5 D impulse mapping in the embryonic heart

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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).