Stilbene derivative as a photosensitive compound to control the excitability of neonatal rat cardiomyocytes

Substances that can be used as photosensitizers for cardiac tissue are very helpful in modeling various excitation patterns in a cardiac tissue culture and may have prospective use in the temporary and permanent ablation of unwanted excitation sources in the heart. The aim of the present work is to study the effect of stilbene derivative c-TAB (2- {4- [(E) -2- (4-ethoxyphenyl) vinyl] phenoxy} ethyl) trimethylammonium bromide) on the cardiomyocyte layers and voltage-gated ion channels in cardiac cells. C-TAB is a structural analog to AzoTAB, reported previously as a photoswitch for cardiac and neural cells, in which the azobenzene moiety is replaced by a stilbene grouping. Such a replacement makes c-TAB less toxic to living cells. c-TAB has been shown to successfully inhibit excitation in cardiac cells in both trans- and cis- forms. The excitation inhibition of cardiac cells under c-TAB is reversible and can be overturned easily by washing out the c-TAB; however, not by light illumination. The irradiation of cardiac cells with near-UV, when the trans- form of c-TAB is applied, changes reversible inhibition to a permanent one that cannot be overturned by a washout.


S3_movie
Then after addition of the trans-c-TAB only the right part of the monolayer (from S2_movie) was irradiated by UV light for one minute. The excitation of the NRVM monolayer was not restored.

S4_movie
Excitability was restored after the c-TAB was washed out of the of NRVM monolayer in the half of the sample that had not been previously illuminated (propagation restored in the left zone after c-TAB wash out from S3_movie) S5_ movie -S11_ Effect of cis-c-TAB on NRVM monolayer: Addition of 30 and 50 µM of cis-c-TAB leads to suppression of the excitation of the NRVM monolayer. Similar results were obtained in three additional samples of NRVM monolayers.

S5_movie
Control of excitable NRVM monolayer. Excitation waves in clear culture

S6_movie
Addition of 30 µM of cis-c-TAB leads to suppression of the excitation of the NRVM monolayer.

S7_movie
The excitation of the NRVM monolayer was restored after wash out

S8_movie
Control of excitable NRVM monolayer. Excitation waves in clear culture

S9_movie
Addition of 50 µM of previously UV-exposed solution c-TAB leads to suppression of the excitation of the NRVM monolayer. The excitation supressed at 50 μM cis-c-TAB (previously UV-exposed solution and then added to the NRVM) S10_movie Additionally irradiated the NRVM by UV-light in the presence of 50 μM cis-c-TAB for 4 minutes by UV-light (~365 nm) S11_movie The suppressed by cis-c-TAB excitation of NRVM (S10_movie) was restored after wash out S_12 -S_18 -Dose-dependency of speed of the excitation propagation for trans-c-TAB 37°C. c-TAB was added to the excitable NRVM monolayer in concentrations of 5-30 µM at 37°C. It can be seen that the excitation of the NRVM monolayer was . The NRVM monolayer in a medium with a constant concentration of c-TAB was exposed to UV light. The propagation speed of the excitation was measured during the illumination. At a concentration of 5 μM, complete suppression was not achieved; at 10 μM, complete suppression was achieved after three minutes of UV exposure; at 15 μM, complete suppression was achieved after one minute of UV exposure.

S12_movie
Control of the excitation waves in clear culture of NRVM at 37°C S13_movie Excitation waves after addition 5 μM of trans-c-TAB at 37°C S14_movie Excitation waves after addition 10 μM of trans-c-TAB at 37°C S15_movie Excitation waves after addition 15 μM of trans-c-TAB at 37°C S16_movie Excitation waves after addition 20 μM of trans-c-TAB at 37°C S17_movie Excitation waves after addition 25 μM of trans-c-TAB at 37°C S18_movie Excitation waves after addition 30 μM of trans-c-TAB at 37°C S19 Fig  Restoring INa+ after washout trans-c-TAB and cis-c-TAB from cells A. Effect of c-TAB on ramp currents in neonatal rat ventricular myocytes. Scaled ramp-evoked currents recorded in response to the same ramp protocol (from -120 to +50 mV, 200 ms) in the control, after the addition of 60 µM trans-c-TAB and washout cells from c-TAB. Three minutes after the application, the current was inhibited by approximately 90% relative to that of the control, and it was restored after washout. Similar results were obtained in more than three additional cells. B. Effect of c-TAB on ramp currents in neonatal rat ventricular myocytes. Scaled ramp-evoked currents recorded in response to the same ramp protocol (from -120 to +50 mV, 200 ms) in the control, after the addition of 60 µM cis-c-TAB and washout cells from c-TAB. Three minutes after the application, the current was inhibited by approximately 90% relative to that of the control, and it was restored after washout. Similar results were obtained in more than three additional cells.

S20 Fig
Restoring ICa, L-type and IK+ after washout cells from trans-c-TAB A. L-type Ca2+ currents obtained in the absence (control), presence of 60 μM trans-с-TAB and after washout cells from c-TAB. Inactivation of INav was achieved by a pre-step from a holding potential HP of -80 mV to -40 mV for 100 ms. Similar results were obtained in more than three additional cells.
B. Whole-cell outward K+ currents of the control in response to 500 ms depolarizing voltage steps from -70 mV to +60 mV, after application of 60 μM c-TAB and after washout cells from c-TAB.  HSQC spectrum of Z-c-TAB: HMBC spectrum of E-c-TAB: Molecule of Z-c-TAB: