的研究为构建生物起搏器提供了新的策略，然而，仍需要进一步研究来确定使用起搏样细胞在体内产生生物起搏器的疗效和安全性。
