天然免疫受体TLR3调控心脏生物电活动的研究

Toll-like receptors (TLRs) are critical innate immunoreceptors involved in host defense against invading microbes. Their non-immune functions remain unclear. In the preliminary study, we observed that TLR3 is highly expressed in mouse ventricular tissue. Its subcellular localization in resting cardiomyocytes mainly locates at the endoplasmic reticulum (ER), in accordance with previous observations in immune cells such as dentritic cells. Notably, we observed that in TLR3-knockout (TLR3-KO) mice, the corrected QT interval (QTc) of ECG and the action potential duration of ventricular myocytes both prolongs. Furthermore, we observed that the Ito current decreases in ventricular myocytes isolated from TLR3-KO mice, whereas all the parameters for activation, inactivation and recovery kinetics of Ito remain unchanged. Given that ER is the first organelle in which newly synthesized proteins are post-translationally processed and further forward-trafficked to plasma membrane through the Golgi apparatus, we hypothesize that TLR3 in cardiomyocyte ER may directly regulate the post-translational processing and/or forward-trafficking of Kv4 (potassium voltage-gated channel subfamily D), the key pore-forming protein underlying Ito current; such regulation subsequently affects the surface expression of Kv4, and therefore, Ito, action potential and QTc interval will be affected. This project intends to systematically study the effects of TLR3 on the whole intracellular journey of Kv4 proteins, including Kv4 gene expression, protein translation, post-translational processing, forward-trafficking and degradation; and study whether TLR3 acts on Kv4 directly, or indirectly through the classic immune signaling pathways. This project will uncover the regulation of TLR3 on plasma membrane expression of Kv4, and clarify its mechanism. On this basis, this project will analyze TLR3 gene of patients with long or short QT syndrome, identify its variants, and verify their function using biological experiments, to reveal the clinical significance of TLR3 gene defects. The present project is expected to discover the regulation of an innate immunoreceptor on cardiac electrical activity and reveal its novel mechanism independent of immune pathways. This will be a breakthrough to the traditional knowledge, bringing novel understanding on the function of innate immunoreceptors.

Toll样受体（TLR）是重要的天然免疫受体，免疫之外的功能并不清楚。预实验发现：小鼠心室肌高表达TLR3；TLR3基因敲除后，心电图QTc间期延长、心室肌细胞动作电位时程延长、Ito电流减少。鉴于TLR3主要分布于内质网，而后者是通道蛋白翻译后加工和转运上膜所经过的第一场所，我们推测：TLR3很可能直接调控负载Ito的钾通道蛋白Kv4，影响其膜表达，进而影响心肌电活动。本项目拟应用电生理学、细胞生物学和影像学等技术，研究TLR3如何影响Kv4蛋白翻译、加工、转运上膜及降解，并研究其效应是否源自直接作用、而非依赖于经典免疫通路，以此阐明TLR3调控Kv4膜表达的功能、作用环节及机制。在此基础上，分析长QT和短QT综合症患者TLR3基因，识别变异体，并进行功能鉴定，揭示TLR3基因缺陷的临床意义。本项目将首次揭示天然免疫受体调控心脏生物电活动功能及其机制，突破传统认知，赋予其新的功能意义。

Toll样受体（TLR）是一类重要的免疫受体，在免疫细胞大量表达，介导天然免疫反应，但其免疫之外的功能尚不清楚。我们前期在预实验中发现：小鼠心室肌高表达TLR3；TLR3基因敲除后，心电图QTc间期延长、心室肌细胞动作电位时程延长、Ito电流减少；提示TLR3参与调控心脏的电活动。在此基础上，本项目系统研究了TLR3如何影响心肌细胞的电活动及其机制。研究结果表明：TLR3能够调控电压依赖性钾通道Kv4蛋白的膜表达（Kv4蛋白是形成心室肌细胞Ito电流的核心通道蛋白），而对钙通道蛋白、钠通道蛋白和Kv4通道辅助性亚基蛋白则无显著调控作用；当TLR3缺陷时（敲低或敲除），Kv4蛋白的膜表达量下降，因而造成Ito电流减小、动作电位时程延长、心电图QTc间期延长；干预TLR3所关联的免疫信号通路，对上述现象无显著影响，提示TLR3对Kv4蛋白膜表达的调控作用不依赖于经典免疫通路。为了研究其机制，我们追踪了Kv4基因转录、翻译、翻译后加工、转运上膜以及蛋白降解的全过程，发现TLR3和Kv4共定位于内质网，TLR3缺陷可导致Kv4蛋白滞留在内质网内，不能被转运上膜，继而通过泛素-蛋白酶体途径被降解，因此出现Kv4膜表达量下降（上述研究结果已在SCI期刊发表）。再进一步，我们通过免疫共沉淀和GST pull-down等实验发现，TLR3的LRR结构域能够直接与Kv4蛋白发生互作，因而能够调控Kv4转运上膜。此外，本项目针对临床上长QT和短QT综合症患者的TLR3基因序列进行了研究，目前已初步识别了其中的变异体，正在进行功能鉴定，以期揭示TLR3基因缺陷的临床意义。综上，本项目首次揭示了TLR受体能够调控心脏的生物电活动，突破了传统认知，赋予了TLR受体新的功能意义。