Recent advances and current limitations of available technology to optically manipulate and observe cardiac electrophysiology.

Optogenetics, utilising light-reactive proteins to manipulate tissue activity, are a relatively novel approach in the field of cardiac electrophysiology. We here provide an overview of light-activated transmembrane channels (optogenetic actuators) currently applied in strategies to modulate cardiac activity, as well as newly developed variants yet to be implemented in the heart. In addition, we touch upon genetically encoded indicators (optogenetic sensors) and fluorescent dyes to monitor tissue activity, including cardiac transmembrane potential and ion homeostasis. The combination of the two allows for all-optical approaches to monitor and manipulate the heart without any physical contact. However, spectral congestion poses a major obstacle, arising due to the overlap of excitation/activation and emission spectra of various optogenetic proteins and/or fluorescent dyes, resulting in optical crosstalk. Therefore, optogenetic proteins and fluorescent dyes should be carefully selected to avoid optical crosstalk and consequent disruptions in readouts and/or cellular activity. We here present a novel approach to simultaneously monitor transmembrane potential and cytosolic calcium, while also performing optogenetic manipulation. For this, we used the novel voltage-sensitive dye ElectroFluor 730p and the cytosolic calcium indicator X-Rhod-1 in mouse hearts expressing channelrhodopsin-2 (ChR2). By exploiting the isosbestic point of ElectroFluor 730p and avoiding the ChR2 activation spectrum, we here introduce a novel optical imaging and manipulation approach with minimal crosstalk. Future developments in both optogenetic proteins and fluorescent dyes will allow for additional and more optimised strategies, promising a bright future for all-optical approaches in the field of cardiac electrophysiology.

Near-infrared voltage-sensitive dyes based on chromene donor.

Voltage-sensitive dyes (VSDs) are used to image electrical activity in cells and tissues with submillisecond time resolution. Most of these fast sensors are constructed from push-pull chromophores whose fluorescence spectra are modulated by the electric field across the cell membrane. It was found that the substitution of naphthalene with chromene produces a 60 to 80 nm red-shift in absorption and emission spectra while maintaining fluorescence quantum efficiency and voltage sensitivity. One dye was applied to ex vivo murine heart with excitation at 730 nm, by far the longest wavelength reported in voltage imaging. This VSD resolves cardiac action potentials in single trials with 12% ΔF/F per action potential. The well-separated excitation spectra between these long-wavelength VSDs and channelrhodopsin (ChR2) enabled monitoring of action potential propagation in ChR2 hearts without any perturbation of electrical dynamics. Importantly, by employing spatially localized optogenetic manipulation, action potential dynamics can be assessed in an all-optical fashion with no artifact related to optical cross-talk between the reporter and actuator. These new environmentally sensitive chromene-based chromophores are also likely to have applications outside voltage imaging.

Optogenetic manipulation of cardiac repolarization gradients using sub-threshold illumination.

Introduction: Mechanisms underlying cardiac arrhythmias are typically driven by abnormalities in cardiac conduction and/or heterogeneities in repolarization time (RT) across the heart. While conduction slowing can be caused by either electrophysiological defects or physical blockade in cardiac tissue, RT heterogeneities are mainly related to action potential (AP) prolongation or abbreviation in specific areas of the heart. Importantly, the size of the area with altered RT and the difference between the short RT and long RT (RT gradient) have been identified as critical determinators of arrhythmogenicity. However, current experimental methods for manipulating RT gradient rely on the use of ion channel inhibitors, which lack spatial and temporal specificity and are commonly only partially reversible. Therefore, the conditions facilitating sustained arrhythmia upon the presence of RT heterogeneities and/or defects in cardiac conduction remain to be elucidated. Methods: We here employ an approach based on optogenetic stimulation in a low-intensity fashion (sub-threshold illumination), to selectively manipulate cardiac electrical activity in defined areas of the heart. Results: As previously described, subthreshold illumination is a robust tool able to prolong action potentials (AP), decrease upstroke velocity as well as slow cardiac conduction, in a fully reversible manner. By applying a patterned sub-threshold illumination in intact mouse hearts constitutively expressing the light-gated ion channel channelrhodopsin-2 (ChR2), we optically manipulate RT gradients and cardiac conduction across the heart in a spatially selective manner. Moreover, in a proof-of-concept assessment we found that in the presence of patterned sub-threshold illumination, mouse hearts were more susceptible to arrhythmias. Hence, this optogenetic-based approach may be able to mimic conduction slowing and RT heterogeneities present in pathophysiological conditions.

Drift and termination of spiral waves in optogenetically modified cardiac tissue at sub-threshold illumination.

The development of new approaches to control cardiac arrhythmias requires a deep understanding of spiral wave dynamics. Optogenetics offers new possibilities for this. Preliminary experiments show that sub-threshold illumination affects electrical wave propagation in the mouse heart. However, a systematic exploration of these effects is technically challenging. Here, we use state-of-the-art computer models to study the dynamic control of spiral waves in a two-dimensional model of the adult mouse ventricle, using stationary and non-stationary patterns of sub-threshold illumination. Our results indicate a light-intensity-dependent increase in cellular resting membrane potentials, which together with diffusive cell-cell coupling leads to the development of spatial voltage gradients over differently illuminated areas. A spiral wave drifts along the positive gradient. These gradients can be strategically applied to ensure drift-induced termination of a spiral wave, both in optogenetics and in conventional methods of electrical defibrillation.

Universal mechanisms for self-termination of rapid cardiac rhythm.

Excitable media sustain circulating waves. In the heart, sustained circulating waves can lead to serious impairment or even death. To investigate factors affecting the stability of such waves, we have used optogenetic techniques to stimulate a region at the apex of a mouse heart at a fixed delay after the detection of excitation at the base of the heart. For long delays, rapid circulating rhythms can be sustained, whereas for shorter delays, there are paroxysmal bursts of activity that start and stop spontaneously. By considering the dependence of the action potential and conduction velocity on the preceding recovery time using restitution curves, as well as the reduced excitability (fatigue) due to the rapid excitation, we model prominent features of the dynamics including alternation of the duration of the excited phases and conduction times, as well as termination of the bursts for short delays. We propose that this illustrates universal mechanisms that exist in biological systems for the self-termination of such activities.

A Software Architecture to Mimic a Ventricular Tachycardia in Intact Murine Hearts by Means of an All-Optical Platform.

Optogenetics is an emerging method that uses light to manipulate electrical activity in excitable cells exploiting the interaction between light and light-sensitive depolarizing ion channels, such as channelrhodopsin-2 (ChR2). Initially used in the neuroscience, it has been adopted in cardiac research where the expression of ChR2 in cardiac preparations allows optical pacing, resynchronization and defibrillation. Recently, optogenetics has been leveraged to manipulate cardiac electrical activity in the intact heart in real-time. This new approach was applied to simulate a re-entrant circuit across the ventricle. In this technical note, we describe the development and the implementation of a new software package for real-time optogenetic intervention. The package consists of a single LabVIEW program that simultaneously captures images at very high frame rates and delivers precisely timed optogenetic stimuli based on the content of the images. The software implementation guarantees closed-loop optical manipulation at high temporal resolution by processing the raw data in workstation memory. We demonstrate that this strategy allows the simulation of a ventricular tachycardia with high stability and with a negligible loss of data with a temporal resolution of up to 1 ms.