Principles of Optogenetic Methods and Their Application to Cardiac Experimental Systems

Abstract

Optogenetic techniques permit studies of excitable tissue through genetically expressed light-gated microbial channels or pumps permitting transmembrane ion movement. Light activation of these proteins modulates cellular excitability with millisecond precision. This review summarizes optogenetic approaches, using examples from neurobiological applications, and then explores their application in cardiac electrophysiology. We review the available opsins, including depolarizing and hyperpolarizing variants, as well as modulators of G-protein coupled intracellular signaling. We discuss the biophysical properties that determine the ability of microbial opsins to evoke reliable, precise stimulation or silencing of electrophysiological activity. We also review spectrally shifted variants offering possibilities for enhanced depth of tissue penetration, combinatorial stimulation for targeting different cell subpopulations, or all-optical read-in and read-out studies. Expression of the chosen optogenetic tool in the cardiac cell of interest then requires, at the single-cell level, introduction of opsin-encoding genes by viral transduction, or coupling “spark cells” to primary cardiomyocytes or a stem-cell derived counterpart. At the system-level, this requires construction of transgenic mice expressing ChR2 in their cardiomyocytes, or in vivo injection (myocardial or systemic) of adenoviral expression systems. Light delivery, by laser or LED, with widespread or multipoint illumination, although relatively straightforward in vitro may be technically challenged by cardiac motion and light-scattering in biological tissue. Physiological read outs from cardiac optogenetic stimulation include single cell patch clamp recordings, multi-unit microarray recordings from cell monolayers or slices, and electrical recordings from isolated Langendorff perfused hearts. Optical readouts of specific cellular events, including ion transients, voltage changes or activity in biochemical signaling cascades, using small detecting molecules or genetically encoded sensors now offer powerful opportunities for all-optical control and monitoring of cellular activity. Use of optogenetics has expanded in cardiac physiology, mainly using optically controlled depolarizing ion channels to control heart rate and for optogenetic defibrillation. ChR2-expressing cardiomyocytes show normal baseline and active excitable membrane and Ca2+ signaling properties and are sensitive even to ~1 ms light pulses. They have been employed in studies of the intrinsic cardiac adrenergic system and of cardiac arrhythmic properties.

The Emergence of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes (hiPSC-CMs) as a Platform to Model Arrhythmogenic Diseases

“…As spontaneous beating can be inconsistent between the cells and wells, it is essential to control it by either electrical stimulation or by optical pacing. The so called optogenetics approach consists of expressing the channelrhodopsin-2 in hiPSC-CMs, which upon activation by a light source triggers electrical activity [42,43]. Combined with the MEA platform, this optical approach allows artifact-free stimulation for pacing cardiomyocytes.…”

“…Others have reviewed the technologies available to perform functional analysis of hiPSC-CMs in more detail [9,43,44]. Despite their differences in electrophysiological characteristics with adult human myocytes, hiPSC-CMs are generally considered as a predictive tool in the context of drug testing [39] and as a promising alternative to animal testing with regard to disease modeling ( Table 1).…”

“…Others have reviewed the technologies available to perform functional analysis of hiPSC-CMs in more detail [9,43,44].…”

Using a bistable animal opsin for switchable and scalable optogenetic inhibition of neurons

“…Inhibitory optogenetic tools are light sensitive proteins or opsins which can be expressed in neurons to cause hyperpolarisation and silencing of spike firing 1,2 . Although several inhibitory optogenetic tools have been developed, there is no consensus regarding the best to use for either basic research or therapeutic applications.…”

Optogenetics: Background, Methodological Advances and Potential Applications for Cardiovascular Research and Medicine

“…Optogenetics is not only limited to the use of light-sensitive ion channels and pumps but the field is also being driven toward other light-sensitive proteins, such as G-protein modulating opsins ( Table 1; Ferenczi et al, 2019). In cardiac research, both natural and engineered versions of these opsins are being investigated to activate cell signaling with high temporal and spatial precision (Ferenczi et al, 2019). A recent study by Makowka et al (2019) has reported the utility of jellyfish opsin (JellyOp) to activate G s signaling in cardiomyocyte embryoid bodies and also in the intact mice hearts.…”