Neural Cell Adhesion Molecule (NCAM-1) is Required for Ventricular Conduction System Development

Cardiac Purkinje cells (PCs) comprise the most distal portion of the ventricular conduction system (VCS) and are essential for synchronous activation of the ventricular myocardium. Contactin-2 (CNTN2), a member of the immunoglobulin superfamily cell adhesion molecules (IgSF-CAMs), was previously identified as a marker of the VCS. Through differential transcriptional profiling we discovered two additional highly enriched IgSF-CAMs in the VCS, NCAM-1 and ALCAM. Immunofluorescence staining showed dynamic expression patterns for each IgSF-CAM during embryonic and early post-natal stages, but ultimately all three proteins became highly enriched in mature PCs. Mice deficient in NCAM-1, but not CNTN2 or ALCAM, exhibited defects in Purkinje cell gene expression and VCS patterning, as well as cardiac conduction disease. Moreover, using ST8sia2 and ST8sia4 knockout mice, we show that inhibition of post-translational modification of NCAM-1 by polysialic acid (PSA) disrupts trafficking of sarcolemmal intercalated disc proteins to Purkinje cell junctional membranes and abnormal expansion of the extracellular space between apposing Purkinje cells. Taken together, our data provide novel insights into the complex developmental biology of the ventricular conduction system. Extracted RNA from Purkinje cells and Ventricular myocytes of male NCAM-1 WT and KO adult mice in triplicate. WT samples used as control心脏浦肯野细胞（Cardiac Purkinje cells, PCs）是心室传导系统（ventricular conduction system, VCS）的最远端部分，对心室心肌的同步激活不可或缺。接触蛋白-2（Contactin-2, CNTN2）作为免疫球蛋白超家族细胞黏附分子（immunoglobulin superfamily cell adhesion molecules, IgSF-CAMs）家族成员，此前已被鉴定为心室传导系统的标志物。本研究通过差异转录组分析，在心室传导系统中发现了另外两种高度富集的免疫球蛋白超家族细胞黏附分子：神经细胞黏附分子-1（NCAM-1）与活化白细胞黏附分子（ALCAM）。免疫荧光染色结果显示，每种免疫球蛋白超家族细胞黏附分子在胚胎期及出生后早期均呈现动态表达模式，但最终三种蛋白在成熟浦肯野细胞中均实现高度富集。NCAM-1缺陷小鼠（而非CNTN2或ALCAM缺陷小鼠）表现出浦肯野细胞基因表达异常、心室传导系统构型缺陷以及心脏传导疾病。此外，本研究利用唾液酸转移酶ST8sia2与ST8sia4基因敲除（knockout, KO）小鼠，证实抑制多唾液酸（polysialic acid, PSA）对NCAM-1的翻译后修饰，会破坏肌膜闰盘蛋白向浦肯野细胞连接膜的转运，并导致相邻浦肯野细胞间细胞外间隙异常扩张。综上，本研究数据为心室传导系统复杂的发育生物学机制提供了全新见解。本研究从雄性NCAM-1野生型（wild type, WT）与基因敲除成年小鼠的浦肯野细胞及心室心肌细胞（Ventricular myocytes）中提取RNA，每组设置三次生物学重复，以野生型样本作为对照。