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Abstract
All-trans-Retinal (ATR) is a photosensitizer, serving as the chromophore for depolarizing and hyperpolarizing light-sensitive ion channels and pumps (opsins), recently employed as fast optical actuators. In mammalian optogenetic applications (in brain and heart), endogenous ATR availability is not considered a limiting factor, yet it is unclear how ATR modulation may affect the response to optical stimulation. We hypothesized that exogenous ATR may improve light responsiveness of cardiac cells modified by Channelrhodopsin2 (ChR2), hence lowering the optical pacing energy. In virally-transduced (Ad-ChR2(H134R)-eYFP) light-sensitive cardiac syncytium in vitro, ATR supplements ≤2 μM improved cardiomyocyte viability and augmented ChR2 membrane expression several-fold, while >4 μM was toxic. Employing integrated optical actuation (470 nm) and optical mapping, we found that 1–2 μM ATR dramatically reduced optical pacing energy (over 30 times) to several μW/mm2, lowest values reported to date, but also caused action potential prolongation, minor changes in calcium transients and no change in conduction. Theoretical analysis helped explain ATR-caused reduction of optical excitation threshold in cardiomyocytes. We conclude that cardiomyocytes operate at non-saturating retinal levels, and carefully-dosed exogenous ATR can enhance the performance of ChR2 in cardiac cells and yield energy benefits over orders of magnitude for optogenetic stimulation.
Highlights
All-trans-Retinal (ATR) is a photosensitizer, serving as the chromophore for depolarizing and hyperpolarizing light-sensitive ion channels and pumps, recently employed as fast optical actuators
We hypothesized that exogenous ATR may improve light responsiveness of cardiac cells modified by Channelrhodopsin[2] (ChR2), lowering the optical pacing energy
Employing integrated optical actuation (470 nm) and optical mapping, we found that 1–2 μM ATR dramatically reduced optical pacing energy to several μW/mm[2], lowest values reported to date, and caused action potential prolongation, minor changes in calcium transients and no change in conduction
Summary
All-trans-Retinal (ATR) is a photosensitizer, serving as the chromophore for depolarizing and hyperpolarizing light-sensitive ion channels and pumps (opsins), recently employed as fast optical actuators. ATR availability can be viewed, simplistically, as a scaler of ChR2 current (IChR2) magnitude by virtue of increasing the average number of functional channels in the cell membrane at any given time, and increasing the maximum macroscopic conductance for ChR2. Considering such purported scaling of IChR2 by ATR availability, it is unclear if supplemental ATR can result in a corresponding improvement of optical excitability when applied to cells and tissues. Black arrows indicate large fold change due to no cell survival above 4μ M. (*) denotes significant difference at p ≪ 0 .001, and (#) indicates significant difference at p < 0 .01, where each group is compared to the control CM at 0 ATR
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