Exploring Mechanisms of Cardiac Pacemaker Cell Fate Determination

心脏起搏细胞命运决定机制的探讨 项目摘要 起搏（PM）细胞位于窦房结（SAN）内，忠实地启动超过30亿次心跳 在人类的一生中。PM功能障碍通常需要植入器械以防止循环衰竭 因为心动过缓尽管心脏PM功能至关重要，但PM细胞 经历血统承诺仍然模糊不清。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。总体目标是 目的是探索PM细胞谱系定型的分子策略。有迫切需要澄清 PM谱系的分子基础致力于了解PM命运的基本生物学， 终止并告知新治疗策略的未来发展。我的实验室最近报告说 Hand 2通过将成纤维细胞转化为诱导的PM肌细胞来调节PM形成的机制 （iPMs）作为一个模型系统。基于这些初步数据，我们的中心假设是，手2相互作用， 与AP-1一起促进亚型多样性，并协同结合基因组靶点以协调PM特异性。 为了检验我们的中心假设，我们提出了以下具体目标：1）定义的机制， Hand 2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， 3）通过组分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重编程系统与基因组占用分析，co-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由此产生的细胞命运。 在目标2中，我们将使用scRNA-seq、细胞命运轨迹作图、ICC、基因组占有率分析和蛋白质- 结合微阵列（PBM）分析谱系调节因子，替代命运阻遏物，和组合的inter- 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。与此同时，我们将从零开始建立PM监管网络， 我们最近开发的新的组合基因组方法。圆满完成亲- 提出的项目将提供关于建立和维护PM小区身份的关键细节。这 贡献将是重大的，因为它将提供详细的洞察细胞命运是如何完成的， 确定PM规范的潜在监管机构和机制。此外，这项研究是创新的- 因为我们独特的实验方法和多学科的研究团队承诺揭示 决定细胞命运的新原则。综上所述，我们预计拟议项目的结果 将提供详细的基因调控网络的知识，推动PM细胞的规范和指导未来 开发新的策略来设计用于窦房结功能障碍的治疗性替代细胞。

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.