School of Life Sciences, The Chinese University of Hong Kong, Hong Kong, China.
School of Biomedical Sciences, The Chinese University of Hong Kong, Hong Kong, China.
J Cell Physiol. 2021 Oct;236(10):6806-6823. doi: 10.1002/jcp.30369. Epub 2021 Mar 30.
Calcium controls the excitation-contraction coupling in cardiomyocytes. Embryonic stem cell-derived cardiomyocytes (ESC-CMs) are an important cardiomyocyte source for regenerative medicine and drug screening. Transient receptor potential vanilloid 1 (TRPV1) channels are nonselective cation channels that permeate sodium and calcium. This study aimed to investigate whether TRPV1 channels regulate the electrophysiological characteristics of ESC-CMs. If yes, what is the mechanism behind? By immunostaining and subcellular fractionation, followed by western blotting, TRPV1 was found to locate intracellularly. The staining pattern of TRPV1 was found to largely overlap with that of the sarco/endoplasmic reticulum Ca -ATPase, the sarcoplasmic reticulum (SR) marker. By electrophysiology and calcium imaging, pharmacological blocker of TRPV1 and the molecular tool TRPV1β (which could functionally knockdown TRPV1) were found to decrease the rate and diastolic depolarization slope of spontaneous action potentials, and the amplitude and frequency of global calcium transients. By calcium imaging, in the absence of external calcium, TRPV1-specific opener increased intracellular calcium; this increase was abolished by preincubation with caffeine, which could deplete SR calcium store. The results suggest that TRPV1 controls calcium release from the SR. By electrophysiology, TRPV1 blockade and functional knockdown of TRPV1 decreased the Na /Ca2 exchanger (NCX) currents from both the forward and reverse modes, suggesting that sodium and calcium through TRPV1 stimulate the NCX activity. Our novel findings suggest that TRPV1 activity is important for regulating the spontaneous activity of ESC-CMs and reveal a novel interplay between TRPV1 and NCX in regulating the physiological functions of ESC-CMs.
钙控制心肌细胞的兴奋-收缩偶联。胚胎干细胞衍生的心肌细胞（ESC-CM）是再生医学和药物筛选的重要心肌细胞来源。瞬时受体电位香草酸 1 型（TRPV1）通道是非选择性阳离子通道，可渗透钠和钙。本研究旨在探讨 TRPV1 通道是否调节 ESC-CM 的电生理特性。如果是，其背后的机制是什么？通过免疫染色和亚细胞分级分离，再进行 Western blot 分析，发现 TRPV1 位于细胞内。TRPV1 的染色模式与肌浆/内质网 Ca -ATP 酶（肌质网（SR）标志物）的染色模式大部分重叠。通过电生理学和钙成像，发现 TRPV1 的药理学阻断剂和分子工具 TRPV1β（可功能性敲低 TRPV1）降低了自发动作电位的频率和舒张去极化斜率，以及整体钙瞬变的幅度和频率。通过钙成像，在没有外部钙的情况下，TRPV1 特异性激动剂增加细胞内钙；这种增加被预先用咖啡因孵育所消除，咖啡因可耗尽 SR 钙库。结果表明，TRPV1 控制着 SR 中的钙释放。通过电生理学，TRPV1 阻断和 TRPV1 的功能敲低降低了正向和反向模式下的钠/钙交换器（NCX）电流，表明钠和钙通过 TRPV1 刺激 NCX 活性。我们的新发现表明，TRPV1 活性对于调节 ESC-CM 的自发活动很重要，并揭示了 TRPV1 和 NCX 之间在调节 ESC-CM 的生理功能方面的新相互作用。
