Quantifying function in the early embryonic heart.

Congenital heart defects arise during the early stages of development, and studies have linked abnormal blood flow and irregular cardiac function to improper cardiac morphogenesis. The embryonic zebrafish offers superb optical access for live imaging of heart development. Here, we build upon previously used techniques to develop a methodology for quantifying cardiac function in the embryonic zebrafish model. Imaging was performed using bright field microscopy at 1500 frames/s at 0.76 µm/pixel. Heart function was manipulated in a wild-type zebrafish at ∼55 h post fertilization (hpf). Blood velocity and luminal diameter were measured at the atrial inlet and atrioventricular junction (AVJ) by analyzing spatiotemporal plots. Control volume analysis was used to estimate the flow rate waveform, retrograde fractions, stroke volume, and cardiac output. The diameter and flow waveforms at the inlet and AVJ are highly repeatable between heart beats. We have developed a methodology for quantifying overall heart function, which can be applied to early stages of zebrafish development.

The Transitional Cardiac Pumping Mechanics in the Embryonic Heart.

Several studies have linked abnormal blood flow dynamics to the formation of congenital heart defects during the early stages of development. The objective of this study is to document the transition of pumping mechanics from the early tube stage to the late looping stage of the embryonic heart. The optically transparent zebrafish embryonic heart was utilized as the in vivo model and was studied using standard bright field microscopy at three relevant stages within the transitional period: (1) tube stage at 30 hours post-fertilization (hpf); (2) early cardiac looping stage at 36 hpf; and (3) late cardiac looping stage at 48 hpf. High-speed videos were collected at 1000 fps at a spatial resolution of 1.1 µm/pixel at each of these stages and were post-processed to yield blood velocity patterns as well as wall kinematics. Results show that several relevant trends exist. Morphological trends from tube through late looping include: (a) ballooning of the chambers, (b) increasing constriction at the atrioventricular junction (AVJ), and (c) repositioning of the ventricle toward the side of the atrium. Blood flow trends include: (a) higher blood velocities, (b) increased AVJ regurgitation, and (c) larger percentages of blood from the upper atrium expelled backward toward the atrial inlet. Pumping mechanics trends include: (a) increasing contraction wave delay at the AVJ, (b) the AVJ begins acting as a rudimentary valve, (c) decreasing chamber constriction during maximum contraction, and (d) a transition in ventricular kinematics from a pronounced propagating wave to an independent, full-chamber contraction. The above results provide new insight into the transitional pumping mechanics from peristalsis-like pumping to a displacement pumping mechanism.