Cardiac optogenetics: shining light on signaling pathways.

In the early 2000s, the field of neuroscience experienced a groundbreaking transformation with the advent of optogenetics. This innovative technique harnesses the properties of naturally occurring and genetically engineered rhodopsins to confer light sensitivity upon target cells. The remarkable spatiotemporal precision offered by optogenetics has provided researchers with unprecedented opportunities to dissect cellular physiology, leading to an entirely new level of investigation. Initially revolutionizing neuroscience, optogenetics quickly piqued the interest of the wider scientific community, and optogenetic applications were expanded to cardiovascular research. Over the past decade, researchers have employed various optical tools to observe, regulate, and steer the membrane potential of excitable cells in the heart. Despite these advancements, achieving control over specific signaling pathways within the heart has remained an elusive goal. Here, we review the optogenetic tools suitable to control cardiac signaling pathways with a focus on GPCR signaling, and delineate potential applications for studying these pathways, both in healthy and diseased hearts. By shedding light on these exciting developments, we hope to contribute to the ongoing progress in basic cardiac research to facilitate the discovery of novel therapeutic possibilities for treating cardiovascular pathologies.

Optogenetic Modulation of Arrhythmia Triggers: Proof-of-Concept from Computational Modeling.

Introduction: Early afterdepolarizations (EADs) are secondary voltage depolarizations associated with reduced repolarization reserve (RRR) that can trigger lethal arrhythmias. Relating EADs to triggered activity is difficult to study, so the ability to suppress or provoke EADs would be experimentally useful. Here, we use computational simulations to assess the feasibility of subthreshold optogenetic stimulation modulating the propensity for EADs (cell-scale) and EAD-associated ectopic beats (organ-scale). Methods: We modified a ventricular ionic model by reducing rapid delayed rectifier potassium (0.25-0.1 × baseline) and increasing L-type calcium (1.0-3.5 × baseline) currents to create RRR conditions with varying severity. We ran simulations in models of single cardiomyocytes and left ventricles from post-myocardial infarction patient MRI scans. Optogenetic stimulation was simulated using either ChR2 (depolarizing) or GtACR1 (repolarizing) opsins. Results: In cell-scale simulations without illumination, EADs were seen for 164 of 416 RRR conditions. Subthreshold stimulation of GtACR1 reduced EAD incidence by up to 84.8% (25/416 RRR conditions; 0.1 µW/mm2); in contrast, subthreshold ChR2 excitation increased EAD incidence by up to 136.6% (388/416 RRR conditions; 50 µW/mm2). At the organ scale, we assumed simultaneous, uniform illumination of the epicardial and endocardial surfaces. GtACR1-mediated suppression (10-50 µW/mm2) and ChR2-mediated unmasking (50-100 µW/mm2) of EAD-associated ectopic beats were feasible in three distinct ventricular models. Conclusions: Our findings suggest that optogenetics could be used to silence or provoke both EADs and EAD-associated ectopic beats. Validation in animal models could lead to exciting new experimental regimes and potentially to novel anti-arrhythmia treatments. Supplementary Information: The online version contains supplementary material available at 10.1007/s12195-023-00781-z.

Optogenetically mediated large volume suppression and synchronized excitation of human ventricular cardiomyocytes.

A major challenge in cardiac optogenetics is to have minimally invasive large volume excitation and suppression for effective cardioversion and treatment of tachycardia. It is important to study the effect of light attenuation on the electrical activity of cells in in vivo cardiac optogenetic experiments. In this computational study, we present a detailed analysis of the effect of light attenuation in different channelrhodopsins (ChRs)-expressing human ventricular cardiomyocytes. The study shows that sustained illumination from the myocardium surface used for suppression, simultaneously results in spurious excitation in deeper tissue regions. Tissue depths of suppressed and excited regions have been determined for different opsin expression levels. It is shown that increasing the expression level by 5-fold enhances the depth of suppressed tissue from 2.24 to 3.73 mm with ChR2(H134R) (ChR2 with a single point mutation at position H134), 3.78 to 5.12 mm with GtACR1 (anion-conducting ChR from cryptophyte algae Guillardia theta) and 6.63 to 9.31 mm with ChRmine (a marine opsin gene from Tiarina fusus). Light attenuation also results in desynchrony in action potentials in different tissue regions under pulsed illumination. It is further shown that gradient-opsin expression not only enables suppression up to the same level of tissue depth but also enables synchronized excitation under pulsed illumination. The study is important for the effective treatment of tachycardia and cardiac pacing and for extending the scale of cardiac optogenetics.

Applications and challenges of rhodopsin-based optogenetics in biomedicine.

Optogenetics is an emerging bioengineering technology that has been rapidly developed in recent years by cross-integrating optics, genetic engineering, electrophysiology, software control, and other disciplines. Since the first demonstration of the millisecond neuromodulation ability of the channelrhodopsin-2 (ChR2), the application of optogenetic technology in basic life science research has been rapidly progressed, especially in neurobiology, which has driven the development of the discipline. As the optogenetic tool protein, microbial rhodopsins have been continuously explored, modified, and optimized, with many variants becoming available, with structural characteristics and functions that are highly diversified. Their applicability has been broadened, encouraging more researchers and clinicians to utilize optogenetics technology in research. In this review, we summarize the species and variant types of the most important class of tool proteins in optogenetic techniques, the microbial rhodopsins, and review the current applications of optogenetics based on rhodopsin qualitative light in biology and other fields. We also review the challenges facing this technology, to ultimately provide an in-depth technical reference to support the application of optogenetics in translational and clinical research.

Ultra-low power deep sustained optogenetic excitation of human ventricular cardiomyocytes with red-shifted opsins: a computational study.

The main challenge in cardiac optogenetics is to have low-power, high-fidelity deep excitation of cells with minimal invasiveness and heating. We present a detailed computational study of optogenetic excitation of human ventricular cardiomyocytes (HVCMs) with new ChRmine, bReaChES and CsChrimson red-shifted opsins to overcome the challenge. Action potentials (APs) in ChRmine-expressing HVCMs can be triggered at 6 µW mm-2 (10 ms pulse) and 0.7 µW mm-2 (100 ms pulse) at 585 nm, which is two orders of magnitude lower than ChR2(H134R). This enables safe sustained excitation of deeply situated cardiac cells with ChRmine (7.46 mm) and with bReaChES (6.21 mm) with the light source at the pericardium surface. Deeper excitation up to 10.2 mm can be achieved with ChRmine by illuminating at 650 nm. Photostimulation conditions for minimum charge transfer during APs have been determined, which is important for tissue health under sustained excitation. The AP duration for all the opsins is constant up to 100 ms pulse width but increases thereafter. Interestingly, the AP frequency increases with irradiance under continuous illumination, but APs are suppressed at higher irradiances. The optimal range of irradiance for each opsin to excite HVCMs has been determined. Under optimal photostimulation conditions, each opsin can precisely excite APs up to 2.5 Hz, while latency and power of light pulse for each AP in a sequence remain most stable and an order of magnitude lower, respectively, in ChRmine-expressing HVCMs. The study highlights the importance of ChRmine and bReaChES for resynchronization, termination of ventricular tachycardia and designing optogenetic cardiac pacemakers with enhanced battery life. KEY POINTS: This work is the formulation of accurate theoretical models of optogenetic control of human ventricular cardiomyocytes (HVCMs) expressed with newly discovered opsins (ChRmine, bReaChES and CsChrimson). Under continuous illumination, action potentials in each opsin-expressing HVCMs can only be evoked in a certain range of irradiances. Action potentials in ChRmine-expressing HVCMs can be triggered at ultra-low power (6 µW mm-2 at 10 ms pulse or 0.7 µW mm-2 at 100 ms pulse at 585 nm), which is two to three orders of magnitude lower than reported results. Ongoing action potentials in ChRmine-expressing HVCMs can be suppressed by continuous illumination of 585 nm light at 2 µW mm-2 . ChRmine enables sustained excitation due to its faster recovery from the desensitized state. Optogenetic excitation of deeply situated cardiac cells is possible up to ∼7.46 and 10.2 mm with ChRmine on illuminating the outer surface of pericardium at safe irradiance at 585 nm and 650 nm, respectively. The study opens up prospects for designing energy-efficient light-induced pacemakers, resynchronization and termination of ventricular tachycardia.

Observing and Manipulating Cell-Specific Cardiac Function with Light.

The heart is a complex multicellular organ comprising both cardiomyocytes (CM), which make up the majority of the cardiac volume, and non-myocytes (NM), which represent the majority of cardiac cells. CM drive the pumping action of the heart, triggered via rhythmic electrical activity. NM, on the other hand, have many essential functions including generating extracellular matrix, regulating CM activity, and aiding in repair following injury. NM include neurons and interstitial, immune, and endothelial cells. Understanding the role of specific cell types and their interactions with one another may be key to developing new therapies with minimal side effects to treat cardiac disease. However, assessing cell-type-specific behavior in situ using standard techniques is challenging. Optogenetics enables population-specific observation and control, facilitating studies into the role of specific cell types and subtypes. Optogenetic models targeting the most important cardiac cell types have been generated and used to investigate non-canonical roles of those cell populations, e.g., to better understand how cardiac pacing occurs and to assess potential translational possibilities of optogenetics. So far, cardiac optogenetic studies have primarily focused on validating models and tools in the healthy heart. The field is now in a position where animal models and tools should be utilized to improve our understanding of the complex heterocellular nature of the heart, how this changes in disease, and from there to enable the development of cell-specific therapies and improved treatments.

A move in the light direction.

Computer simulations show how low-intensity illumination can be used to terminate cardiac arrhythmias.

Patterned Illumination Techniques in Optogenetics: An Insight Into Decelerating Murine Hearts.

Much has been reported about optogenetic based cardiac arrhythmia treatment and the corresponding characterization of photostimulation parameters, but still, our capacity to interact with the underlying spatiotemporal excitation patterns relies mainly on electrical and/or pharmacological approaches. However, these well-established treatments have always been an object of somehow heated discussions. Though being acutely life-saving, they often come with potential side-effects leading to a decreased functionality of the complex cardiac system. Recent optogenetic studies showed the feasibility of the usage of photostimulation as a defibrillation method with comparatively high success rates. Although, these studies mainly concentrated on the description as well as on the comparison of single photodefibrillation approaches, such as locally focused light application and global illumination, less effort was spent on the description of excitation patterns during actual photostimulation. In this study, the authors implemented a multi-site photodefibrillation technique in combination with Multi-Lead electrocardiograms (ECGs). The technical connection of real-time heart rhythm measurements and the arrhythmia counteracting light control provides a further step toward automated arrhythmia classification, which can lead to adaptive photodefibrillation methods. In order to show the power effectiveness of the new approach, transgenic murine hearts expressing channelrhodopsin-2 ex vivo were investigated using circumferential micro-LED and ECG arrays. Thus, combining the best of two methods by giving the possibility to illuminate either locally or globally with differing pulse parameters. The optical technique presented here addresses a number of challenges of technical cardiac optogenetics and is discussed in the context of arrhythmic development during photostimulation.

Channelrhodopsins for Cell-Type Specific Illumination of Cardiac Electrophysiology.

Optogenetic approaches have evolved as potent means to investigate cardiac electrophysiology, with research ranging from the study of arrhythmia mechanisms to effects of cardiac innervation and heterocellular structural and functional interactions, both in healthy and diseased myocardium. Most commonly, these studies use channelrhodopsin-2 (ChR2)-expressing murine models that enable light-activated depolarization of the target cell population. However, each newly generated mouse line requires thorough characterization, as cell-type specific ChR2 expression cannot be taken for granted, and the electrophysiological response of its activation in the target cell should be evaluated. In this chapter, we describe detailed protocols for assessing ChR2 specificity using immunohistochemistry, isolation of specific cell populations to analyze electrophysiological effects of ChR2 activation with the patch-clamp technique, and whole-heart experiments to assess in situ effects of optical stimulation.

Modulation of cardiac optogenetics by vitamin A.

Cardiac optogenetics is an emergent research area and refers to the delivery of light-activated proteins to excitable heart tissue and the subsequent use of light for controlling the electrical function with high spatial and temporal resolution. Channelrhodopsin-2 (ChR2) is a light-sensitive ion channel with the chromophore, all trans retinal, derived from vitamin A (all-trans-retinol; retinol). In this study, we explored whether exogenous vitamin A can be a limiting factor in the light responsiveness of cardiomyocytes-expressing ChR2. We showed that in cardiomyocytes virally transduced with ChR2 (H134R)-enhanced yellow fluorescent protein, vitamin A supplements lower than 10 µM significantly increased ChR2 expression. Adding 1 µM vitamin A changed light-induced transmembrane potential difference significantly, whereas 5 µM dramatically induced membrane depolarization and triggered intracellular calcium elevation. We concluded that vitamin A supplementation can modulate the efficiency of ChR2 and provide a complementary strategy for improving the performance of optogenetic tools.

Adeno-Associated Virus Mediated Gene Delivery: Implications for Scalable in vitro and in vivo Cardiac Optogenetic Models.

Adeno-associated viruses (AAVs) provide advantages in long-term, cardiac-specific gene expression. However, AAV serotype specificity data is lacking in experimental models relevant to cardiac electrophysiology and cardiac optogenetics. We aimed to identify the optimal AAV serotype (1, 6, or 9) in pursuit of scalable rodent and human models using genetic modifications in cardiac electrophysiology and optogenetics, in particular, as well as to elucidate the mechanism of virus uptake. In vitro syncytia of primary neonatal rat ventricular cardiomyocytes (NRVMs) and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were infected with AAVs 1, 6, and 9 containing the transgene for eGFP or channelrhodopsin-2 (ChR2) fused to mCherry. In vivo adult rats were intravenously injected with AAV1 and 9 containing ChR2-mCherry. Transgene expression profiles of rat and human cells in vitro revealed that AAV1 and 6 significantly outperformed AAV9. In contrast, systemic delivery of AAV9 in adult rat hearts yielded significantly higher levels of ChR2-mCherry expression and optogenetic responsiveness. We tracked the mechanism of virus uptake to purported receptor-mediators for AAV1/6 (cell surface sialic acid) and AAV9 (37/67 kDa laminin receptor, LamR). In vitro desialylation of NRVMs and hiPSC-CMs with neuraminidase (NM) significantly decreased AAV1,6-mediated gene expression, but interestingly, desialylation of hiPSC-CMs increased AAV9-mediated expression. In fact, only very high viral doses of AAV9-ChR2-mCherry, combined with NM treatment, yielded consistent optogenetic responsiveness in hiPSC-CMs. Differences between the in vitro and in vivo performance of AAV9 could be correlated to robust LamR expression in the intact heart (neonatal rat hearts as well as adult human and rat hearts), but no expression in vitro in cultured cells (primary rat cells and hiPS-CMs). The dynamic nature of LamR expression and its dependence on environmental factors was further corroborated in intact adult human ventricular tissue. The combined transgene expression and cell surface receptor data may explain the preferential efficiency of AAV1/6 in vitro and AAV9 in vivo for cardiac delivery and mechanistic knowledge of their action can help guide cardiac optogenetic efforts. More broadly, these findings are relevant to future efforts in gene therapy for cardiac electrophysiology abnormalities in vivo as well as for genetic modifications of cardiomyocytes by viral means in vitro applications such as disease modeling or high-throughput drug testing.

Sudden Heart Rate Reduction Upon Optogenetic Release of Acetylcholine From Cardiac Parasympathetic Neurons in Perfused Hearts.

The balance of sympathetic and parasympathetic tone provides exquisite control of heart rate and contractility and has also been shown to modulate coronary flow and inflammation. Understanding how autonomic balance is altered by cardiac disease is an active area of research, and developing new ways to control this balance provides insights into disease therapies. However, achieving acute neuron-specific stimulation of autonomic neurons can be difficult in experiments that measure the acute effects of nerve stimulation on the heart. Conventional electrical and pharmacological approaches can be spatially and temporally non-selective. Cell-specific expression of light-activated channels (channelrhodopsin, ChR2) is a powerful approach that enables control of the timing and distribution of cellular stimulation using light. We present such an optogenetic approach where parasympathetic cardiac neurons are selectively photoactivated at high temporal precision to initiate cholinergic-mediated slowing of heart rate. Mice were crossbred to express ChR2 in peripheral cholinergic neurons using Cre-Lox recombination driven by a choline acetyltransferase (ChAT) promoter. Hearts from adult mice were excised, perfused, and the epicardium was illuminated (peak 460-465 nm) to photoactivate ChR2. In one set of studies, hearts were illuminated using a large-field LED light source. In other studies, a micro LED was placed on the right atrium to selectively illuminate the junction of the superior vena cava (SVC) and right atrium. The ECG was acquired before, during, and after tissue illumination to measure changes in heart rate. Upon illumination, hearts exhibited sudden and dramatic reductions in heart rate with restoration of normal heart rate after cessation of illumination. Delays in atrioventricular conduction were also observed. Heart rate reductions at the highest irradiance levels were similar to heart rate reductions caused by application of bethanechol (10 µM) or acetylcholine (800 µM). Atropine (50 nM) completely blocked the effect of ChR2 photoactivation, confirming cholinergic mediation. Optogenetic activation of intrinsic parasympathetic neurons reduced heart rate in an immediate, dose-dependent fashion, resembling the slowing of sinus rate in response to acetylcholine. Our results demonstrate a new approach for controlling parasympathetic modulation of cardiac function by selectively activating the endogenous release of acetylcholine from intrinsic cardiac cholinergic neurons. Key Message: Optogenetic photoactivation of intrinsic cardiac neurons provides immediate, tissue-specific stimulation with minimal cross-reactivity. Our results demonstrate that selective expression of channelrhodopsin within cardiac cholinergic neurons enables photoactivated release of acetylcholine, thereby instantaneously slowing sinus rate and altering atrioventricular conduction. This provides for in-depth examination of the endogenous interplay between cardiac autonomic neurons and the functional outcomes of downstream post-synaptic receptor activation.

Cardiac Optogenetics: 2018.

Cardiac optogenetics is an emergent research area involving the delivery of light-sensitive proteins (opsins) to excitable heart tissue to enable optical modulation of cardiac electrical function. Optogenetic stimulation has many noteworthy advantages over conventional electrical methods, including selective electrophysiological modulation in specifically targeted cell subpopulations, high-resolution spatiotemporal control via patterned illumination, and use of different opsins to elicit inward or outward transmembrane current. This review summarizes developments achieved since the inception of cardiac optogenetics research, which has spanned nearly a decade. The authors first provide an overview of recent methodological advances in opsin engineering, light sensitization of cardiac tissue, strategies for illuminating the heart, and frameworks for simulating optogenetics in realistic computational models of patient hearts. They then review recent cardiac optogenetics applications, including: 1) all-optical, high-throughput, contactless assays for quantification of electrophysiological properties; 2) optogenetic perturbation of cardiac tissue to unveil mechanistic insights on the initiation, perpetuation, and termination of arrhythmia; and 3) disruptive translational innovations such as light-based pacemaking and defibrillation.

Termination of re-entrant atrial tachycardia via optogenetic stimulation with optimized spatial targeting: insights from computational models.

KEY POINTS: Optogenetics has emerged as a potential alternative to electrotherapy for treating heart rhythm disorders, but its applicability for terminating atrial arrhythmias remains largely unexplored. We used computational models reconstructed from clinical MRI scans of fibrotic patient atria to explore the feasibility of optogenetic termination of atrial tachycardia (AT), comparing two different illumination strategies: distributed vs. targeted. We show that targeted optogenetic stimulation based on automated, non-invasive flow-network analysis of patient-specific re-entry morphology may be a reliable approach for identifying the optimal illumination target in each individual (i.e. the critical AT isthmus). The above-described approach yields very high success rates (up to 100%) and requires dramatically less input power than distributed illumination We conclude that simulations in patient-specific models show that targeted light pulses lasting longer than the AT cycle length can efficiently and reliably terminate AT if the human atria can be successfully light-sensitized via gene delivery of ChR2. ABSTRACT: Optogenetics has emerged as a potential alternative to electrotherapy for treating arrhythmia, but feasibility studies have been limited to ventricular defibrillation via epicardial light application. Here, we assess the efficacy of optogenetic atrial tachycardia (AT) termination in human hearts using a strategy that targets for illumination specific regions identified in an automated manner. In three patient-specific models reconstructed from late gadolinium-enhanced MRI scans, we simulated channelrhodopsin-2 (ChR2) expression via gene delivery. In all three models, we attempted to terminate re-entrant AT (induced via rapid pacing) via optogenetic stimulation. We compared two strategies: (1) distributed illumination of the endocardium by multi-optrode grids (number of optrodes, Nopt  = 64, 128, 256) and (2) targeted illumination of the critical isthmus, which was identified via analysis of simulated activation patterns using an algorithm based on flow networks. The illuminated area and input power were smaller for the targeted approach (19-57.8 mm2 ; 0.6-1.8 W) compared to the sparsest distributed arrays (Nopt  = 64; 124.9 ± 6.3 mm2 ; 3.9 ± 0.2 W). AT termination rates for distributed illumination were low, ranging from <5% for short pulses (1/10 ms long) to ∼20% for longer stimuli (100/1000 ms). When we attempted to terminate the same AT episodes with targeted illumination, outcomes were similar for short pulses (1/10 ms long: 0% success) but improved for longer stimuli (100 ms: 54% success; 1000 ms: 90% success). We conclude that simulations in patient-specific models show that light pulses lasting longer than the AT cycle length can efficiently and reliably terminate AT in atria light-sensitized via gene delivery. We show that targeted optogenetic stimulation based on analysis of AT morphology may be a reliable approach for defibrillation and requires less power than distributed illumination.

Will cardiac optogenetics find the way through the obscure angles of heart physiology?

Optogenetics is a technique exploded in the last 10 years, which revolutionized several areas of biological research. The brightest side of this technology is the use of light to modulate non-invasively, with high spatial resolution and millisecond time scale, excitable cells genetically modified to express light-sensitive microbial ion channels (opsins). Neuroscience has first benefited from such fascinating strategy, in intact organisms. By shining light to specific neuronal subpopulations, optogenetics allowed unearth the mechanisms involved in cell-to-cell communication within the context of intact organs, such as the brain, formed by complex neuronal circuits. More recently, scientists looked at optogenetics as a tool to answer some of the questions, remained in the dark, of cardiovascular physiology. In this review, we focus on the application of optogenetics in the study of the heart, a complex multicellular organ, homing different populations of excitable cells, spatially and functionally interconnected. Moving from the first proof-of-principle works, published in 2010, to the present time, we discuss the in vitro and in vivo applications of optogenetics for the study of electrophysiology of the different cardiac cell types, and for the dissection of cellular mechanisms underlying arrhythmias. We also present how molecular biology and technology foster the evolution of cardiac optogenetics, with the aim to further our understanding of fundamental questions in cardiac physiology and pathology. Finally, we confer about the therapeutic potential of such biotechnological strategy for the treatment of heart rhythm disturbances (e.g. cardiac pacing, cardioversion).

Computational modeling of cardiac optogenetics: Methodology overview & review of findings from simulations.

Cardiac optogenetics is emerging as an exciting new potential avenue to enable spatiotemporally precise control of excitable cells and tissue in the heart with low-energy optical stimuli. This approach involves the expression of exogenous light-sensitive proteins (opsins) in target heart tissue via viral gene or cell delivery. Preliminary experiments in optogenetically-modified cells, tissue, and organisms have made great strides towards demonstrating the feasibility of basic applications, including the use of light stimuli to pace or disrupt reentrant activity. However, it remains unknown whether techniques based on this intriguing technology could be scaled up and used in humans for novel clinical applications, such as pain-free optical defibrillation or dynamic modulation of action potential shape. A key step towards answering such questions is to explore potential optogenetics-based therapies using sophisticated computer simulation tools capable of realistically representing opsin delivery and light stimulation in biophysically detailed, patient-specific models of the human heart. This review provides (1) a detailed overview of the methodological developments necessary to represent optogenetics-based solutions in existing virtual heart platforms and (2) a survey of findings that have been derived from such simulations and a critical assessment of their significance with respect to the progress of the field.

Optogenetics-enabled dynamic modulation of action potential duration in atrial tissue: feasibility of a novel therapeutic approach.

AIMS: Diseases that abbreviate the cardiac action potential (AP) by increasing the strength of repolarizing transmembrane currents are highly arrhythmogenic. It has been proposed that optogenetic tools could be used to restore normal AP duration (APD) in the heart under such disease conditions. This study aims to evaluate the efficacy of an optogenetic treatment modality for prolonging pathologically shortened APs in a detailed computational model of short QT syndrome (SQTS) in the human atria, and compare it to drug treatment. METHODS AND RESULTS: We used a human atrial myocyte model with faster repolarization caused by SQTS; light sensitivity was inscribed via the presence of channelrhodopsin-2 (ChR2). We conducted simulations in single cells and in a magnetic resonance imaging-based model of the human left atrium (LA). Application of an appropriate optical stimulus to a diseased cell dynamically increased APD, producing an excellent match to control AP (<1.5 mV deviation); treatment of a diseased cell with an AP-prolonging drug (chloroquine) also increased APD, but the match to control AP was worse (>5 mV deviation). Under idealized conditions in the LA (uniform ChR2-expressing cell distribution, no light attenuation), optogenetics-based therapy outperformed chloroquine treatment (APD increased to 87% and 81% of control). However, when non-uniform ChR2-expressing cell distribution and light attenuation were incorporated, optogenetics-based treatment was less effective (APD only increased to 55%). CONCLUSION: This study demonstrates proof of concept for optogenetics-based treatment of diseases that alter atrial AP shape. We identified key practical obstacles intrinsic to the optogenetic approach that must be overcome before such treatments can be realized.