Peli1调控心肌细胞外泌体microRNA在压力过负荷所致心肌纤维化中的作用机制研究

Development of cardiac fibrosis and heart failure is controlled by the activity of communication between cardiomyocytes and fibroblasts in the heart. However, the underlying mechanisms of cardiomyocyte-fibroblast communication are not yet fully understood. We have previously reported that E3 ligase protein Peli1 plays a critical role in cardiac remodeling. Our preliminary data showed that exosomes from mechanical stretch treated cardiomyocytes may induce cardiac fibroblasts activation through modulating the content of exosomes, such as microRNA. And fortunately, we identified miR-468-3p and miR-494-3p as suitable candidate for their probable effect on post-transcriptional regulation of BAMBI gene according to a series of experiments and documents. We hypothesized that Peli1 regulated cardiac fibrosis induced by pressure overload via cardiomyocyte exosomal–derived miR-468-3p and miR-494-3p. To critically evaluate our hypotheses, a series of experiments both in vivo and in vitro have been scheduled, such as Peli1 cardiomyocytes conditional or general gene knockout and miR-468-3p and miR-494-3p mimic or inhibitor transfection et, al. These studies will provide an understanding of the mechanisms of myocyte-fibroblast communication and their effects on cardiac fibrosis induced by pressure overload. It may also be possible to apply this knowledge in a practical fashion to identify new and novel therapeutic approaches to prevent or manage cardiac fibrosis and heart failure.

心肌细胞与心肌成纤维细胞之间的信息传递在心肌纤维化和心衰的发展进程中具有重要作用，但有关心肌细胞旁分泌及其与心肌成纤维细胞之间信息传递的分子机制，尚未阐明。我们的研究表明Peli1在心肌重塑的发生机制中具有重要作用，预实验发现机械过度牵张心肌细胞分泌的外泌体可通过旁分泌促进心肌成纤维细胞活化，而且 Peli1可以调控心肌细胞外泌体MicroRNA，并筛选出miR-468-3p及miR-494-3p可能为主要靶标。本项目拟在此基础上，从整体动物与体外细胞两个方面，采用Peli1心肌条件性敲除小鼠、AAV9-miR-468-3p/miR-494-3p 拟似物或抑制剂心肌特异性转染、心肌细胞外泌体处理心肌成纤维细胞等方法，明确Peli1是否可通过影响心肌细胞外泌体MicroRNA调控心肌成纤维细胞活化及压力过负荷所致的心肌纤维化，并阐释其调控的分子机制，为探究心肌纤维化及心衰的防治提供新的线索。

心肌细胞与心肌成纤维细胞之间的信息传递在心肌纤维化和心衰的发展进程中具有重要作用，但有关心肌细胞旁分泌及其与心肌成纤维细胞之间信息传递的分子机制，尚未阐明。我们提出假说：Peli1可能通过影响心肌细胞外泌体microRNA，调控心肌成纤维细胞活化和压力过负荷所致心肌纤维化的发展进程，其主要机制可能与心肌细胞Peli1通过影响外泌体miR-494-3p旁分泌调控心肌成纤维细胞活化密切相关。本项目在动物与细胞两个水平，进行了以下研究：①明确心肌细胞Peli1及miR-494-3p在压力过负荷所致心肌纤维化中的作用：发现心肌细胞特异性敲除Peli1可显著改善压力过负荷所致的心肌肥大和心肌纤维化；rAAV9介导的特异性抑制心肌组织miR-494-3p可以改善压力过负荷所致的心肌纤维化和心功能障碍；②阐明Peli1调控心肌细胞外泌体及其携带的miR-494-3p对心肌成纤维细胞活化的影响：发现机械过度牵张可显著增加体外培养心肌细胞中Peli1表达并上调其分泌的外泌体中miR-494-3p表达，而且心肌细胞外泌体可被心肌成纤维细胞吞噬，并诱导心肌成纤维细胞向肌成纤维细胞分化。心肌细胞Peli1敲除可下调miR-494-3p表达并显著抑制心肌细胞外泌体诱导的心肌成纤维细胞活化，而心肌成纤维细胞中过表达miR-494-3p mimics则可显著诱导心肌成纤维细胞活化；③揭示心肌细胞外泌体miR-494-3p调控心肌成纤维细胞活化的分子机制：心肌组织特异性抑制miR-494-3p可以显著逆转压力过负荷诱导的左室心肌组织PTEN蛋白表达下调及AKT、Smad2/3和ERK磷酸化水平的上调；机械过度牵张诱导的心肌细胞外泌体miR-494-3p亦可通过旁分泌进入心肌成纤维细胞调控PTEN蛋白表达及AKT、Smad2/3和ERK信号通路的激活。综上，证实Peli1可通过影响心肌细胞外泌体miR-494-3p调控心肌成纤维细胞活化及压力过负荷所致的心肌纤维化，其机制与Peli1通过调节miR-494-3p 参与心肌成纤维细胞PTEN、AKT/Smad2/3/ERK信号通路的调控密切相关。上述研究可为临床寻找基于外泌体-miRNA治疗心肌纤维化和心力衰竭的潜在靶点提供重要的理论依据。