Optical stimulation enables paced electrophysiological studies in embryonic hearts

Abstract

Cardiac electrophysiology plays a critical role in the development and function of the heart. Studies of early embryonic electrical activity have lacked a viable point stimulation technique to pace in vitro samples. Here, optical pacing by high-precision infrared stimulation is used to pace excised embryonic hearts, allowing electrophysiological parameters to be quantified during pacing at varying rates with optical mapping. Combined optical pacing and optical mapping enables electrophysiological studies in embryos under more physiological conditions and at varying heart rates, allowing detection of abnormal conduction and comparisons between normal and pathological electrical activity during development in various models.

Keywords: (120.3890) Medical optics instrumentation; (140.3460) Lasers; (140.6810) Thermal effects; (170.2520) Fluorescence microscopy.

Fig. 1

Demonstration of high-precision optical pacing. Bright-field images of a 2-day quail heart that was paced at 2.00 Hz (left, Media 1) and an E9.5-day mouse heart that was paced at 1.00 Hz (right, Media 2). The hearts were initially unpaced and optical pacing was turned on, off, and then on again. Traces are of pixel intensity at an arbitrary point on the edge of the hearts, providing an indication of contraction, but the degree and direction of the change has no physiological meaning. Red bars indicate when the laser was on. Scale bars are 500 µm.

Fig. 2

Effect of spot size and pulse width on relative pacing threshold. Spot size (A, B; n = 7) was varied from 12 µm to 480 µm. Pulse width (C, D; n = 3) was varied from 5 ms to 100 ms. The threshold required to achieve 1:1 capture are plotted as pulse amplitude (A, C) and radiant exposure per pulse (B, D) normalized to the value at 12 µm and 20 ms (red points) to allow for aggregation of hearts. * p < 0.05 compared to 12 µm/20 ms (red points).

Fig. 3

Quantification of artifact observed in optical mapping during optical pacing. An example excised 2-day (HH 14) quail embryo heart was optically mapped (A) with a 500-µm scale bar. The optical mapping recordings from the single pixel at the green marker on the ventricle are shown while unpaced at 0.52 Hz (B) and during pacing at 1.00 Hz (C). An optical artifact was observed near the pacing site which manifested as a darkening. That area, indicated by the red box, is enlarged in (D) with a portion of the optical mapping recording shown for the pixel on the atrioventricular junction at the blue marker. The area marked by the red arrows in (D) show the darkening between a frame taken before the stimulus (solid red arrow and line) and during the stimulus (dotted red arrow and line). The entire optical mapping recording at the blue marker in (A) is shown in (E) with the sharp downward spikes of the artifact. Spatially, the artifact has a Gaussian distribution as shown for 3 different energies per pulse in (F), with the full width half maximum diameter (FWHM) listed.

Fig. 4

Optical mapping of unpaced and optically paced hearts. Representative data from 2-day, with no binning (top rows), and 5-day, with 4x4 binning (bottom rows), excised embryonic quail hearts. Fluorescence images of the hearts (left) have a blue marker on the atrioventricular junction (2-day) or atrium (5-day) and a green marker on the ventricle. The purple marker indicates the position of the pacing laser. Scale bars are 500 µm. Representative recordings of electrical activity (center-left) are shown for the matching colored markers. Activation maps (center-right) are shown with the color map in seconds and the isochrones are 10 ms apart for the 2-day heart and 1 ms apart for the 5-day heart. Action potential duration maps (APD90, right) are shown with the color map in seconds.

Fig. 5

Frequency-dependent effects. In older embryonic hearts, action potential duration (APD90) was frequency-dependent as illustrated by the example 7-day heart shown in (A), with the blue marker on the atrium and the green marker on the ventricle. Electrical recordings in matching colors from pixels at those marker positions are shown in (B) when unpaced at 1.72 Hz (left) and during optical pacing at 3.00 Hz (right). Aggregate data for the APD90 across the atria and the ventricles are shown in (C), with p < 0.001 between the frequencies in both cases. A 2-day heart that showed frequency-dependent conduction block is shown in (D), with the green marker on the ventricle and the red marker on the outflow tract. Electrical recordings in matching colors from pixels at those marker positions are shown in (E) when optically paced at 2.00 Hz (left) and at 2.50 Hz (right). The purple markers in (A) and (D) indicate the position of the laser.