5 D impulse mapping in the embryonic heart

项目摘要/摘要 先天性心脏病是最常见和最具破坏性的出生缺陷之一。异常 心脏传导的发展往往与冠心病的病因有关。调查……的发展 心脏传导对于我们理解这些情况背后的机制是至关重要的。 心脏传导系统(CCS)的发展是一个复杂的转变 通过分子信号、结构特性和生理功能之间的相互作用，包括 血流动力学和电生理学。虽然对分子网络的理解已经取得了快速的进展 近几十年来，追踪有助于发育和分化的生理因素的工具 CCS仍然存在缺陷。填补这一空白非常重要。连接分子和功能信息是 了解传导系统的病理并指导潜在的治疗策略。 心脏跨膜电压或细胞内钙动力学的光学标测(OM) 电压或钙敏感的荧光染料是研究心脏电生理学的有力工具，并已 被改编成早期胚胎心脏的成像取得了巨大的成功。然而，目前OM仅限于成像 摘除胚胎心脏，用兴奋-收缩-解偶联药物静止。拆除易碎的肾小管 心脏从胚胎的结构和血流动力学负荷中分离出来，并在染料和药物中孵育会产生干扰 与正常生理和不允许纵向研究超过发展阶段。 这个项目的目标是开发技术，使全面的，纵向成像 近低温条件下培养的早期禽类胚胎活心脏跳动的电生理功能 生理条件。我们将以1-2天的活跃形态发生为目标 均质传导速度到异质传导速度需要三项关键技术开发来实现 做到这一点。(A)需要荧光电压和钙指示器的荧光、容量、快速成像，以 成像完整的活胚胎，并捕捉传导动力学(目标1)。(B)需要运动校正 在不使用兴奋-收缩-解偶联药物的情况下，实现心脏跳动的传导标测(目的 1)。(C)需要在心肌细胞中表达钙和电压报告的胚胎鹌鹑模型 实现电生理的活体和纵向成像(目标2)。 建议的技术将能够在两个时间尺度上同时进行3D传导映射， 心跳和心脏发育(5D脉冲标测)。再加上定量的3D鱼类，这将允许 传导数据和基因/蛋白质表达之间的点对点3D配准，目前不是 可用，使研究更好地了解传导功能、功能障碍和 发展(目标3)。

Project Summary/Abstract Congenital heart defects (CHDs) are among the most common and devastating of birth defects. Abnormal development of cardiac conduction often associates with CHD etiology. Investigating the development of cardiac conduction is essential for us to understand the mechanisms behind such conditions. Development of the cardiac conduction system (CCS) is a complicated transformation that comes about through interplay between molecular signaling, structural properties, and physiological function, including hemodynamics and electrophysiology. While an understanding of the molecular networks has progressed rapidly in recent decades, tools to follow the physiological factors contributing to development and differentiation of the CCS remain deficient. This gap is important to fill. Connecting molecular and functional information is key for understanding pathologies of the conduction system and for guiding potential therapeutic strategies. Optical mapping (OM) of transmembrane voltage or intracellular calcium dynamics in the heart using voltage- or calcium-sensitive fluorescent dyes is a powerful tool for studying cardiac electrophysiology, and has been adapted for imaging early embryonic hearts with great success. However currently, OM is limited to imaging excised embryonic hearts which are stilled with excitation-contraction-uncoupler drugs. Removing fragile tubular hearts from the structure and hemodynamic load of the embryo, and incubating them in dyes and drugs interferes with the normal physiology and does not allow longitudinal study over stages of development. The goal of this project is to develop technology to enable comprehensive, longitudinal imaging of the electrophysiological function of the living, beating heart of the early avian embryo, cultured under near- physiological conditions. We will target 1-2 days of active morphogenesis through the transition from homogeneous to heterogeneous conduction velocity Three key technology developments are needed to achieve this. (A) Episcopic, volumetric, fast imaging of fluorescent voltage and calcium indicators is needed to image the intact, living embryo, and to capture conduction dynamics (Aim 1). (B) Motion correction is needed to enable conduction mapping of the beating heart, without using excitation-contraction-uncoupling drugs (Aim 1). (C) Embryonic quail models with calcium and voltage reporters expressed in cardiomyocytes are needed to enable in vivo and longitudinal imaging of electrophysiology (Aim 2). The proposed technology will enable simultaneous 3D conduction mapping over two time scales, the heartbeat, and heart development (5D impulse mapping). Coupled with quantitative 3D FISH, this will allow point-to-point 3D registration between conduction data and gene/protein expression, which is not currently available, enabling studies to better understand mechanisms of conduction function, dysfunction and development (Aim 3).