未折叠蛋白反应在人多能干细胞向心肌细胞分化中的作用和分子机制研究

The capacity of human pluripotent stem cells (hPSCs) to differentiate into cardiomyocytes enables the study of human cardiomyogenesis, thus provides new insight into how the heart develops and how dysregulation of heart development leads to congenital heart disease. To cope with the dramatic cell fate change during differentiation, the differentiating cells must acquire a considerable capacity for new protein synthesis and also the machinery for the proper modification, folding, quality-control, and transport of newly synthesized proteins. However, how the differentiating cells achieves this and relieve the stress remains poorly understood. Our previous study has suggested that endoplasmic reticulum stress and subsequent activation of the unfolded protein response (UPR) may enable the hPSCs to resolve the stress and differentiate into cardiomyocytes. Based on these findings, we will first use pharmacological and genetic approaches to disrupt the three UPR signaling pathways, including IRE1, PERK and ATF6 and investigate the role of UPR in regulating cardiac differentiation and cardiomyocyte function; Secondly, we will use RNA-seq and other biochemistry methods to assay the dynamic changes of UPR-related genes during differentiation, as well as to compare the gene expression profiles of control and UPR-inhibited groups. Based on this knowledge, we will then perform bioinformatic analysis to investigate the possible function of UPR in cardiac differentiation of hPSCs; Thirdly, we will use ChIP-seq, immunoprecipitation, and mass spectrometry to identify the major downstream genes of UPR and their interacting co-factors, with particular emphasis on analyzing their relationship to master cardiac transcriptional factors. By utilizing this data, we will clarify the molecular mechanism of how UPR involves and regulates cardiac differentiation of hPSCs. In summary, this study will extend our understanding of human heart development and facilitate the establishment of new diagnostic and therapeutic strategies for congenital heart disease.

人多能干细胞向心肌细胞（CMs）分化系统为阐明人心脏发育规律、理解先心病发病机理提供了重要模型。这一细胞命运剧变的过程伴随着大量新蛋白质合成、修饰、折叠、分泌及其质量监控。然而，细胞如何调控和应对这一压力的机制仍知之甚少。本课题组前期研究表明内质网应激及其引起的未折叠蛋白反应（UPR）可能参与调控上述过程并在CMs分化中发挥了重要作用。研究拟1）以3条核心UPR信号通路为切入点，通过药理和基因操作手段抑制特定UPR通路，明确其对CMs分化和功能的影响；2）通过RNA-seq和生化技术揭示分化过程中UPR相关基因的动态变化，并比较特定UPR通路抑制前后基因表达谱的差异，探讨UPR在分化中的功能与机制；3）通过ChIP-seq、IP-MS等技术发现UPR下游靶基因和协同作用蛋白，阐明其调节CMs分化和功能的分子机理。本研究将加深我们对人心脏发育规律的理解，为建立新的先心病防治策略提供理论依据。

胚胎干细胞（embryonic stem cells, ESCs）是具有自我更新（self-renewal）能力和分化全能性（pluripotency）的稳定细胞系。人ESCs（human ESCs, hESCs）可通过细胞因子联合小分子化合物诱导，高效地分化为心肌细胞（cardiomyocytes, CMs）。CMs分化发育过程涉及大量的新生蛋白质合成、修饰加工以及转运。.内质网（endoplasmic reticulum, ER）是真核细胞内蛋白质合成、折叠、修饰和转运的主要场所。正确折叠修饰的蛋白质会被ER及时地运送出腔室以发挥相应的功能，未折叠或错误折叠蛋白质过多时会聚集形成不溶的“蛋白多聚体”颗粒（Aggregation），激活未折叠蛋白反应 (unfolded protein response, UPR)。UPR包括ATF6、PERK和IRE1三条信号通路，主要功能是维持细胞内蛋白质组稳态以保证细胞内环境的稳定。.RNA-seq数据分析和药理学抑制实验结果显示：PERK在CMs分化发育早期具有重要作用，在中内胚层阶段抑制PERK活性可显著阻滞心肌分化。另外，PERK敲除细胞分化至Day1时蛋白多聚体显著升高；而在PERK敲除细胞株上对PERK进行阶段性过表达拯救后可显著降低蛋白多聚体，并挽救心肌分化缺陷。.进一步分析RNA-seq数据结合实验验证，发现在CMs分化的中内胚层期转录因子ATF4是PERK的关键下游效应基因。通过ATF4 ChIP-seq分析，我们系统描述了ATF4在心肌分化早期（Day0, Day1和Day2）调节蛋白质稳态维持的分子作用网络和可能的关键靶基因。实验证实在CMs分化早期WARS1是PERK-ATF4通路的主要效应基因。阶段性上调WARS表达可有效挽救PERK敲除导致的蛋白多聚体累积和心肌分化障碍。在H1细胞株中敲降WARS后，细胞分化至Day1时蛋白多聚体累积显著升高，其向CMs分化的潜能亦被显著的抑制。.本研究系统阐明了PERK-ATF4-WARS信号通路在hESCs向CMs分化发育的过程中，通过维持蛋白质稳态保护细胞内环境的稳定，进而促进心肌分化的分子机制。上述研究丰富了我们对CMs分化过程中蛋白质稳态机制的认识，加深了我们对人心脏发育规律的理解，有望为建立新的先心病防治策略提供理论依据，具有重要的生物学和医学研究意义。