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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

Theoretical explanation of optical excitability and sensitivity to supplemental ATR.(A) Two possible paths to the observed increase in optical excitability by the addition of ATR. (B) Electric field thresholds for excitation are not significantly altered by adding ATR, thereby eliminating the first possibility in (A), while indirect evidence exists for the second possibility (Fig. 2). (C) Light stimulation triggers retinal’s isomerization from trans to cis form, which in turn leads to ChR2 opening and net inward current. Several possibilities exist for the relationship between IChR2 and [ATR]suppl, including simple linear scaling (1), saturating curve (2), as indirectly suggested by our ChR2 experimental data, or a hypothetical non-monotonic parabolic relationship with an optimal ATR concentration (3). (D) From assumed monotonic relationship between IChR2 and [ATR]suppl (we used the linear curve type 1), we calculate the optical Eth to stimulate human cardiomyocytes by light as function of [ATR]suppl (in silico data); overlaid is a simple hyperbolic curve fit. Two distinct regions exist—of high sensitivity to ATR (if the endogenous ATR levels are low, possibly like found in cardiac tissue), and low sensitivity to ATR (for saturating ATR levels, possibly like neuronal or retinal tissue). Note that a saturating curve (type 2 in panel C) would yield a similar but steeper relationship. The inset replots our experimental data from Fig. 3, and red arrows indicate that the theoretical curve and the experimental data differ after ATR = 1 μM. This is likely due to cytotoxicity caused by excess ATR, independent of the ATR-ChR2 interactions, e.g. cell viability change as seen in Fig. 1 (replotted upper right panel) or, less likely, due to a non-monotonic parabolic relationship between IChR2 and [ATR]suppl, which would mean that functional IChR2 data does not follow the expression levels (see overlaid data from Fig. 2 in the lower right panel).
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f5: Theoretical explanation of optical excitability and sensitivity to supplemental ATR.(A) Two possible paths to the observed increase in optical excitability by the addition of ATR. (B) Electric field thresholds for excitation are not significantly altered by adding ATR, thereby eliminating the first possibility in (A), while indirect evidence exists for the second possibility (Fig. 2). (C) Light stimulation triggers retinal’s isomerization from trans to cis form, which in turn leads to ChR2 opening and net inward current. Several possibilities exist for the relationship between IChR2 and [ATR]suppl, including simple linear scaling (1), saturating curve (2), as indirectly suggested by our ChR2 experimental data, or a hypothetical non-monotonic parabolic relationship with an optimal ATR concentration (3). (D) From assumed monotonic relationship between IChR2 and [ATR]suppl (we used the linear curve type 1), we calculate the optical Eth to stimulate human cardiomyocytes by light as function of [ATR]suppl (in silico data); overlaid is a simple hyperbolic curve fit. Two distinct regions exist—of high sensitivity to ATR (if the endogenous ATR levels are low, possibly like found in cardiac tissue), and low sensitivity to ATR (for saturating ATR levels, possibly like neuronal or retinal tissue). Note that a saturating curve (type 2 in panel C) would yield a similar but steeper relationship. The inset replots our experimental data from Fig. 3, and red arrows indicate that the theoretical curve and the experimental data differ after ATR = 1 μM. This is likely due to cytotoxicity caused by excess ATR, independent of the ATR-ChR2 interactions, e.g. cell viability change as seen in Fig. 1 (replotted upper right panel) or, less likely, due to a non-monotonic parabolic relationship between IChR2 and [ATR]suppl, which would mean that functional IChR2 data does not follow the expression levels (see overlaid data from Fig. 2 in the lower right panel).

Mentions: Theoretically, an increase in the ATR-mediated optical excitability in our experimental system can result in two ways (Fig. 5A): 1) exogenous ATR may act as a sensitizer of the cardiomyocytes, increasing their intrinsic excitability, independent of ChR2, and therefore reducing the opposing currents (to ChR2) and/or altering cell-cell coupling to trigger activity; and 2) exogenous ATR may augment the macroscopic ChR2 photocurrent, potentially by stabilizing more functional ChR2 molecules in the membrane at any given time, as suggested by the expression levels shown in Fig. 2. Using electrical stimulation, we tested the first possibility and found no evidence for supplemental ATR increasing the intrinsic excitability of the cardiac syncytium (Fig. 5B). Similarly, there was no evidence of increased self-oscillatory activity in the ATR-treated samples (CM or ChR2-CM), which would have been consistent with enhanced excitability. Therefore, indirectly, by elimination, we believe that the most plausible factor leading to increased optical excitability is indeed an ATR-augmented ChR2 photocurrent, likely by a previously proposed stabilizing mechanism for the ChR2 channels28. Even though our fluorescent data support such mechanism, a limitation of this study is that we did not directly measure the ChR2 photocurrents in cardiomyocytes under different ATR treatments.


Cardiac Optogenetics: Enhancement by All-trans-Retinal.

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

Theoretical explanation of optical excitability and sensitivity to supplemental ATR.(A) Two possible paths to the observed increase in optical excitability by the addition of ATR. (B) Electric field thresholds for excitation are not significantly altered by adding ATR, thereby eliminating the first possibility in (A), while indirect evidence exists for the second possibility (Fig. 2). (C) Light stimulation triggers retinal’s isomerization from trans to cis form, which in turn leads to ChR2 opening and net inward current. Several possibilities exist for the relationship between IChR2 and [ATR]suppl, including simple linear scaling (1), saturating curve (2), as indirectly suggested by our ChR2 experimental data, or a hypothetical non-monotonic parabolic relationship with an optimal ATR concentration (3). (D) From assumed monotonic relationship between IChR2 and [ATR]suppl (we used the linear curve type 1), we calculate the optical Eth to stimulate human cardiomyocytes by light as function of [ATR]suppl (in silico data); overlaid is a simple hyperbolic curve fit. Two distinct regions exist—of high sensitivity to ATR (if the endogenous ATR levels are low, possibly like found in cardiac tissue), and low sensitivity to ATR (for saturating ATR levels, possibly like neuronal or retinal tissue). Note that a saturating curve (type 2 in panel C) would yield a similar but steeper relationship. The inset replots our experimental data from Fig. 3, and red arrows indicate that the theoretical curve and the experimental data differ after ATR = 1 μM. This is likely due to cytotoxicity caused by excess ATR, independent of the ATR-ChR2 interactions, e.g. cell viability change as seen in Fig. 1 (replotted upper right panel) or, less likely, due to a non-monotonic parabolic relationship between IChR2 and [ATR]suppl, which would mean that functional IChR2 data does not follow the expression levels (see overlaid data from Fig. 2 in the lower right panel).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: Theoretical explanation of optical excitability and sensitivity to supplemental ATR.(A) Two possible paths to the observed increase in optical excitability by the addition of ATR. (B) Electric field thresholds for excitation are not significantly altered by adding ATR, thereby eliminating the first possibility in (A), while indirect evidence exists for the second possibility (Fig. 2). (C) Light stimulation triggers retinal’s isomerization from trans to cis form, which in turn leads to ChR2 opening and net inward current. Several possibilities exist for the relationship between IChR2 and [ATR]suppl, including simple linear scaling (1), saturating curve (2), as indirectly suggested by our ChR2 experimental data, or a hypothetical non-monotonic parabolic relationship with an optimal ATR concentration (3). (D) From assumed monotonic relationship between IChR2 and [ATR]suppl (we used the linear curve type 1), we calculate the optical Eth to stimulate human cardiomyocytes by light as function of [ATR]suppl (in silico data); overlaid is a simple hyperbolic curve fit. Two distinct regions exist—of high sensitivity to ATR (if the endogenous ATR levels are low, possibly like found in cardiac tissue), and low sensitivity to ATR (for saturating ATR levels, possibly like neuronal or retinal tissue). Note that a saturating curve (type 2 in panel C) would yield a similar but steeper relationship. The inset replots our experimental data from Fig. 3, and red arrows indicate that the theoretical curve and the experimental data differ after ATR = 1 μM. This is likely due to cytotoxicity caused by excess ATR, independent of the ATR-ChR2 interactions, e.g. cell viability change as seen in Fig. 1 (replotted upper right panel) or, less likely, due to a non-monotonic parabolic relationship between IChR2 and [ATR]suppl, which would mean that functional IChR2 data does not follow the expression levels (see overlaid data from Fig. 2 in the lower right panel).
Mentions: Theoretically, an increase in the ATR-mediated optical excitability in our experimental system can result in two ways (Fig. 5A): 1) exogenous ATR may act as a sensitizer of the cardiomyocytes, increasing their intrinsic excitability, independent of ChR2, and therefore reducing the opposing currents (to ChR2) and/or altering cell-cell coupling to trigger activity; and 2) exogenous ATR may augment the macroscopic ChR2 photocurrent, potentially by stabilizing more functional ChR2 molecules in the membrane at any given time, as suggested by the expression levels shown in Fig. 2. Using electrical stimulation, we tested the first possibility and found no evidence for supplemental ATR increasing the intrinsic excitability of the cardiac syncytium (Fig. 5B). Similarly, there was no evidence of increased self-oscillatory activity in the ATR-treated samples (CM or ChR2-CM), which would have been consistent with enhanced excitability. Therefore, indirectly, by elimination, we believe that the most plausible factor leading to increased optical excitability is indeed an ATR-augmented ChR2 photocurrent, likely by a previously proposed stabilizing mechanism for the ChR2 channels28. Even though our fluorescent data support such mechanism, a limitation of this study is that we did not directly measure the ChR2 photocurrents in cardiomyocytes under different ATR treatments.

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