用人类多能干细胞衍生心肌细胞研究芪苈强心胶囊对胞质/胞核钙信号的作用

Cardiovascular diseases are the leading cause of death globally, and heart failure represents the severe stage of various cardiovascular diseases with similar risk of numerous types of cancer. Qili qiangxin capsules (QL) are a traditional Chinese medicine extract containing a number of types of herbs. QL has been approved for heart failure treatment in China, and its clinical therapeutic effect has been verified worldwide recently. However, how QL affect cardiac function and gene transcription via cytosolic and nuclear calcium signaling, respectively, is largely unknown. Moreover, published mechanistic studies of QL are all based on animal model or primary animal cardiomyocytes. Our preliminary experiments have established methods for cytosolic and nuclear calcium measurement in human pluripotent stem cell-derived cardiomyocytes. We hypothesize QL can enhance cardiac contractility and regulate cardiac gene transcription via calcium signalling in cytosol and nucleus, respectively. We propose to study the mechanistic role of QL on calcium signaling and gene transcription using techniques such as confocal microscope-based calcium imaging and microarray. Moreover, induced pluripotent stem cells with different genetic backgrounds will also be employed to examine the possible effect of QL on genetic heart diseases in vitro. In sum, this study will illustrate the calcium signaling mechanisms of QL in heart failure treatment, and provide new strategy for traditional Chinese medicine research.

心血管疾病是全球头号死因。心力衰竭是各种心血管疾病的严重阶段，其5年存活率与恶性肿瘤相当。芪苈强心胶囊抗心衰疗效已被多中心临床试验证实，但该药对代表心肌收缩功能的胞质钙信号及调控心肌基因转录的胞核钙信号的作用机制尚不明确。此外，现有芪苈强心胶囊的机制研究均基于动物模型或细胞。本课题预实验已经建立在人类多能干细胞衍生心肌细胞中测定胞质/胞核钙信号的实验方法，可用于研究药物的作用机制。我们认为芪苈强心胶囊可通过胞质/胞核钙信号增强心肌收缩功能/调控心肌基因转录。本课题拟采用激光共聚焦显微镜钙成像、基因表达谱芯片等实验技术，阐明芪苈强心胶囊对人源心肌细胞钙信号和基因转录的作用及其机制；利用有心血管疾病遗传背景的诱导多能干细胞衍生心肌细胞，明确芪苈强心胶囊通过钙信号通路对相应疾病模型的影响。本课题用干细胞技术为中药研究提供新思路和新方法，对于芪苈强心胶囊治疗心衰的机制研究及其科学内涵具有重要意义。

心血管疾病是全球首要死因，心力衰竭是各种心血管疾病的严重阶段。芪苈强心胶囊抗心衰的临床疗效显著，但其对代表心肌收缩功能的胞质钙信号及调控心肌基因转录的胞核钙信号的作用机制尚不明确。鉴于此，课题组通过活细胞钙成像技术，系统地研究了芪苈强心胶囊对人类多能干细胞衍生心肌细胞胞质/胞核钙信号的作用，结果显示，芪苈强心胶囊可显著降低自发钙瞬变频率，但不影响电刺激诱导下的钙瞬变振幅。激光共聚焦显微镜线扫描模式下的钙火花测定结果表明，芪苈强心胶囊增加了胞质和胞核中的钙火花频率，但肌质网钙库的钙含量未受到显著影响。在有长QT综合征遗传背景的人源心肌细胞中，芪苈强心胶囊缓解了连续早期后除极导致的钙震荡。课题组通过慢病毒介导的小清蛋白细胞核定位表达、转录组学、实时荧光定量PCR和转录因子预测，明确了芪苈强心胶囊可调控心肌细胞基因转录，并阐明了胞核钙信号在其中的作用。此外，课题组通过网络药理学方法探究了芪苈强心胶囊主要药对的作用靶点，构建了“中药-成分-靶点-通路-疾病”网络，揭示其可通过多条信号通路发挥作用。同时，课题组建立了基于高内涵成像和心肌钙信号测定的药物筛选平台，可对芪苈强心胶囊活性成分进行高通量评价。因此，本课题解析了芪苈强心胶囊对人源心肌细胞钙信号的影响，并进一步探讨其作用机制，为芪苈强心胶囊的临床应用提供科学依据。