Energy-Reduced Arrhythmia Termination Using Global Photostimulation in Optogenetic Murine Hearts

Abstract

Complex spatiotemporal non-linearity as observed during cardiac arrhythmia strongly correlates with vortex-like excitation wavelengths and tissue characteristics. Therefore, the control of arrhythmic patterns requires fundamental understanding of dependencies between onset and perpetuation of arrhythmia and substrate instabilities. Available treatments, such as drug application or high-energy electrical shocks, are discussed for potential side effects resulting in prognosis worsening due to the lack of specificity and spatiotemporal precision. In contrast, cardiac optogenetics relies on light sensitive ion channels stimulated to trigger excitation of cardiomyocytes solely making use of the inner cell mechanisms. This enables low-energy, non-damaging optical control of cardiac excitation with high resolution. Recently, the capability of optogenetic cardioversion was shown in Channelrhodopsin-2 (ChR2) transgenic mice. But these studies used mainly structured and local illumination for cardiac stimulation. In addition, since optogenetic and electrical stimulus work on different principles to control the electrical activity of cardiac tissue, a better understanding of the phenomena behind optogenetic cardioversion is still needed. The present study aims to investigate global illumination with regard to parameter characterization and its potential for cardioversion. Our results show that by tuning the light intensity without exceeding 1.10 mW mm-2, a single pulse in the range of 10-1,000 ms is sufficient to reliably reset the heart into sinus rhythm. The combination of our panoramic low-intensity photostimulation with optical mapping techniques visualized wave collision resulting in annihilation as well as propagation perturbations as mechanisms leading to optogenetic cardioversion, which seem to base on other processes than electrical defibrillation. This study contributes to the understanding of the roles played by epicardial illumination, pulse duration and light intensity in optogenetic cardioversion, which are the main variables influencing cardiac optogenetic control, highlighting the advantages and insights of global stimulation. Therefore, the presented results can be modules in the design of novel illumination technologies with specific energy requirements on the way toward tissue-protective defibrillation techniques.

Keywords: cardiac arrhythmia; channelrhodopsin-2; energy-reduced defibrillation; global illumination; optogenetics; photostimulation.

Figure 1

Global illumination and optical mapping setup. (A) The heart is perfused in a bath surrounded by three blue-LED for panoramic photostimulation. Light of a 625 nm LED is reflected via a dichroic mirror onto the heart surface. Emission was recorded through a 775±70 nm bandpass filter and an EMCCD camera at 1 kHz. Global illumination is reached via overlapping light cylinders. (B) Activation maps in posterior view of the heart of spontaneous sinus rhythm and stimulation examples electrically (electrode position is marked with an asterisk) as well as by global illumination. (C) Exemplary activation maps in anterior view of the heart of induced cardiac arrhythmia and collision of waves leading to annihilation of arrhythmic pattern. Color-bars indicate activation time in ms and the scale-bars mark 2 mm. A movie of the wave collision is available as (Supplementary Material Movie 1). LV - Left Ventricle, RV - Right Ventricle.

Figure 2

Dependency of pulse duration and light intensity as well as pulse energy in global optogenetic pacing. (A) The three-dimensional plot illustrates the averaged success rate ± SEM of 1:1 capture in dependency of investigated combinations of light intensity and pulse duration (as indicated in Table 1). In this view the variation of measured capture rates is clearly visible, but still approving the dependency of successful 1:1 capture of light intensity and pulse duration. In the x-y section are the most efficient combinations marked, which is visualized in a plain view in the next panel. (B) Shown are the most efficient combinations of required light intensity and energy for different pulse duration values (N = 6). The analysis shows a nearly constant energy, but an increase in intensity when the pulse duration is shortened.

Figure 3

Arrhythmia classification on the basis of electrical signal analysis. Shown are exemplary traces of MAP measurements (in red) for (A) sinus rhythm, (B) non-sustained and (C) sustained arrhythmia. Green overlay indicates part of the electrical pacing used to induce arrhythmia.

Figure 4

Global illumination for cardioversion. (A) Shows the pseudoECG signal during arrhythmia evocation via electrical pacing and termination using global illumination (green overlay indicates electrical pacing at 50 Hz, 30 pulse). The arrhythmic conditions were terminated with a 1 s stimulation pulse (indicated in blue), during which the normal sinus rhythm already returned. (B) Summary of the influence of intensity and pulse duration on cardioversion attempts. Successful optogenetic defibrillation rates (percentage of successful attempts reported as mean ± SEM, N = 6). (C) Visualizes the effect of the termination pulse shown in (A) in more detail. Two time points were defined in order to characterize the moment of arrhythmia termination for the 1 s pulses: t_last (green arrow), which denotes the last peak of the arrhythmia and t_sin (brown arrow), which denotes the first peak of the sinus rhythm during optogenetic stimulation. (D) Charts indicating the effect of intensity on cardioversion times in percentage of arrhythmia terminated in those periods of t_last (left) and t_sin (right) for 0.56 mW mm-2 (N = 47), 0.25 mW mm-2 (N = 40), 0.08 mW mm-2 (N = 33).

Figure 5

Wave dynamics during short global light pulse (10 ms, 0.25 mW mm-2, highlighted in blue). Shown are the MAP recording (in red) and optical traces of several local spots on the ventricles (in black, as indicated in panel I). Optical mapping analysis revealed vortex-like electrical activity on the heart surface (image sequence I), which is disturbed by global illumination (image sequence II) and thus provoking wave collision resulting in annihilation (image sequence III). Sinus activity follows after a peak corresponding to depolarization of the whole heart (image sequence IV). The corresponding movie is available as (Supplementary Material Movie 2).

Figure 6

Optical determination of the second identified mechanism, which was only observed in 1 s pulses (0.25 mW mm-2, highlighted in blue). Here the locally stable spiral activity (image sequence I) is disrupted by the illumination resulting in extrusion of the arrhythmic wave pattern to the boundaries (image sequence II). Already during photostimulation the sinus rhythm resets (image sequence III) and continues after switching off (image sequence IV). The scale-bars indicate 2 mm and are equal for all images in the corresponding panel. Shown are the MAP recording (in red) and optical traces of several local spots on the ventricles (in black, as indicated in panel I). The corresponding movie is available as (Supplementary Material Movie 3).