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Cardiac Optogenetics: Enhancement by All-trans-Retinal.

Yu J, Chen K, Lucero RV, Ambrosi CM, Entcheva E - Sci Rep (2015)

Bottom Line: 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/mm(2), 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, Stony Brook University, Stony Brook, NY.

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/mm(2), 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.

No MeSH data available.


Related in: MedlinePlus

ATR effects on cardiomyocyte electrophysiology.(A) Action potentials in response to electrical pacing were imaged optically (using di4-ANBDQBS) and presented as mean ± SEM at each point for each group. (B) Quantified APD80 for control CM and ChR2-CM without ATR and with 1 μM ATR (highest optical excitability). Both A and B had n = 3–4 samples per experimental group. (C) Calcium transients in response to electrical pacing were imaged optically (using Rhod4-AM) and presented as mean ± SEM at each point for each group. (D) Quantified CTD80 for control CM and ChR2-CM at different ATR supplements. Both C and D had n = 7–33 samples for each of the eight experimental groups. (E) Example activation maps of ChR2-CM, following point electrical stimulation at the bottom; isochrones are 10 ms apart. Scale bar is 5 mm. (F) Quantified conduction velocity from the activation maps (n = 7–14 per group). (*) indicates significant difference at p < 0.05 compared to the respective control (zero ATR) or as indicated by the brackets.
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f4: ATR effects on cardiomyocyte electrophysiology.(A) Action potentials in response to electrical pacing were imaged optically (using di4-ANBDQBS) and presented as mean ± SEM at each point for each group. (B) Quantified APD80 for control CM and ChR2-CM without ATR and with 1 μM ATR (highest optical excitability). Both A and B had n = 3–4 samples per experimental group. (C) Calcium transients in response to electrical pacing were imaged optically (using Rhod4-AM) and presented as mean ± SEM at each point for each group. (D) Quantified CTD80 for control CM and ChR2-CM at different ATR supplements. Both C and D had n = 7–33 samples for each of the eight experimental groups. (E) Example activation maps of ChR2-CM, following point electrical stimulation at the bottom; isochrones are 10 ms apart. Scale bar is 5 mm. (F) Quantified conduction velocity from the activation maps (n = 7–14 per group). (*) indicates significant difference at p < 0.05 compared to the respective control (zero ATR) or as indicated by the brackets.

Mentions: We further examined the effects of ATR supplementation on CM electrophysiology by quantifying action potentials, calcium transients, and conduction properties. We compared optically-measured (via Di-4-ANBDQBS) action potential durations (APDs) of ChR2-CM to CM with the same treatment, Fig. 4A. At zero ATR, CM and ChR2-CM showed overlapping AP and similar APD80 of 260 ms and 270 ms, respectively; reassuring of the benign nature of the opsin expression and optogenetic activation. Addition of 1 μM ATR significantly prolonged the APD80 in ChR2-CMs by 44% (p < 0.05) compared to ChR2-CM without ATR (Fig. 4B). Similarly, at zero ATR, no significant differences were seen in calcium transient morphology between the CM and ChR2-CM groups across the tested conditions (Fig. 4C); CTD80 of CM and ChR2-CM were 552 ms and 530 ms, respectively (Fig. 4D). Supplementation with ATR of 1, 2, and 4 μM in control CM led to: 13.8% (p < 0.05), 13.3% (p < 0.05), and 4% (n.s.) prolongation of CTD80, respectively. In ChR2-CM samples, exogenous ATR supplementation also led to small but significant prolongation of CTD80 by approximately 10.9%, 11.6%, and non-significant 10% for the same tested concentrations (Fig. 4D). Examination of propagation and activation maps for the different ChR2-CM groups (Fig. 4E) showed that ATR supplementation at 1 and 2 μM yielded smooth and fast propagation with similar CV to ChR2-CM control: 19.1 cm/s, 17.0 cm/s, and 17.0 cm/s for 0, 1, and 2 μM, respectively, while 4 μM resulted in small conduction disturbances (likely due to increased number of dead myocytes) and a drop in CV to 14.7 cm/s (Fig. 4F). In control CM, ATR had no effect on conduction velocity.


Cardiac Optogenetics: Enhancement by All-trans-Retinal.

Yu J, Chen K, Lucero RV, Ambrosi CM, Entcheva E - Sci Rep (2015)

ATR effects on cardiomyocyte electrophysiology.(A) Action potentials in response to electrical pacing were imaged optically (using di4-ANBDQBS) and presented as mean ± SEM at each point for each group. (B) Quantified APD80 for control CM and ChR2-CM without ATR and with 1 μM ATR (highest optical excitability). Both A and B had n = 3–4 samples per experimental group. (C) Calcium transients in response to electrical pacing were imaged optically (using Rhod4-AM) and presented as mean ± SEM at each point for each group. (D) Quantified CTD80 for control CM and ChR2-CM at different ATR supplements. Both C and D had n = 7–33 samples for each of the eight experimental groups. (E) Example activation maps of ChR2-CM, following point electrical stimulation at the bottom; isochrones are 10 ms apart. Scale bar is 5 mm. (F) Quantified conduction velocity from the activation maps (n = 7–14 per group). (*) indicates significant difference at p < 0.05 compared to the respective control (zero ATR) or as indicated by the brackets.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4644984&req=5

f4: ATR effects on cardiomyocyte electrophysiology.(A) Action potentials in response to electrical pacing were imaged optically (using di4-ANBDQBS) and presented as mean ± SEM at each point for each group. (B) Quantified APD80 for control CM and ChR2-CM without ATR and with 1 μM ATR (highest optical excitability). Both A and B had n = 3–4 samples per experimental group. (C) Calcium transients in response to electrical pacing were imaged optically (using Rhod4-AM) and presented as mean ± SEM at each point for each group. (D) Quantified CTD80 for control CM and ChR2-CM at different ATR supplements. Both C and D had n = 7–33 samples for each of the eight experimental groups. (E) Example activation maps of ChR2-CM, following point electrical stimulation at the bottom; isochrones are 10 ms apart. Scale bar is 5 mm. (F) Quantified conduction velocity from the activation maps (n = 7–14 per group). (*) indicates significant difference at p < 0.05 compared to the respective control (zero ATR) or as indicated by the brackets.
Mentions: We further examined the effects of ATR supplementation on CM electrophysiology by quantifying action potentials, calcium transients, and conduction properties. We compared optically-measured (via Di-4-ANBDQBS) action potential durations (APDs) of ChR2-CM to CM with the same treatment, Fig. 4A. At zero ATR, CM and ChR2-CM showed overlapping AP and similar APD80 of 260 ms and 270 ms, respectively; reassuring of the benign nature of the opsin expression and optogenetic activation. Addition of 1 μM ATR significantly prolonged the APD80 in ChR2-CMs by 44% (p < 0.05) compared to ChR2-CM without ATR (Fig. 4B). Similarly, at zero ATR, no significant differences were seen in calcium transient morphology between the CM and ChR2-CM groups across the tested conditions (Fig. 4C); CTD80 of CM and ChR2-CM were 552 ms and 530 ms, respectively (Fig. 4D). Supplementation with ATR of 1, 2, and 4 μM in control CM led to: 13.8% (p < 0.05), 13.3% (p < 0.05), and 4% (n.s.) prolongation of CTD80, respectively. In ChR2-CM samples, exogenous ATR supplementation also led to small but significant prolongation of CTD80 by approximately 10.9%, 11.6%, and non-significant 10% for the same tested concentrations (Fig. 4D). Examination of propagation and activation maps for the different ChR2-CM groups (Fig. 4E) showed that ATR supplementation at 1 and 2 μM yielded smooth and fast propagation with similar CV to ChR2-CM control: 19.1 cm/s, 17.0 cm/s, and 17.0 cm/s for 0, 1, and 2 μM, respectively, while 4 μM resulted in small conduction disturbances (likely due to increased number of dead myocytes) and a drop in CV to 14.7 cm/s (Fig. 4F). In control CM, ATR had no effect on conduction velocity.

Bottom Line: 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/mm(2), 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, Stony Brook University, Stony Brook, NY.

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/mm(2), 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.

No MeSH data available.


Related in: MedlinePlus