Optical pacing of the embryonic heart

Abstract

Light has been used to noninvasively alter the excitability of both neural and cardiac tissue 1-10. Recently, pulsed laser light has been shown to be capable of eliciting action potentials in peripheral nerves and in cultured cardiomyocytes 7-10. Here, we demonstrate for the first time optical pacing (OP) of an intact heart in vivo. Pulsed 1.875 μm infrared laser light was employed to lock the heart rate to the pulse frequency of the laser. A laser Doppler velocimetry (LDV) signal was used to verify the pacing. At low radiant exposures, embryonic quail hearts were reliably paced in vivo without detectable damage to the tissue, indicating that OP has great potential as a tool to study embryonic cardiac dynamics and development. In particular, OP can be utilized to control the heart rate, and thereby alter stresses and mechanically transduced signaling.

Figures

Figure 1. OP setup

a, Photograph of a 53 hour quail embryo in the New culture. b, Block diagram of the setup of the optical pacing experiment. Embryos in the Petri dish under the microscope were stimulated by a pulsed infrared laser, while a laser Doppler velocimeter probe measured blood flow. The LDV output and trigger pulse from the laser were recorded to verify pacing. Also, the trigger pulse from the laser activated a white light LED which was observable in the video. The video output from the microscope camera was recorded at video rate (29.97 fps) by a laptop computer.

Figure 2. Pacing of the embryonic quail heart

a,b, The trigger pulse (blue) from the pulsed laser is superimposed on the LDV recordings (red) of heart rate. (a) presents a recording from a stage 17 (59 hour) quail embryo in a New culture paced at 2 Hz. The laser pulse duration was 1 ms and the radiant exposure was 0.92 J/cm2 per pulse. The laser pulses were directed toward the inflow region of the heart tube. The laser pulses were turned on and off several times to demonstrate the robustness of optical pacing. The heart rate increased from 0.634 to 2 Hz when the stimulation laser pulses were started and decreased to 0.693 Hz by the end of the trace in 2a. (b) The dashed box in 2a is expanded for a close up view in 2b. Clearly the trigger pulse and LDV signal were synchronized with each laser pulse eliciting a heartbeat. (c) presents a recording from a stage 14 (53 hour) quail embryo in a New culture. The laser pulse duration was 2 ms and the radiant exposure was 0.84 mJ/cm2 per pulse. The frequency of the laser pulses were varied from 2 Hz to 3 Hz and back to 2 Hz to demonstrate the ability of the embryo heart to follow the pulse frequency. Laser stimulation (blue) and heart rate (red) traces were calculated from the video by plotting the pixel intensity of LED flashes triggered by the laser and intensity at the edge of the heart wall.

Figure 3. Threshold measurement

25 embryos in New cultures were exposed to varying radiant exposures and the results were plotted in terms of pacing probability (successful pacing = 1, unsuccessful pacing = 0). The data were fit to a normal cumulative distribution function (blue line). The threshold (50% probability point) was 0.81+/−0.01 J/cm2 with a t-value of 79.9 and 95% confidence interval between 0.794 and 0.836 J/cm2. The standard deviation of the distribution was 0.036+/−0.016 with a t-value of 2.32.

Figure 4. Transmission electron microscopy (TEM) after the OP procedure

a-c, The TEM images show a typical cardiomyocyte in the inflow (sinoatrial) region of the heart tube of three different quail embryos, where laser light was directed. Each embryo was between 52 to 58 hours of development. The torso of each embryo was excised and fixed immediately after OP. a, Control embryo. The embryo was exposed to the OP experimental procedure, except that the pacing laser was not turned on during the experiment. b, Embryo paced slightly above threshold. The embryo heart was paced for approximately 20 seconds with 2 ms pulses (2.64 mJ/pulse) at 2 Hz. c, Embryo paced well above threshold. The embryo heart was paced for approximately 20 seconds with 4 ms pulses (13 mJ/pulse) at 2 Hz. The micrograph image was taken from a border region separating severely damaged tissue (partially ablated) from apparently healthy tissue. The mitochondria are vacuolated and the nuclear envelope is swollen, as are elements of the rough ER (large arrow). The cells and mitochondria in 4a and b have a similar appearance and show no signs of damage from the pulsed laser light. Some subtle abnormalities in the cristae present in cells from both threshold paced and unpaced control hearts are due to the unavoidable delay in excising the embryonic torso. N -nucleus, M - mitochondria.