Ahmad Azmi A, Streiff Molly E, Hunter Chris, Sachse Frank B
Nora Eccles Harrison Cardiovascular Research and Training Institute, University of Utah, Salt Lake City, UT, United States.
Department of Biomedical Engineering, University of Utah, Salt Lake City, UT, United States.
Front Physiol. 2020 Feb 14;11:44. doi: 10.3389/fphys.2020.00044. eCollection 2020.
Transient receptor potential canonical 6 (TRPC6) channels are non-selective cation channels that are thought to underlie mechano-modulation of calcium signaling in cardiomyocytes. TRPC6 channels are involved in development of cardiac hypertrophy and related calcineurin-nuclear factor of activated T cells (NFAT) signaling. However, the exact location and roles of TRPC6 channels remain ill-defined in cardiomyocytes. We used an expression system based on neonatal rat ventricular myocytes (NRVMs) to investigate the location of TRPC6 channels and their role in calcium signaling. NRVMs isolated from 1- to 2-day-old animals were cultured and infected with an adenoviral vector to express enhanced-green fluorescent protein (eGFP) or TRPC6-eGFP. After 3 days, NRVMs were fixed, immunolabeled, and imaged with confocal and super-resolution microscopy to determine TRPC6 localization. Cytosolic calcium transients at 0.5 and 1 Hz pacing rates were recorded in NRVMs using indo-1, a ratio-metric calcium dye. Confocal and super-resolution microscopy suggested that TRPC6-eGFP localized to the sarcolemma. NRVMs infected with TRPC6-eGFP exhibited higher diastolic and systolic cytosolic calcium concentration as well as increased sarcoplasmic reticulum (SR) calcium load compared to eGFP infected cells. We applied a computer model comprising sarcolemmal TRPC6 current to explain our experimental findings. Altogether, our studies indicate that TRPC6 channels play a role in sarcolemmal and intracellular calcium signaling in cardiomyocytes. Our findings support the hypothesis that upregulation or activation of TRPC6 channels, e.g., in disease, leads to sustained elevation of the cytosolic calcium concentration, which is thought to activate calcineurin-NFAT signaling and cardiac hypertrophic remodeling. Also, our findings support the hypothesis that mechanosensitivity of TRPC6 channels modulates cytosolic calcium transients and SR calcium load.
瞬时受体电位经典型6（TRPC6）通道是非选择性阳离子通道，被认为是心肌细胞中钙信号机械调节的基础。TRPC6通道参与心脏肥大的发展以及相关的钙调神经磷酸酶-活化T细胞核因子（NFAT）信号传导。然而，TRPC6通道在心肌细胞中的具体位置和作用仍不明确。我们使用基于新生大鼠心室肌细胞（NRVMs）的表达系统来研究TRPC6通道的位置及其在钙信号传导中的作用。从1至2日龄动物分离的NRVMs进行培养，并感染腺病毒载体以表达增强型绿色荧光蛋白（eGFP）或TRPC6-eGFP。3天后，将NRVMs固定、免疫标记，并用共聚焦和超分辨率显微镜成像以确定TRPC6的定位。使用比率型钙染料indo-1在NRVMs中记录0.5和1 Hz起搏频率下的胞质钙瞬变。共聚焦和超分辨率显微镜显示TRPC6-eGFP定位于肌膜。与感染eGFP的细胞相比，感染TRPC6-eGFP的NRVMs表现出更高的舒张期和收缩期胞质钙浓度以及增加的肌浆网（SR）钙负荷。我们应用了一个包含肌膜TRPC6电流的计算机模型来解释我们的实验结果。总之，我们的研究表明TRPC6通道在心肌细胞的肌膜和细胞内钙信号传导中起作用。我们的研究结果支持这样的假设，即TRPC6通道的上调或激活，例如在疾病中，会导致胞质钙浓度持续升高，这被认为会激活钙调神经磷酸酶-NFAT信号传导和心脏肥大重塑。此外，我们的研究结果支持这样的假设，即TRPC6通道的机械敏感性调节胞质钙瞬变和SR钙负荷。
