Determining the role of macrophages in the developing cardiac conduction system

项目摘要/摘要 巨噬细胞是协调细胞损伤反应的前哨免疫细胞。 和感染。然而，新出现的证据表明，巨噬细胞具有新的、非典型的 对发育调节、组织动态平衡和再生至关重要的功能。同样，心脏 巨噬细胞在冠状动脉血管构型、瓣膜重塑和心肌梗死中具有重要作用。 调节成人心脏传导。考虑到与以下疾病相关的大量发病率和经济负担 异常心脏发生和心律失常，进一步阐明心脏巨噬细胞在正常心脏的作用 发展和功能是设计新的治疗策略的关键。 在成年哺乳动物心脏中，巨噬细胞与房室结处的心肌细胞电偶联。 通过连接蛋白43，一个缝隙连接蛋白。这些巨噬细胞直接调节结节的电活动。 心肌细胞，从而心脏传导。然而，目前尚不清楚是否需要巨噬细胞(1) 胚胎发育以建立适当的传导或(2)巨噬细胞来源的信号是否调节心脏功能 发育中的心脏。因此，这项提议将解决心脏领域的一个关键知识差距。 两个目标的发展。在目标1中，我将建立胚胎巨噬细胞的电势来调节 胎儿心脏传导。在目标2中，我将确定胚胎巨噬细胞的丢失如何影响传导和 成人心脏健康。在我的方法中，我利用了斑马鱼，这是一种成熟的发展模型，其显著特点是 特点包括快速的体外发育，光学透明，以及对基因操作的高度适应性。 这项建议将在布朗大学进行，这是一所具有广泛代表性的模范学术研究机构 可以使用先进的仪器。在她的赞助人杰西卡·普拉维基博士的指导和指导下，以及 共同赞助人，克里斯·摩尔博士，申请者已经准备好并准备好执行这项奖学金。 我令人兴奋的初步数据揭示了之前未被描述的巨噬细胞在胚胎心脏中的作用 传导和心脏发生。利用巨噬细胞特异性表达转基因斑马鱼的研究 编码的钙指示剂GCaMP6s，我发现种子巨噬细胞有同步的钙爆发 随着室性搏动而及时活动。在光遗传学实验中，我证明了幼虫的心率是可以调节的 通过巨噬细胞特异性表达光门离子来刺激或沉默巨噬细胞的电活动 频道。这提出了一个耐人寻味的问题：发育性心律失常是否可以通过 专门针对巨噬细胞。通过药物诱导的消融或在胚胎中丢失胚胎巨噬细胞 斑马鱼胚胎和成体的巨噬细胞突变体改变了脑室的形成和功能 心肌细胞致密和心脏大体形态。成功完成这项奖学金将澄清和 扩展我们对发育中的心脏中非典型巨噬细胞功能的知识，以及提供 对调节胚胎心脏功能的细胞类型有价值的见解。

PROJECT SUMMARY/ABSTRACT Macrophages are well-characterized as sentinel immune cells that coordinate cellular responses to injury and infection. However, emerging evidence demonstrates that macrophages have novel, non-canonical functions critical for developmental regulation, tissue homeostasis, and regeneration. Likewise, cardiac macrophages have essential functions in patterning the coronary vasculature, valvular remodeling, and in modulating adult heart conduction. Considering the substantial morbidity and economic burden associated with abnormal cardiogenesis and arrhythmias, further elucidating the roles of cardiac macrophages in normal heart development and function is critical for devising novel therapeutic strategies. In adult mammalian hearts, macrophages electrically couple to cardiomyocytes at the atrioventricular node via Connexin 43, a gap junction protein. These macrophages directly modulate electrical activity of nodal cardiomyocytes, and thus cardiac conduction. However, it is not known if macrophages (1) are required during embryogenesis to establish proper conduction or (2) if macrophage-derived signals modulate heart function in the developing heart. Therefore, this proposal will address a critical knowledge gap in the field of cardiac development in two aims. In Aim 1, I will establish the electrical potential of embryonic macrophages to modulate fetal cardiac conduction. In Aim 2, I will determine how loss of embryonic macrophage affects conduction and adult heart health. In my approach, I utilize zebrafish, a well-established developmental model whose salient features include rapid ex vivo development, optical transparency, and high amenability to genetic manipulation. This proposal will be carried out at Brown University, an exemplar academic research institution with extensive access to advanced instrumentation. With the guidance and mentorship of her sponsor, Dr. Jessica Plavicki, and co-sponsor, Dr. Chris Moore, the applicant is prepared and equipped to carryout this fellowship. My exciting preliminary data reveal previously undescribed roles of macrophages in embryonic cardiac conduction and cardiogenesis. Using transgenic zebrafish with macrophage-specific expression of a genetically encoded calcium indicator, GCaMP6s, I found that seeded macrophages have synchronous bursts of calcium activity in time with ventricular beating. In optogenetic experiments, I show that larval heart rate can be modulated by stimulating or silencing macrophage electrical activity via macrophage-specific expression of light-gated ion channels. This poses an intriguing question of whether developmental arrythmias could be corrected by specifically targeting macrophages. Loss of embryonic macrophages, via drug-inducible ablation or in embryonic macrophage mutants, altered ventricular chamber formation and function in embryonic zebrafish, as well as adult cardiomyocyte compaction and gross heart morphology. Successful completion of this fellowship will clarify and expand our knowledge of non-canonical macrophage functions in the developing heart, as well as provide valuable insight into the cell types modulating embryonic heart function.