Enlightening the heartbeat: photo-switchable compounds for optical modulation of cardiac bioelectricity

The use of light represents a powerful approach for the control of cellular behavior, offering exceptional spatio-temporal precision. Beyond fundamental applications in cell biology, light is increasingly being explored as an alternative approach to address rhythm disorders and electrical instability in cardiomyocytes (CMs). However, because CMs are intrinsically weakly photosensitive, optical control requires molecular tools capable of converting light stimuli into electrophysiological responses. This thesis investigates three novel membrane-targeting azobenzene derivatives: Ziapin2, MTP2, and SZ, as photoactuators for the non-genetic optical modulation of cardiac bioelectricity. A multidisciplinary approach integrating photophysical analysis, computational modeling, tissue engineering, and functional characterization was employed to elucidate the mechanisms underlying their light responsiveness and impact on cellular behavior. i) Ziapin2 acts as an opto-mechanical actuator: once integrated into the sarcolemma, it forms dimers that reduce membrane thickness and increase capacitance. Upon visible-light exposure, trans-cis isomerization reverses this effect, inducing a transient hyperpolarization followed by a rebound depolarization. In human induced pluripotent stem cell-derived CMs, this membrane potential (Vm) modulation culminates in action potential (AP) generation coupled with synchronized intracellular Ca²⁺ transients and contractions, demonstrating precise optical control of excitation–contraction coupling. Ziapin2 also enables non-contact electrophysiology when integrated into high-throughput platforms combining laser stimulation and multielectrode arrays. Mechanistic studies in adult mouse CMs, supported by in silico simulations, revealed that stretch-activated Ca²⁺ channels mediate the conversion of membrane capacitance changes into electrical excitation, facilitating the depolarization required to reach the threshold for Na⁺ channel activation and AP generation. At the tissue level, we demonstrated that Ziapin2 modulates Ca²⁺ dynamics in engineered cardiac microtissues with precision comparable to electrical pacing and can terminate reentrant activity in arrhythmic constructs, providing proof-of-principle for light-mediated rhythm control. ii) MTP2 presents enhanced solubility in water, no activity in the dark and a monophasic response on Vm upon light stimulation. The compound operates primarily through dipole-driven surface charge modulation, inducing light-dependent depolarization via charge displacement across the membrane. Although the observed Vm variation is subthreshold for AP initiation, MTP2- induced depolarization can influence cellular excitability enabling fine tuning of the electrical response and potentially destabilize arrhythmic circuits. iii) SZ was designed to combine the capacitance modulation of Ziapin2 with the dipolar efficiency and solubility of MTP2. Once inserted into the plasma membrane, SZ undergoes reversible photoisomerization that simultaneously modifies membrane capacitance and surface charge. The combined action of these mechanisms produces strong and reproducible biphasic Vm perturbation, enabling deterministic one-to-one optical pacing in hiPSC-derived CMs with excellent biocompatibility. Overall, these results demonstrate that small photoresponsive azo derivatives can spontaneously integrate into the plasma membrane, preserve cell viability, and reproducibly modulate Vm, from single cells to tissue level. Ziapin2 and SZ enable suprathreshold optical pacing with Ziapin2 successfully mediating optically driven arrhythmia termination in 2D in vitro models, while MTP2 allows subthreshold modulation potentially enabling fine control over excitability and conduction. Although still in the proof-of-concept stage, this thesis proposes three novel light-based tools for control of cellular activity, with potential applications in cardiac rhythm management and cardioversion.