Fig. 6: Modulation of cardiac activity mediated by triplet-sensitized photoswitching of muscarinic drug PAI in the phototherapeutic window.

a Apparatus to optically record cardiac activity in frog tadpoles, including a binocular microscope equipped with a camera and LEDs with 365, 455, and 730 nm wavelengths. b Freely swimming tadpoles at stages 46–48. c Image representing the application of 0.23 mM pancuronium dibromide solution to immobilize the tadpole (stage 47) for long-term imaging of cardiac activity under dim light at 32 frames per second (fps) video recording. A region of interest (ROI) is placed at the edge of the heart to record the beating. d Image representing the setup for applying compounds to the medium and illuminating at wavelengths of interest (365, 455 and 730 nm LED in the photo). e Time course of the mean ROI intensity of heartbeat under the conditions indicated in the labels: control in the absence of PAI and light, inactive 365 nm-preilluminated (I_o = 10.9 mW cm⁻²) 30 µM PAI together with 10 µM ZnPc1 photosensitizer in the dark, and effect of illuminating the mix with 730 nm light (I_o = 42 mW cm⁻²) in consecutive 15 min periods. f Time course of the amplitude of cardiac contraction during controls, compound application in the dark, and three consecutive 10 min periods of 730 nm illumination (shaded in red). The black line represents inactive 365 nm-preilluminated 30 µM PAI with 10 µM ZnPc1, and 2% PF127 in 0.1X MBS diluted buffer, the gray line shows the control experiment in the absence of the triplet photosensitizer ZnPc1, and the dotted line represents the control corresponding to the triplet sensitizer alone (10 µM ZnPc1 and 2% PF127 in 0.1X MBS). The corresponding frequency plots are shown in the Supplementary Information. g Quantification of the experiments in panels (e, f). White bars represent the mean amplitude of contraction measured at intervals of 15 s during each experimental phase (baseline, application, 730 nm 10 min, 20 min, and 30 min) following treatment with the sensitizer (ZnPc1) alone (n = 3 animals, biological replicates). Gray bars represent measurements with PAI alone (n = 2 animals, biological replicates), and black bars represent the combination of ZnPc1 and PAI (n = 3 animals, biological replicates). Each data point shown represents independent biological replicates derived from separate animals. Due to ethical considerations regarding animal use, the number of biological replicates was kept minimal. Error bars indicate the standard error of the mean (SEM). Statistical significance (*) indicates p < 0.05 when comparing the combination treatment (PAI + sensitizer) to sensitizer alone, multiple unpaired t test. “nd” indicates no significant difference.