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Advanced Cardiac Rhythm Management by Applying Optogenetic Multi-Site Photostimulation in Murine Hearts

Ventricular tachyarrhythmias are a major cause of mortality and morbidity worldwide. Electrical defibrillation using high-energy electric shocks is ...

Max Planck Institute for Dynamics and Self-Organization

University Medical Center Goettingen

DZHK e.V., partner site Goettingen

German Primate Center Leibniz Institute for Primate Research

Extrasystoles lead to several consequences, ranging from uneventful palpitations to lethal ventricular arrhythmias, in the presence of pathologies, such as myocardial ischemia. The role of working versus conducting cardiomyocytes, as well as the tissue requirements (minimal cell number) for the generation of extrasystoles, and the properties leading ectopies to become arrhythmia triggers (topology), in the normal and diseased heart, have not been determined directly in vivo. Here, we used optogenetics in transgenic mice expressing ChannelRhodopsin-2 selectively in either cardiomyocytes or the conduction system to achieve cell type-specific, noninvasive control of heart activity with high spatial and temporal resolution. By combining measurement of optogenetic tissue activation in vivo and epicardial voltage mapping in Langendorff-perfused hearts, we demonstrated that focal ectopies require, in the normal mouse heart, the simultaneous depolarization of at least 1,300-1,800 working cardiomyocytes or 90-160 Purkinje fibers. The optogenetic assay identified specific areas in the heart that were highly susceptible to forming extrasystolic foci, and such properties were correlated to the local organization of the Purkinje fiber network, which was imaged in three dimensions using optical projection tomography. Interestingly, during the acute phase of myocardial ischemia, focal ectopies arising from this location, and including both Purkinje fibers and the surrounding working cardiomyocytes, have the highest propensity to trigger sustained arrhythmias. In conclusion, we used cell-specific optogenetics to determine with high spatial resolution and cell type specificity the requirements for the generation of extrasystoles and the factors causing ectopies to be arrhythmia triggers during myocardial ischemia. 

Optogenetic determination of the myocardial requirements for extrasystoles by cell type-specific targeting of ChannelRhodopsin-2.

The control of spatiotemporal dynamics in biological systems is a fundamental problem in nonlinear sciences and has important applications in engineering and medicine. Optogenetic tools combined with advanced optical technologies provide unique opportunities to develop and validate novel approaches to control spatiotemporal complexity in neuronal and cardiac systems. Understanding of the mechanisms and instabilities underlying the onset, perpetuation, and control of cardiac arrhythmias will enable the development and translation of novel therapeutic approaches. Here we describe in detail the preparation and optical mapping of transgenic channelrhodopsin-2 (ChR2) mouse hearts, cardiac cell cultures, and the optical setup for photostimulation using digital light processing. 

Optogenetic Light Crafting Tools for the Control of Cardiac Arrhythmias.

Increased sarcoplasmic reticulum (SR) Ca leak via the cardiac ryanodine receptor (RyR2) has been suggested to play a mechanistic role in the development of heart failure (HF) and cardiac arrhythmia. Mice treated with a selective RyR2 stabilizer, rycal S36, showed normalization of SR Ca leak and improved survival in pressure overload (PO) and myocardial infarction (MI) models. The development of HF, measured by echocardiography and molecular markers, showed no difference in rycal S36- versus placebo-treated mice. Reduction of SR Ca leak in the PO model by the rycal-unrelated RyR2 stabilizer dantrolene did not mitigate HF progression. Development of HF was not aggravated by increased SR Ca leak due to RyR2 mutation (R2474S) in volume overload, an SR Ca leak-independent HF model. Arrhythmia episodes were reduced by rycal S36 treatment in PO and MI mice in vivo and ex vivo in Langendorff-perfused hearts. Isolated cardiomyocytes from murine failing hearts and human ventricular failing and atrial nonfailing myocardium showed reductions in delayed afterdepolarizations, in spontaneous and induced Ca waves, and in triggered activity in rycal S36 versus placebo cells, whereas the Ca transient, SR Ca load, SR Ca adenosine triphosphatase function, and action potential duration were not affected. Rycal S36 treatment of human induced pluripotent stem cells isolated from a patient with catecholaminergic polymorphic ventricular tachycardia could rescue the leaky RyR2 receptor. These results suggest that SR Ca leak does not primarily influence contractile HF progression, whereas rycal S36 treatment markedly reduces ventricular arrhythmias, thereby improving survival in mice.

Sarcoplasmic reticulum calcium leak contributes to arrhythmia but not to heart failure progression.

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.

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

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.

Article	Year
Optogenetic determination of the myocardial requirements for extrasystoles by cell type-specific targeting of ChannelRhodopsin-2. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 26204914	2015
Optogenetic Light Crafting Tools for the Control of Cardiac Arrhythmias. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 26965131	2016
Sarcoplasmic reticulum calcium leak contributes to arrhythmia but not to heart failure progression. Science translational medicine\| PubMed ID: 30209242	2018
Energy-Reduced Arrhythmia Termination Using Global Photostimulation in Optogenetic Murine Hearts. Frontiers in physiology\| PubMed ID: 30542292	2018
Drift and termination of spiral waves in optogenetically modified cardiac tissue at sub-threshold illumination. eLife\| PubMed ID: 33502313	2021

Claudia Richter

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