BiPOLES for Bidirectional Optogenetic Control of Cardiomyocytes and Non-Myocytes

Optogenetics has revolutionized the control of excitable cells by enabling millisecond and micrometre scale modulation of their activity using light. BiPOLES (Bidirectional Pair of Opsins for Light-induced Excitation and Silencing) is a dual-function actuator that combines two opsins responsive to different wavelengths, allowing bidirectional modulation—activation and silencing—of the same cell population, including cardiomyocytes (CM).

Despite its potential, the application of BiPOLES to modulate membrane potential (Vm) in non-excitable cells remains largely unexplored. In the ventricular myocardium, interstitial non-myocytes (NM) are electrically coupled to CM and are exposed to rhythmic Vm fluctuations. However, the impact of these fluctuations on NM structure and function is still poorly understood. Exploring Vm modulation in NM may offer novel strategies to preserve cardiac function in disease. In this context, BiPOLES presents a promising tool to mimic or modulate Vm fluctuations in NM, providing insights into cardiac physiology and potential therapeutic strategies beyond rhythm control. 

We virally introduced a BiPOLES construct combining the red-light-gated (635 nm) cation channel f-Chrimson with a fast variant of the blue-light-gated (435 nm) K⁺ channel WiChR, and assessed its ability to modulate Vm in both CM and NM using patch-clamp recordings. The construct was first expressed in (a) Human Embryonic Kidney (HEK) cells for biophysical characterization, and subsequently in (b) immortalized Human Atrial Fibroblasts (HAF) and (c) hiPSC-derived atrial CM.

In HEK cells, both blue and red light evoked substantial photocurrents and enabled bidirectional Vm modulation (resulting in Vm shifts of approximately -20 mV and 10 mV upon stimulation at 435 and 635 nm, respectively). Repeated stimulation elicited reproducible responses resembling action potential (AP)-like dynamics. HAF cells exhibited similar responses, displaying robust photocurrents upon stimulation with both wavelengths. Light-induced currents effectively modulated Vm with high temporal precision, and the combined application of the two light stimuli reproduced rhythmic depolarization–repolarization cycles (Fig.1a) closely resembling those experienced by fibroblasts (FB) electrically coupled to CM in vivo. These results suggest that BiPOLES can mimic effects of electrical coupling, useful for investigating FB electrophysiology and their role in cardiac pathophysiology.

In hiPSC-derived CM expressing BiPOLES, red-light evoked a rapid inward photocurrent (of approximately -10 pA/pF) at resting Vm, leading to effective depolarization and reliable induction of AP. Conversely, blue-light triggered large, sustained outward photocurrents (in the order of 30 pA/pF at resting Vm), hyperpolarizing the membrane and suppressing both spontaneous and red-light-evoked AP. Notably, simultaneous exposure to both wavelengths and variable light intensities allowed precise and dynamic tuning of Vm, enabling bidirectional control of CM excitability (Fig.1b).

Our findings establish the proposed BiPOLES variant as a versatile tool for bidirectional optogenetic control of cardiac cells. Their utility in finely modulating Vm in FB provides a novel approach to investigate their electrophysiological role in cardiac remodelling and arrhythmogenesis, enhancing our understanding of NM contributions to heart function and disease.