Cardiac Optogenetics: Enhancement by All-trans-Retinal

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.

Scientific RepoRts | 5:16542 | DOI: 10.1038/srep16542 cells and tissues in vitro and in vivo, availability of retinal has not been viewed as a limiting factor and optical excitation has been possible without ATR supplementation 6 or with other supplements that included vitamin A 25 . Nevertheless, considering that ATR in nonretinal and nonembryonic tissues is only present in small amounts (< 0.5 nmol/g) 26 , it is feasible to expect that exogenous ATR may modulate the optogenetic response, at least in some tissues, such as cardiac muscle, which was found to contain lower amounts of endogenous retinoids compared to liver, kidney, adipose and brain tissue, based on tissue measurements with chromatography and UV techniques (HPLC/UV) 26,27 . Given ATR's diverse roles in different tissues, it is important to investigate its specific effects on cardiac electrophysiology and the possibility of its use as a potent light sensitizer in applications of optogenetics to the heart. Therefore, in this study, we set out to explore the in vitro use of exogenous ATR as a photosensitizer in cardiomyocytes (CMs), genetically modified to express ChR2, and its potential to substantially reduce optical stimulation energy.

Results
Cardiomyocyte viability upon ChR2 expression and ATR treatment. We first confirmed that the genetic modifications of cardiomyocytes with ChR2 (Ad-ChR2-eYFP virus and its infection process) were benign. Using optimized multiplicity of infection 11 , ChR2-CM with no exogenous ATR had the same high viability rate as CM control, i.e. about 11% death as indicated by PI fluorescence image analysis.
Initial exploration of dosing with exogenous ATR revealed a narrow window of viable concentrations (Fig. 1). ATR supplementation greater than 4 μ M was toxic to the cardiomyocytes, regardless of whether they expressed ChR2 or not, and caused complete cell death above 8 μ M. However, ATR supplements less than 4 μ M appeared to paradoxically improve cell viability even though control samples were quite healthy (  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 28 , we examined ChR2 expression efficiency (by eYFP reporter) in the viable range of ATR supplements, i.e. 0-4 μ M. Employing previously reported infection methods 11 without the addition of exogenous ATR, stable and robust transgene expression (> 98%) was achieved within 48 hours 11,5 (Fig. 2A, 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 ( Fig. 2A, 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 ( Fig. 2A, middle row). Higher resolution images and lowered gain to optimize imaging of the ChR2-CM samples with 4 μ M are also shown ( Fig. 2A 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 μ M ATR compared to ChR2-CM without retinal, p ≪ 0.001 in all cases (Fig. 2B). The eYFP fluorescence indicates the total number of expressed channels, including non-functional channelopsins without ATR, therefore the increases shown in Fig. 2 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.
Augmentation of optical excitability of cardiac syncytia with ATR supplementation. To assess in detail the effects of ATR supplementation on modulating optical excitability, we conducted functional analysis in a total of 12 experimental groups, using six ATR doses in CM and ChR2-CM: 0, 0.1, 0.5, 1, 2, and 4 μ M ATR. We quantified the minimal optical power (irradiance in mW/mm 2 ) needed to elicit global excitation in cardiac syncytia in vitro at different light pulse durations (1 to 90 ms) by constructing strength-duration curves for optical excitation for each of the six ChR2-CM groups (Fig. 3). Irradiance thresholds were determined for successful capture of at least 10 consecutive beats by optical pacing.
The ChR2-CM group without retinal supplementation showed excellent excitability (less than 0.5 mW/mm 2 for all pulse durations), on par with previous values for cell delivery to drive cardiac syncytia by light 15 . Gradual monotonic drop in E th (increase in optical excitability) across pulse durations was observed as the ChR2-CM samples were dosed with 0.1, 0.5 and 1 μ M of ATR, achieving a reduction in E th of over 30 times for 1 μ M ATR. Interestingly, increasing ATR concentration to 4 μ M did not further reduce the threshold but reversed the trend towards the levels seen in un-supplemented ChR2-CM, despite increased ChR2 expression as seen in Fig. 2. This non-monotonic effect of ATR on optical excitability (see inset in Fig. 3) was in line with our findings on cell viability in Fig. 1. Detailed statistical comparison for all studied cases can be seen in Supplementary Table S1. In an alternative view, the optical strength-duration curves were further fitted to extract rheobase (the asymptotic irradiance at long pulse durations, i.e. minimal irradiance to stimulate) and chronaxie (a parameter related to the response kinetics of the biological system), Supplementary. Fig. S1. While rheobase followed the same trend of significant reduction in the 1 and 2 μ M ATR groups, chronaxie did not reveal a distinct pattern.
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 Strength-duration curves (presented on a log-log scale) for ChR2-CM (at different ATR concentrations), linking the minimal irradiance, E th , needed to optically trigger propagating waves in cardiac syncytia at different light pulse durations. Data from n = 3 to 13 samples per data point, presented as mean ± SEM. The inset plot (non-log scale) illustrates the nonmonotonic dependence of E th on exogenous ATR concentration using data from 1 and 10 ms pulses. Highest optical excitability in ChR2-CM, or lowest E th , was achieved with 1 or 2 μ M ATR. Two way ANOVA analysis (independent factors are pulse duration and ATR concentrations), setting α level at 0.05, produced 36 interaction comparisons (see Supplementary. Table S1).
Scientific RepoRts | 5:16542 | DOI: 10.1038/srep16542 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 μ M 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 (*) indicates significant difference at p < 0.05 compared to the respective control (zero ATR) or as indicated by the brackets. 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 μ M in control CM led to: 13.8% (p < 0.05), 13.3% (p < 0.05), and 4% (n.s.) prolongation of CTD 80 , respectively. In ChR2-CM samples, exogenous ATR supplementation also led to small but significant prolongation of CTD 80 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 μ M 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 μ M, respectively, while 4 μ M 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.
Overall, low concentrations of ATR (up to 2 μ M) 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.

Discussion
Dramatic optical energy benefits of ATR supplementation in cardiac cells. Means to manipulate cell electrophysiology optogenetically using low light are of great interest because of simpler setups (LED light sources vs. lasers) and avoidance of potential side effects on the cells, e.g. thermal effects caused by higher-intensity light (see Supplement in our previous publication 14 ). So far, mutagenesis has produced a number of opsins with enhanced photocurrents and/or enhanced light sensitivity, including the recent super-sensitive ChR2-XXL, but in all cases, these optical enhancements come with a sacrifice in kinetics 29 . Such slow kinetics may not be ideal for pacing applications. Enhancement of optogenetic response by ATR or synthetic ATR mutants 30 , on the other hand, can theoretically yield low-light operation with less interference with opsin kinetics. When supplemented exogenously, as nutrients, Vitamin A derivatives can be transported via the vasculature into tissues 31 , and the photosensitizer ATR can be recruited to the cytoplasm to help form functional ChR2 complexes by covalently binding channelopsins 20 . Such diet-mediated delivery has worked in C. elegans 21 and D. melanogaster 24 . As ATR is the chromophore for mammalian and microbial opsins, higher ATR concentrations are expected to increase light responsiveness, in general. Yet modulation of optogenetic responsiveness of mammalian cells by ATR has not been systematically studied before.
The baseline optical excitability of the cardiac syncytia, used in this study, without exogenous ATR, was excellent, i.e. using adenoviral delivery in cardiomyocytes we achieved irradiances needed to excite well under 1 mW/mm 2 even for short pulses. Yet, the application of very low ATR doses (< 2 μ M) led to a further significant reduction in irradiance for excitation. While the photosensitizing effect of ATR on the ChR2 operation was expected based on prior studies in cells and organisms lacking endogenous retinal 21,24 , the surprising finding here is the extent of such enhancement in optical excitability of cardiomyocytes -1-2 orders of magnitude across light pulse durations (Fig. 3). In contrast, in previous studies in neurons, ATR supplementation was not considered a factor in optical excitability 25 , presumably because of sufficient/saturating levels of ATR for ChR2 operation in neural cells, grown in B-27 supplemented medium, which contains some (undisclosed) amount of retinyl acetate. The implications of our findings are that when ATR is supplemented in low concentrations, extremely low light levels may be suitable for cardiac applications (this is the first study to report light levels needed to excite ChR2(H134R) down to a couple of μ W/mm 2 ), including optical pacing and cardioversion/defibrillation. This is important because such low threshold light levels are in the range of a variety of newer miniaturized and implantable low-power and/or remote-powered solutions for potential in vivo operation 32,33 , though such in vivo testing was not demonstrated here and is beyond the scope of this report. Further studies need to be conducted in adult cardiac tissue, which is denser, better coupled and presumably harder to excite, to see if controlled ATR supplements may help reduce the required light levels. Since all current optogenetic actuators of voltage (depolarizing and hyperpolarizing) use ATR as a chromophore, our findings have broader implications beyond the effects on ChR2 alone.

From augmentation of ChR2 expression to improvement of optical excitability. Recently,
Nagel and colleagues have elucidated the stabilizing role of ATR as a cofactor in the formation of functional ChR2 channels by showing that the absence of retinal results in faster degradation of ChR2 28 . Here in cardiomyocytes, we demonstrate that addition of very low concentrations of ATR increases ChR2 expression several-fold, as measured by the reporter eYFP (Fig. 2). This 5-6 times increase in ChR2 expression (likely through increase in the number of functional ChR2 channels in the membrane) then seems to result in much larger (over 30 times) augmentation of optical excitability for supplementation with 1-2 μ M ATR (Fig. 3), as discussed above.
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 channels 28 . 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.
Furthermore, we considered the relationship between the optical energy required for tissue excitation and the ChR2 photocurrent, I ChR2 (and its augmentation by ATR supplement), Fig. 5C,D, i.e. how does a five-fold increase in ChR2 expression translate into 1-2 orders of magnitude increase in optical excitability? Using a computational model of ChR2 in (human) cardiomyocytes, we have examined the relationship between irradiance needed to trigger an action potential and ChR2 conductance (see Supplement in our previous publication 14 ). Current biophysical models of ChR2 14,22 consider a simple scaling effect of ATR on ChR2 conductance (g ChR2 ), i.e. the g ChR2 can be alternatively viewed as ATR concentration (linear curve 1 in the bottom panel of Fig. 5C); a more biologically-realistic curve would be a non-linear saturating increase (curve 2 in Fig. 5C, which is also suggested by the ChR2 expression  (2), as indirectly suggested by our ChR2 experimental data, or a hypothetical nonmonotonic parabolic relationship with an optimal ATR concentration (3). (D) From assumed monotonic relationship between I ChR2 and [ATR] suppl (we used the linear curve type 1), we calculate the optical E th 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 μ M. 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 I ChR2 and [ATR] suppl , which would mean that functional I ChR2 data does not follow the expression levels (see overlaid data from Fig. 2

in the lower right panel).
Scientific RepoRts | 5:16542 | DOI: 10.1038/srep16542 data from Fig. 2); a third possibility is a purely hypothetical non-monotonic parabolic curve (curve 3 in Fig. 5C), where an optimal ATR concentration may exist, beyond which excess ATR actually interferes with ChR2 function and reduces I ChR2 .
Using the linear curve 1, we show the model-generated dependence of the threshold irradiance for cardiac excitation, E th , on supplemental ATR, Fig. 5D; note that using a nonlinear saturating curve 2 would yield a similar but steeper monotonically decreasing relationship. A hyperbolic curve is fit for the E th as function of ATR -such theoretical (1/x) dependence is expected for an ideal rectangular current, where a lower ChR2 conductance (lower peak current) would require a corresponding increase of stimulus strength to maintain the same charge needed to excite. For the light-and voltage-dependent ChR2 current, such a fit is only qualitative because the provided current pulse is not rectangular, i.e. the ChR2 current waveform is feedback-regulated by the membrane voltage 12 . Two distinct regions of sensitivity of E th to ChR2 conductance (or indirectly to ATR availability) are seen in Fig. 5D: 1) a high sensitivity region (for low endogenous ATR levels, as perhaps seen here for cardiomyocytes) and 2) a low sensitivity region (for saturating ATR levels, as perhaps seen in retinal and neural tissues). Operation in the steep-slope region (high sensitivity to ATR) can explain how a 5-6 times increase in ChR2 conductance may yield 30 times decrease in irradiance needed to excite cardiac cells, as reported here. Comparison of our experimental data for E th (replotted example from Fig. 3 into the inset of Fig. 5D) to the theoretical predictions reveals similarities in the steep declining portion, but also differences -an increase in E th beyond 1 μ M ATR (red arrows). A likely explanation for this rise in E th comes from the ATR-related cytotoxicity data (replotted from Fig. 1). Akin to receptor-mediated hormonal action 34 , ATR seems to operate within a well-defined optimal range in cardiac cells (see below), and a decrease in cell viability is seen > 2 μ M ATR, which can explain the reduced optical excitability. Such cytotoxic effects are not related to ChR2-ATR interactions per se and are not part of the model predictions. An alternative, less likely, cause for the deviation between experimental and theoretical data is the possibility for a non-monotonic parabolic relationship between supplemental ATR and I ChR2 (hypothetical curve 3 in Fig. 5C), which would be in contrast to the expression data from Fig. 2, showing total channelopsin expression with or without bound ATR. A more mechanistic study to test such proposition would require extensive photocurrent characterization under different conditions. ATR effects on cardiac electrophysiology: narrow range of safe ATR supplementation in cardiac tissue. This study presents a first glimpse at the electrophysiological effects of optogenetically modifying cardiomyocytes (directly by adenoviruses) to express the light-sensitive actuator ChR2. Similar to our earlier findings using dedicated ChR2-expressing donor cells coupled to non-modified CMs, employing the "tandem-cell-unit" 15 , we find here no significant alterations in the action potentials, calcium transients or conduction velocity between control CMs and genetically modified ChR2-CMs (Fig. 4). This is an important baseline characterization in order to use optical actuation as a "clean" tool for affecting cardiac function.
Furthermore, in this study, we quantified the effects of supplemental ATR on cardiomyocyte electrophysiology. For low doses of supplemented ATR (< 4 μ M), the ChR2-CM exhibited significant prolongation of APD80 (44%) and small CTD80 prolongation (< 15%), without significant effects on conduction velocity, Fig 4. The APD prolongation was only seen in ChR2-CMs and not in the control CM samples supplemented with 1 μ M ATR, which points to ChR2-dependent effects of ATR on repolarization. One possibility is that the enhanced light sensitivity by ATR supplementation affects the entire spectrum of ChR2 excitation, bringing about some unexpected responsiveness to the longer wavelength (660 nm) used for voltage dye imaging, at which normally ChR2 should not have any absorption. If so, some small residual depolarizing current via ChR2 may indeed prolong the APD. However, further studies are needed to fully understand the mechanism of these electrophysiological changes and the potential ion currents involved.
Overall, our results indicate high sensitivity of cardiomyomyocytes to exogenous ATR -in this in vitro system, the optimal range was determined to be 1-2 μ M, with a sharp increase in toxicity above 4 μ M (Figs 1 and 4), regardless whether the cells expressed an opsin or not, but this ATR-mediated cytotoxicity seems to be enhanced for ChR2-CM compared to CM (Fig. 1). This is not surprising considering the critical role retinoids play as potent regulators of gene expression and cell fate. Believed to control over 500 genes 35 , retinoids play a major role in cell growth, differentiation in embryogenesis, and in the adult stage. Among other effects, they also have been shown to augment differentiation of embryonic stem cells into ventricular cardiomyocytes 36 ; in zebra fish, immediately after ventricular injury, an upregulation in the cardiac production of retinoic acid was found, essential for heart regeneration 37 . The concentrations of intermediary products in the retinoid metabolism are likely tightly regulated to avoid undesired effects. For example, excess exogenous ATR is likely converted to retinoic acid (RA) and can result in elevated RA levels. Endogenous RA levels are maintained in a narrow range (1-4 nM) and excess RA is cytotoxic 38 , known to engage apoptosis-related signaling pathways. Therefore, excess RA is a likely toxicity driver in our results with ATR concentrations higher than 4 μ M (Fig. 1). Alternatively, excess ATR itself (not used by the opsins and not converted to RA) may be toxic. ATR has been reported as a competitive blocker of cyclic-nucleotide-gated (CNG) ion channels 39 . Previously characterized are cGMP-gated (rod) ion channels, showing full inhibition at 4 μ M ATR, coincidentally, on par with concentrations at which we detected cytotoxicity here. One can speculate that, at sufficiently high doses, ATR action may extend to other ion channels relevant to cardiac electrophysiology (cyclic-nucleotide-gated, e.g. HCN, or not). Either of these can explain the ATR's impact on cardiac electrophysiology and its clear toxicity outside of the narrow range of 1-2 μ M, found here. A limitation of this study is that only in vitro testing was conducted using multicellular cardiac samples prepared with neonatal rat cells, which have different electrophysiological characteristics than adult cardiomyocytes. Future studies in intact adult animals are needed to confirm our predictions (obtained in vitro, in neonatal myocytes) for ATR's potent light-sensitizing effects at small doses to lower the optical energies needed in cardiac applications of optogenetics. We conclude that cardiomyocytes, as tested in this in vitro study, seem to operate at non-saturating retinal levels, and carefully-dosed exogenous ATR can enhance the performance of ChR2 in cardiac cells and yield orders of magnitude energy benefits for optogenetic stimulation.
ChR2-CM were plated on fibronectin-treated glass bottom dishes (14 mm in diameter) at a density of 400 k/cm 2 to form well-connected CM monolayers (syncytia), and incubated in 5% CO 2 at 37 °C. On day 1, all samples were washed with PBS for 5 minutes with gentle shaking in dark and continued to be incubated in ATR-supplemented media with 10% FBS until day 3, when the ATR culture medium was switched to 2% FBS and exchanged every other day. All functional electrophysiological experiments were conducted on day 4 with Tyrode's perfusion maintained at 30 °C throughout data acquisition, as done before 15 . Samples used for imaging (of cell viability or ChR2 expression) were also utilized on the same day.

All-trans-Retinal (ATR) treatment. All-trans-Retinal (Sigma) was first dissolved in filtered DMSO
(Sigma) to a stock concentration of 100 mM, and further diluted in M199 growth medium (GIBCO) with 10% or 2% FBS to final concentration of 1 to 10 μ M. Prior literature shows that concentrations of < 0.02% DMSO do not have undesirable effects on neurons nor cardiomyocytes 42,43 . The growth media supplemented with ATR was filtered again before use. CMs were grown in ATR-containing medium with replenishment as described, up to the time of viability assay and experimentation. The experimental groups included both control and Ad-ChR2(H134R)-eYFP-infected samples, treated with the selected ATR concentration (0, 0.1, 0.5, 1, 2, 4, 8, and 10 μ M), i.e. 16 groups total.
Quantification of cell viability and ChR2 expression by image analysis. Images were acquired using the Olympus FluoView TM FV1000 confocal system, maintaining similar gain and exposure values across conditions for further processing. Image analysis and quantification were done using custom-developed software in Matlab (Mathworks, Natick, MA).
Cell viability was assessed by detecting dead cells' DNA fragments by staining with 2 μ g/ml Propidium Iodide (PI) (GIBCO) in Tyrode's solution on the 4 th day after isolation and infection. Quantification of cell death was done by removing image background based on a constant threshold for all groups and summing up the PI fluorescence pixels. Summed PI pixels of images of CM control with no ATR (18 FOV) were then averaged, and the mean served as a reference of normalization for other conditions. The summed PI pixels of images from all groups were then normalized to CM control PI average to calculate fold change.
Scientific RepoRts | 5:16542 | DOI: 10.1038/srep16542 ChR2 expression was judged by eYFP fluorescence, after the samples were fixed in 3.7% formaldehyde on the 4 th day after infection, followed by permeablization in 0.02% Triton X-100 and nuclei stain with 1 μ g/ml 4′ ,6-diamidino-2-phenylindole (DAPI). ChR2 expression was quantified by eYFP fluorescence as follows: Images of control CMs treated with different concentrations of ATR were processed to determine a noise threshold (for each ATR treatment), below which all green pixels were considered to be caused by autofluorescence. These noise thresholds were then used (by subtraction) to correct for background in ChR2-CM images of the corresponding ATR concentration. Thus corrected eYFP readouts were normalized by cell number from DAPI staining in each image. Finally, to present the data as fold-changes, results were pooled from multiple cultures, and the corrected and cell-normalized values for each ChR2-CM group, treated by ATR, were further divided by the mean value from the zero ATR ChR2-CM group.
Electrophysiological characterization: electrical stimulation, optical actuation and optical mapping. In order to characterize wave propagation in the CM monolayers, electrical stimulation (pulses 10 V, 0.005 s, 1 Hz) was delivered through a bipolar platinum electrode connected to a pulse generator (IonOptix, Milton, MA). Electric field stimulation (with a pair of line electrodes, spaced 8 mm apart) was used for testing the electrical excitability (variable field strength and pulse duration, 1 Hz pacing). Optical stimulation (470 nm, variable pulse width, 1 Hz) was applied as global illumination of the sample from underneath (area of illumination about 10 mm in diameter), using a collimated beam generated by an optical fiber-coupled laser (Shanghai Laser, Shanghai, China) driven by a TTL pulse generator (IonOptix). Optical excitation thresholds were determined as the minimum optical energy needed to induce ChR2-CM activation at different pulse durations. Optical energy (mW/mm 2 ) was measured by a digital power meter (Thorlab, Newton, New Jersey) with a sensor (diameter 9.5 mm) behind the same type of glass bottom dish.
Data was acquired continuously using CamWare interface (pco) or NIS-Elements AR (Nikon Instruments; Melville, NY), and analyzed using custom-developed software in Matlab that automatically detects events (action potentials or calcium transients) and characteristic parameters associated with these; furthermore, it generates activation maps and Hilbert-phase movies of propagation 44 . Data pre-processing included baseline removal, contrast improvement by scaling to full dynamic range, a Bartlett spatial filter (3 × 3 window) and a temporal Savitzky-Golay filter (second order, 7 frames window). Action potential duration and calcium transient duration at 80% (APD80 and CTD80) were determined as the time difference between the beginning of a transient and the point of 80% recovery from the peak. Each sample's APD80 and CTD80 are an average of 3-4 beats. Activation maps were generated based on automatic detection of the time of maximum rise. Isochrones were marked by the frames with 10 ms difference. Conduction velocity (CV) was calculated by sampling points along straight paths in the activation maps (distance traveled divided by the timing between frames).
Statistics. Image analysis of ChR2 expression, PI, and other functional. Threshold irradiance data on strength duration curves were analyzed using 2-way ANOVA.