Exploring Mechanisms of Cardiac Pacemaker Cell Fate Determination

心脏起搏细胞命运决定机制的探讨 项目总结 起搏器(PM)细胞驻留在窦房结(SAN)内，它忠实地引发超过30亿次心跳 在人类的一生中。PM功能障碍通常需要植入装置以防止循环衰竭 心动过缓。尽管心脏质膜功能至关重要，但PM细胞的机制 经历世系承诺仍然鲜为人知。我们研究计划的长期目标是了解 心脏传导系统内细胞命运决定的机制基础。这个项目的总体目标是 建议探索PM细胞谱系承诺的分子策略。有迫切的需要澄清 PM血统致力于了解PM命运的基本生物学基础的分子基础 终止治疗，并告知未来新治疗策略的发展。我的实验室最近报告了Key Hand2通过成纤维细胞转化为诱导PM肌细胞调节PM形成的机制 (IPMS)作为一个示范系统。基于这个初步数据，我们的中心假设是Hand2相互作用 与AP-1合作促进亚型多样性，并协同结合基因组目标以协调PM规范。 为了检验我们的中心假设，我们提出了以下具体目标：1)定义 Hand2确保心脏亚型多样性，2)探索心脏PM谱系承诺和交替的基础。 主动命运限制，以及3)通过组件注释和组合扰动来促进iPM重编程。 在目标1中，我们将使用我们的iPM重新编程系统，结合基因组占有率分析，co-im-im. 免疫沉淀、免疫细胞化学(ICC)、单细胞RNA测序(scRNA-seq)和共聚焦显微镜 复制以定义生化相互作用，扰乱心脏重编程，并表征所产生的细胞命运。 在目标2中，我们将使用scRNA-seq、细胞命运轨迹图、ICC、基因组占位分析和蛋白质- 结合微阵列(PBM)用于分析谱系调节因子、替代命运抑制因子和组合间相互作用 IPM重新编程期间的操作。在目标3中，我们将系统地注释从我们的 初步数据和文献。同时，我们将使用以下工具从头开始构建PM监管网络 我们最近开发的新的组合基因组学方法。顺利完成PRO- 提出的项目将提供有关建立和维护PM单元身份的关键细节。这 贡献将是重大的，因为它将提供对细胞命运是如何完成的详细洞察，以及 确定PM规范的潜在调节器和机制。此外，拟议的研究是不可能的- 创新是因为我们独特的实验方法和多学科研究团队承诺 细胞命运测定的新原理。综上所述，我们预计拟议项目的结果 将提供驱动PM细胞规范并指导未来的基因调控网络的详细知识 开发用于治疗窦房结功能障碍的治疗替代细胞的新策略。

Exploring mechanisms of cardiac pacemaker cell fate determination PROJECT SUMMARY Pacemaker (PM) cells reside within the sinoatrial node (SAN), which faithfully initiates over 3 billion heartbeats during the human lifespan. PM dysfunction often necessitates device implantation to prevent circulatory collapse from bradycardia. Despite the critical importance of cardiac PM function, the mechanisms by which PM cells undergo lineage commitment remain obscure. The long-term goal of our research program is to understand the mechanistic basis for cell fate determination within the cardiac conduction system. The overall objective for this proposal is to explore molecular strategies for PM cell lineage commitment. There is an urgent need to elucidate the molecular underpinnings of PM lineage commitment to understand the fundamental biology of PM fate de- termination and to inform future development of new therapeutic strategies. My lab recently reported on key mechanisms by which Hand2 regulates PM formation using conversion of fibroblasts into induced PM myocytes (iPMs) as a model system. Building upon this preliminary data, our central hypothesis is that Hand2 interacts with AP-1 to promote subtype diversity and cooperatively binds genomic targets to orchestrate PM specification. To test our central hypothesis, we propose the following Specific Aims: 1) Define the mechanisms by which Hand2 ensures cardiac subtype diversity, 2) Explore the basis for cardiac PM lineage commitment and alterna- tive fate restriction, and 3) Boost iPM reprogramming by component annotation and combinatorial perturbation. In Aim 1, we will use our iPM reprogramming system in conjunction with genomic occupancy analysis, co-im- munoprecipitation, immunocytochemistry (ICC), single-cell RNA sequencing (scRNA-seq), and confocal micros- copy to define biochemical interactions, perturb cardiac reprogramming, and characterize the resulting cell fates. In Aim 2, we will use scRNA-seq, cell fate trajectory mapping, ICC, genomic occupancy analysis, and protein- binding microarrays (PBMs) to analyze lineage regulators, alternative fate repressors, and combinatorial inter- actions during iPM reprogramming. In Aim 3, we will systematically annotate candidate factors curated from our preliminary data and the literature. In parallel, we will build PM regulatory networks from the ground-up using novel combinatorial genomic approaches that we have recently developed. Successful completion of the pro- posed project will provide critical details regarding the establishment and maintenance of PM cell identity. This contribution will be significant because it will provide detailed insight into how cell fate is accomplished and identify potential regulators and mechanisms of PM specification. Furthermore, the proposed research is inno- vative because our unique experimental approaches and multi-disciplinary research team promise to uncover new principles in cell fate determination. Taken together, we anticipate that the results of the proposed project will provide detailed knowledge of the gene regulatory networks that drive PM cell specification and guide future development of novel strategies to engineer therapeutic replacement cells for sinus node dysfunction.