窦房结功能障碍和起搏细胞诱导分化机制的研究

The sinus node (SAN) is the dominant pacemaker of the heart. Sinus node dysfunction (SND) is a group of arrhythmias, commonly associated with heart failure and aging. So far, we know very little about pacemaker cell proliferation and turnover, gene expression pattern and epigenetic landscape of pacemaker cells, and mechanisms of SND. We have generated a number of SAN specific mouse lines (HCN4-CreERT2, -nLacZ/GFP), and performed RNA-seq and ChIP-seq analyses on FACS-purified embryonic and neonatal pacemaker cells. In this proposal, we will: 1) perform RNA-seq and epigenetic analyses of SAN cells at different stages of SAN development and in aging and Angiotensin II-induced SND, to uncover molecular mechanisms of SAN formation and function, and SND; 2) perform lineage tracing and BrdU labeling analysis to investigate pacemaker cell proliferation and turnover; 3) generate transgenic mouse lines overexpressing key SAN transcription factors fused with a myc tag to facilitate immunoprecipitation. We will perform ChIP-seq and chromatin conformation capture analysis to identify direct targets of these transcription factors, and remote promoter-enhancer/repressor interactions mediated by these transcription factors, and together with RNA-seq analysis to reveal genetic networks regulated by these transcription factors; 4) study the ability of these transcription factors, when overexpressed alone and in combination, to induce SAN gene program in the working cardiomyocytes or fibroblasts and reprogram these cells into pacemaker cells, and study the underlying mechanisms. Results of proposed studies will give insight into SAN formation, and will enhance our understanding of the pathology of SND and facilitate the development of biological pacemakers.

窦房结是心脏的起搏器官，心衰和老龄化常伴发窦房结功能障碍（SND）。但是对窦房结起搏细胞的更新，基因表达和表观特征及其调控机制，以及SND的机制知之甚少。前期研究中，我们构建了多种窦房结起搏细胞谱系标记小鼠模型，流式分选了起搏细胞并进行了初步的转录组分析。本研究将通过谱系追踪和BrdU标记，研究窦房结起搏细胞的增殖和更新；分离纯化起搏细胞，进行转录和表观组学分析，阐明不同时期以及SND的起搏细胞特异性基因表达谱和表观特征，探讨窦房结的形成和功能的调控机制以及SND的发病机制；构建窦房结关键转录因子-Myc融合蛋白条件过表达的小鼠模型，通过ChIP-seq和染色质相互作用分析，揭示关键转录因子的直接作用靶点和调控网络；在体内和体外，联合过表达上述关键转录因子，诱导产生窦房结起搏细胞和组织，并探讨起搏细胞转分化机制，为生物起搏器的研发奠定基础。

窦房结是心脏的起搏器官，其功能的异常可以引起心衰和老龄化常伴发窦房结功能的障碍（SND）。本研究的目包括：窦房结起搏细胞的更新；基因表达和表观特征及其调控机制，为临床相关疾病的防治奠定理论基础。通过构建了多种窦房结起搏细胞谱系标记小鼠模型，流式分选了起搏细胞并进行了一系列的高通量分析，了解起搏器器官细胞的来源和调控。通过谱系追踪和BrdU标记，阐明了窦房结起搏细胞的增殖和更新；分离纯化起搏细胞，进行转录和表观组学分析，阐明不同时期以及SND的起搏细胞特异性基因表达谱和表观特征，揭示窦房结的形成和功能的调控机制以及SND的发病机制；构建窦房结关键转录因子-Myc融合蛋白条件过表达的小鼠模型，通过ChIP-seq和染色质相互作用分析，揭示关键转录因子的直接作用靶点和调控网络；在体内和体外，联合过表达上述关键转录因子，诱导产生窦房结起搏细胞和组织，并阐明起搏细胞转分化机制，为生物起搏器的研发提供理论基础。