Qi Zenghua, Wong Chun Kit, Suen Chi Ho, Wang Jinzhao, Long Cheng, Sauer Heinrich, Yao Xiaoqiang, Tsang Suk Ying
School of Life Sciences, The Chinese University of Hong Kong, Hong Kong.
School of Life Sciences, South China Normal University, Guangzhou, PR China.
Int J Cardiol. 2016 Jan 15;203:169-81. doi: 10.1016/j.ijcard.2015.10.018. Epub 2015 Oct 9.
Cardiac pacemaking is a complex phenomenon that is not completely understood. Canonical transient receptor potential isoform 3 (TRPC3) channel is a cation channel that permeates both Ca(2+) and Na(+). TRPC3 was previously found to express in adult cardiomyocytes. However, its role in cardiac pacemaking is unexplored. Here we used mouse embryonic stem cell-derived cardiomyocytes (mESC-CMs) to investigate whether TRPC3 regulates the spontaneous automaticity and the underlying mechanism involved.
Immunocytochemistry results showed that TRPC3 is expressed at the T-tubules of mESC-CMs. Whole-cell patch clamping showed that single mESC-CMs contain TRPC3 current. Confocal Ca(2+) imaging showed that the TRPC3-specific blocker Pyr3 decreased Ca(2+) transients and local Ca(2+) release (LCR) of mESC-CMs. Combined current and voltage clamp recordings from the same cell showed that reducing the TRPC3 current, either by Pyr3 or a dominant negative (loss-of-function) construct of TRPC3, decreased the pacemaker activity of mESC-CMs as reflected by a decrease in action potential rate, a depolarized maximum diastolic potential and a decrease in slope of phase 4 diastolic depolarization. Furthermore, decreasing the TRPC3 current diminished, while increasing the TRPC3 current augmented the sodium-calcium exchanger (NCX) current in mESC-CMs. Lastly, decrease in TRPC3 current decreased the phosphorylation of ryanodine receptor isoform 2 at Ser2809 and phospholamban at Thr17.
TRPC3 positively regulates diastolic depolarization of spontaneous action potential by increasing LCR and NCX current and therefore is an important determinant in pacemaking of mESC-CMs.
心脏起搏是一种尚未完全被理解的复杂现象。典型瞬时受体电位3型（TRPC3）通道是一种可通透Ca(2+)和Na(+)的阳离子通道。此前发现TRPC3在成年心肌细胞中表达。然而，其在心脏起搏中的作用尚未得到探索。在此，我们使用小鼠胚胎干细胞衍生的心肌细胞（mESC-CMs）来研究TRPC3是否调节自发自律性及其潜在机制。
免疫细胞化学结果显示，TRPC3在mESC-CMs的T小管中表达。全细胞膜片钳记录表明单个mESC-CMs含有TRPC3电流。共聚焦Ca(2+)成像显示，TRPC3特异性阻滞剂Pyr3可降低mESC-CMs的Ca(2+)瞬变和局部Ca(2+)释放（LCR）。来自同一细胞的电流和电压钳联合记录显示，通过Pyr3或TRPC3的显性负性（功能丧失）构建体降低TRPC3电流，会降低mESC-CMs的起搏活性，表现为动作电位频率降低、最大舒张电位去极化以及4期舒张期去极化斜率降低。此外，降低TRPC3电流会减少，而增加TRPC3电流会增强mESC-CMs中的钠钙交换体（NCX）电流。最后，TRPC3电流降低会降低兰尼碱受体2型在Ser2809位点的磷酸化以及受磷蛋白在Thr17位点的磷酸化。
TRPC3通过增加LCR和NCX电流正向调节自发动作电位的舒张期去极化，因此是mESC-CMs起搏的重要决定因素。
