Characterization of embryonic cardiac pacemaker and atrioventricular conduction physiology in Xenopus laevis using noninvasive imaging

Abstract

Congenital heart defects often include altered conduction as well as morphological changes. Model organisms, like the frog Xenopus laevis, offer practical advantages for the study of congenital heart disease. X. laevis embryos are easily obtained free living, and the developing heart is readily visualized. Functional and morphological evidence for a conduction system is available for adult frog hearts, but information on the normal properties of embryonic heart contraction is lacking, especially in intact animals. With the use of fine glass microelectrodes, we were able to obtain cardiac recordings and make standard electrophysiological measurements in 1-wk-old embryos (stage 46). In addition, a system using digital analysis of video images was adapted for measurement of the standard cardiac intervals and compared with invasive measurements. Video images were obtained of the heart in live, pharmacologically paralyzed, stage 46 X. laevis embryos. Normal values for the timing of the cardiac cycle were established. Intervals determined by video analysis (n = 53), including the atrial and ventricular cycle lengths (473 +/- 10 ms and 464 +/- 19 ms, respectively) and the atrioventricular interval (169 +/- 5 ms) were not statistically different from those determined by intrathoracic cardiac recordings. We also present the data obtained from embryos treated with standard medications that affect the human conduction system. We conclude that the physiology of embryonic X. laevis cardiac conduction can be noninvasively studied by using digital video imaging. Additionally, we show the response of X. laevis embryonic hearts to chronotropic agents is similar but not identical to the response of the human heart.

Fig. 1

Intrathoracic extracellular cardiac recording of a stage 46 Xenopus laevis embryo. A and V, atrial and ventricular waveform in millivolt scale over time.

Fig. 2

Still images captured from digitized video of the stage 46 X. laevis embryonic heart. Major cardiac structures are traced in white and labeled. White circles, areas in which the grayscale value was measured to depict filling and contraction of the cardiac chambers. A: image during ventricular systole with filled atria and a contracted ventricle. Note the darkness in the circular atrial area. B: still image during ventricular diastole with contracted atria and a filled ventricle. Note the lighter color of the circular atrial area of measurement with concurrent darkening of the ventricular area of measurement. Diagram to the right depicts the size and gross morphological features of a stage 46 X. laevis embryo (17). Lat, lateral view; Ventr, ventral view.

Fig. 3

Waveforms of grayscale values of the cardiac chambers relative to time, which is calculated from the video acquisition frame rate. A higher value represents a value closer to white or a relatively empty cardiac chamber. A lower value represents a darker color of a filled cardiac chamber. Vertical solid lines denote where measurements were taken. Measurements bracketed by the horizontal double lines include the atrial cycle length (AA interval), the duration of the atrial contraction (A dur), the AV interval or the delay between atrial and ventricular contractions, the duration of the ventricular contraction (V dur), and the ventricular cycle length (VV interval). Note that as the atria contract, the ventricle completes filling.

Fig. 4

Comparison between the effect of the paralytic agents, cisatracurium and pancuronium, on the atrial cycle length in individual stage 46 X. laevis embryos. The drugs were tested sequentially in 10 embryos. The response of the atrial cycle length in individual embryos is plotted relative to the paralytic treatment.

Fig. 5

Cardiac intervals in stage 46 X. laevis embryos treated with chronotropic medications as measured by video analysis. *Statistically significant difference (P < 0.05 by Tukey’s F test) compared with the cisatracurium paralyzed control group (n = 53; open bars). Left, time scale AA interval and VV interval. Right, time scale for the A duration, V duration, and AV interval. †No measurable AV interval due to second- or third-degree heart block.