Nora Eccles Harrison Cardiovascular Research and Training Institute, University of Utah, Salt Lake City, UT 84112, USA; Department of Cardiovascular Surgery, Xiangya Hospital, Central-South University, Changsha, Hunan 410078, China.
Nora Eccles Harrison Cardiovascular Research and Training Institute, University of Utah, Salt Lake City, UT 84112, USA; Department of Biomedical Engineering, University of Utah, Salt Lake City, UT 84112, USA.
J Mol Cell Cardiol. 2020 Feb;139:113-123. doi: 10.1016/j.yjmcc.2020.01.008. Epub 2020 Jan 23.
Transient receptor potential canonical 1 (TRPC1) protein is abundantly expressed in cardiomyocytes. While TRPC1 is supposed to be critically involved in cardiac hypertrophy, its physiological role in cardiomyocytes is poorly understood. We investigated the subcellular location of TRPC1 and its contribution to Ca signaling in mammalian ventricular myocytes. Immunolabeling, three-dimensional scanning confocal microscopy and quantitative colocalization analysis revealed an abundant intracellular location of TRPC1 in neonatal rat ventricular myocytes (NRVMs) and adult rabbit ventricular myocytes. TRPC1 was colocalized with intracellular proteins including sarco/endoplasmic reticulum Ca ATPase 2 in the sarcoplasmic reticulum (SR). Colocalization with wheat germ agglutinin, which labels the glycocalyx and thus marks the sarcolemma including the transverse tubular system, was low. Super-resolution and immunoelectron microscopy supported the intracellular location of TRPC1. We investigated Ca signaling in NRVMs after adenoviral TRPC1 overexpression or silencing. In NRVMs bathed in Na and Ca free solution, TRPC1 overexpression and silencing was associated with a decreased and increased SR Ca content, respectively. In isolated rabbit cardiomyocytes bathed in Na and Ca free solution, we found an increased decay of the cytosolic Ca concentration [Ca] and increased SR Ca content in the presence of the TRPC channel blocker SKF-96365. In a computational model of rabbit ventricular myocytes at physiological pacing rates, Ca leak through SR TRPC channels increased the systolic and diastolic [Ca] with only minor effects on the action potential and SR Ca content. Our studies suggest that TRPC1 channels are localized in the SR, and not present in the sarcolemma of ventricular myocytes. The studies provide evidence for a role of TRPC1 as a contributor to SR Ca leak in cardiomyocytes, which was previously explained by ryanodine receptors only. We propose that the findings will guide us to an understanding of TRPC1 channels as modulators of [Ca] and contractility in cardiomyocytes.
瞬时受体电位经典型 1（TRPC1）蛋白在心肌细胞中大量表达。虽然 TRPC1 被认为在心脏肥大中起着关键作用，但它在心肌细胞中的生理作用还知之甚少。我们研究了 TRPC1 的亚细胞定位及其在哺乳动物心室肌细胞中的钙信号转导中的作用。免疫标记、三维扫描共聚焦显微镜和定量共定位分析显示，TRPC1 在新生大鼠心室肌细胞（NRVMs）和成年兔心室肌细胞中存在丰富的细胞内定位。TRPC1 与包括肌浆网（SR）中的肌浆网 Ca-ATP 酶 2 在内的细胞内蛋白共定位。与标记糖萼并因此标记包括横管系统在内的肌膜的麦胚凝集素的共定位较低。超分辨率和免疫电子显微镜支持 TRPC1 的细胞内定位。我们研究了 NRVMs 中腺病毒 TRPC1 过表达或沉默后的钙信号转导。在浴液中含有 Na 和无 Ca 的 NRVMs 中，TRPC1 过表达和沉默分别与 SR Ca 含量减少和增加有关。在浴液中含有 Na 和无 Ca 的分离的兔心肌细胞中，我们发现 TRPC 通道阻断剂 SKF-96365 的存在会增加胞质 Ca 浓度[Ca]的衰减和 SR Ca 含量。在生理起搏率下的兔心室肌细胞的计算模型中，SR TRPC 通道的 Ca 泄漏增加了收缩期和舒张期[Ca]，对动作电位和 SR Ca 含量的影响很小。我们的研究表明，TRPC1 通道定位于 SR，而不存在于心室肌细胞的肌膜中。这些研究为 TRPC1 作为心肌细胞中 SR Ca 泄漏的贡献者提供了证据，此前这一现象仅归因于兰尼碱受体。我们提出，这些发现将指导我们理解 TRPC1 通道作为心肌细胞中[Ca]和收缩性的调节剂。
