Läs källbundna sammanställningar.

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.

Cardiomyocyte viability upon ChR2 expression and ATR treatment

Initial exploration of dosing with exogenous ATR revealed a narrow window of viable concentrations (). ATR supplementation greater than 4 μM was toxic to the cardiomyocytes, regardless of whether they expressed ChR2 or not, and caused complete cell death above 8 μM. However, ATR supplements less than 4 μM appeared to paradoxically improve cell viability even though control samples were quite healthy (): CM with 1 and 2 μM ATR supplements had only 0.1 × and 0.15 × the dead cell amount of CM control (virtually eliminating dead cells, i.e. <2% cell death), p ≪ 0.001 for both, and ChR2-CM with 1 and 2 μM ATR supplements had only 0.38 × and 0.45 × the dead cell amount of CM control, p < 0.01 for both. Intermediate ATR concentration of 4 μM increased cell death compared to cells without ATR supplementation, significant in both CM (1.8 × increase) and ChR-CM cells (4.4 × increase), p < 0.01 for both. Furthermore, it is worth noting that when ATR was present, the ChR2-CM samples had consistently lower viability than the CM samples at the same ATR, even though this was not the case for non-supplemented samples.

ChR2 expression with ATR supplementation

Since ChR2 stabilization/degradation had been reported to be dependent on ATR availability, we examined ChR2 expression efficiency (by eYFP reporter) in the viable range of ATR supplements, i.e. 0–4 μM. Employing previously reported infection methods without the addition of exogenous ATR, stable and robust transgene expression (>98%) was achieved within 48 hours, (, middle row, first image). We confirmed that in confocal image acquisition of fluorescence (eYFP), greater ATR concentrations did not result in an increase in autofluorescence (, top row). ATR supplementation in ChR2-CM samples led to significant enhancement of ChR2 expression, indicated by the saturation of eYFP fluorescence at the higher doses, despite our attempt to select a gain that maximizes the dynamic range and accommodates all ATR dosing groups (, middle row). Higher resolution images and lowered gain to optimize imaging of the ChR2-CM samples with 4 μM are also shown ( bottom row). Image analysis of eYFP-positive pixels (normalized per cell) showed 3.8 ×, 5 × and 5.9 × increase in ChR2 expression by the addition of 1, 2, and 4 μM ATR compared to ChR2-CM without retinal, p ≪ 0.001 in all cases (). The eYFP fluorescence indicates the total number of expressed channels, including non-functional channelopsins without ATR, therefore the increases shown in represent an upper bound for functional ChR2 channels. We assume that the data indicate a proportionally increasing number of stabilized functional ChR2 channels (fraction of the total reported channels), though increase in actual ChR2 current may be more modest.

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.

Functional electrophysiology effects of ChR2 expression and ATR supplementation in cardiomyocytes

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, . 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 (). Similarly, at zero ATR, no significant differences were seen in calcium transient morphology between the CM and ChR2-CM groups across the tested conditions (); CTD80 of CM and ChR2-CM were 552 ms and 530 ms, respectively (). 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 (). Examination of propagation and activation maps for the different ChR2-CM groups () 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 (). In control CM, ATR had no effect on conduction velocity.

Functional electrophysiology effects of ChR2 expression and ATR supplementation in cardiomyocytes

Overall, low concentrations of ATR (up to 2 μM) helped improve survival of cardiomyocytes in standard culturing conditions, did not alter conduction and resulted in significant APD prolongation but only slight prolongation of intracellular calcium transients in the ChR2-expressing cardiomyocytes.

Abstract

All‐trans‐retinal (atRAL) is a highly reactive carbonyl specie, known for its reactivity on cellular phosphatidylethanolamine in photoreceptor. It is generated by photoisomerization of 11‐cis‐retinal chromophore linked to opsin by the Schiff's base reaction. In ABCA4‐associated autosomal recessive Stargardt macular dystrophy, atRAL results in carbonyl and oxidative stress, which leads to bisretinoid A2E, accumulation in the retinal pigment epithelium (RPE). This A2E‐accumulation presents as lipofuscin fluorescent pigment, and its photooxidation causes subsequent damage. Here we describe protection against a lethal dose of atRAL in both photoreceptors and RPE in primary cultures by a lipidic polyphenol derivative, an isopropyl‐phloroglucinol linked to DHA, referred to as IP‐DHA. Next, we addressed the cellular and molecular defence mechanisms in commonly used human ARPE‐19 cells. We determined that both polyunsaturated fatty acid and isopropyl substituents bond to phloroglucinol are essential to confer the highest protection. IP‐DHA responds rapidly against the toxicity of atRAL and its protective effect persists. This healthy effect of IP‐DHA applies to the mitochondrial respiration. IP‐DHA also rescues RPE cells subjected to the toxic effects of A2E after blue light exposure. Together, our findings suggest that the beneficial role of IP‐DHA in retinal cells involves both anti‐carbonyl and anti‐oxidative capacities.

INTRODUCTION

In the present study, we investigated the protective effect of the medicinal chemical compound, isopropyl‐phloroglucinol‐DHA (IP‐DHA), also called lipophenol, against atRAL‐related carbonyl and oxidative stresses (COS). We first analysed primary cultures of outer retina to demonstrate the dose‐dependent protective effect against lethal dose of atRAL. We then used ARPE‐19 cells as a standard cellular model to study the mechanisms of cell death and protection. We demonstrate that each of the structural part, that is isopropyl and PUFA, is essential for the full action of the lipophenol with selectivity for LA and DHA. We compared the capacities of phloroglucinol and IP‐DHA to reverse the effects of atRAL and finally discuss how to understand the enhanced protective effects of IP‐DHA compared to phloroglucinol.

Cell cultures and atRAL treatment

Primary rat RPE and mouse neural retina (NR) cultures were obtained as previously described. RPE cells were cultured for 3 days until they reached 80%‐85% confluency, and NR was cultured for 10 days until glial cells were confluent in the bottom layer and neural cells with a neurite outgrowth in the upper layer. Human RPE‐like cells, ARPE‐19, were seeded at 100 000 cells/cm2 and grown to confluency before being assayed as instruct by ATCC.

Cell cultures and atRAL treatment

Pre‐ and co‐treatment procedures with lipophenol were carried out. During pre‐treatments, rat RPE primary cultures received a medium containing lipophenol at different concentrations (40‐320 μmol/L) for 24 hours. The medium was then removed and replaced by a serum‐free culture medium containing 25 μmol/L atRAL for 4 hours. During co‐treatments, RPE and NR cells received a serum‐free medium containing 25 μmol/L atRAL and/or IP‐DHA (10‐320 μmol/L) for 4 hours. NR cells were refreshed with serum‐free medium the next day.

A2E treatment

ARPE‐19 cells were plated into 96‐well plates (4 × 104 cells/well) and cultured for 24 hours to confluence prior to lipophenol treatment. The cell cultures were treated with serum‐free DMEM/F12 medium without phenol red containing IP‐DHA at different concentrations (0‐80 μmol/L) for 1 hour. A2E was then added to a final concentration of 20 μmol/L for 6 hours before rinsing with medium. Control cells were incubated with 0.2% DMSO with or without A2E. The cells were exposed to intense blue light (4600 lux) for 30 minutes to induce phototoxicity of A2E and incubated at 37°C. Irradiation was achieved using a LED device with blue emission wavelengths from 430 to 470 nm and a dimmable luminance (Roleadro lighting). Control ambient white light was less than 300 lux. Without blue light exposure, A2E‐loaded cell survival was not affected. The cell viability was determined 16‐20 hours later using a MTT colorimetric assay. Results are expressed in percentage of viable cells normalized to control conditions in the absence of lipophenol and stressor.

Formation of bisretinoid fluorophores by reaction of vitamin a aldehyde with lipid

Incubation of bovine outer segments with all-trans-retinal mimics bisretinoid formation in vivo, and by optimizing the detection of these bisretinoid species, their formation by all-trans-retinal reactivity was confirmed (). Indeed, adjusting the ratio of all-trans-retinal to PE in synthetic mixtures to determine how the two precursors affect bisretinoid production showed that the yield of bisretinoid saturates when the ratio of equivalents of all-trans-retinal to PE (egg-PE as starting material) is 4:2; no further increase occurred with eight equivalents of all-trans-retinal (8:2 ratio) (). Conversely, increasing the concentration of PE from an equivalent ratio of 4:1 through 4:8 (all-trans-retinal to PE) was associated with a steady increase in bisretinoid product. These findings indicated that the concentration of PE in the environment of the outer segment membrane has a marked effect on the rate of bisretinoid synthesis. Formation of NRPE, the first step in the synthetic pathway, is likely the most facile reaction. In the Abca4−/− mouse, a mouse model exhibiting accelerated bisretinoid formation, the level of PE in the outer segment membrane was reported to be increased (). It has been suggested that a higher level of PE in Abca4−/− mice may facilitate sequestering of all-trans-retinal as NRPE, thereby accelerating recovery of the rod photoresponse after a 30–40% bleach ().

Formation of bisretinoid fluorophores by reaction of vitamin a aldehyde with lipid

The unsaturated fatty acid 22:6n-3 (DHA) is abundant in the outer segments of photoreceptor cells, and DHA content has been shown to have several effects on retina (), including susceptibility to light damage. When 22:6n-3-containing PE was used as precursor in the synthetic reaction mixture versus egg-PE (a mixture of fatty acid moieties), phosphatidyl-pyridinium bisretinoid (A2PE) production was found to be greater with the latter. Then again, if free DHA was added to the egg-PE/all-trans-retinal reaction mixture, the yield of A2PE was increased. Under deprotonating conditions associated with the addition of base (triethylamine) to the reactants, the generation of A2PE was increased. Altogether, not only is the phospholipid PE the reactant that combines with all-trans-retinal to form bisretinoid, the lipid composition of outer segments may also impact the extent to which the reaction may be favored.