High Speed Imaging of Single Cardiomyocyte Action Potentials Using a Far-red Genetically Encoded Voltage Sensor

The ability to image cardiac membrane potentials allows for the observation of cellular communication and electrical activity, both of which are important to maintain cardiac syncytium; these can be altered in diseases (e.g. Long QT Syndrome). Traditionally voltage dyes such as Di-8-ANEPPS have been used to optically measure action potentials (APs). However, these dyes express transiently, have poor signal to noise ratios, and are toxic. More recently, genetically encoded voltage indicators (GEVIs) have been developed to replace state-of-the-art voltage dyes, but sensors currently used within the cardiac field exhibit poor kinetics and/or low signal to noise ratios (SNR). Recently, Archon1, a new genetically encoded voltage sensor, was developed in the neuroscience field; this sensor exhibits excellent membrane localization, temporal sensitivity, and SNR, enabling the optical detection of individual spikes in neurons. Here we use Archon1 for the first time in cardiac cells, to monitor single cell cardiac APs in 2D and 3D in vitro systems in response to different environmental stimuli. Human induced pluripotent stem cell-derived cardiomyocytes were infected with Archon1 and imaged using a one-photon fluorescence microscope equipped with a high speed sCMOS camera to demonstrate cardiac AP tracings. The kinetics and SNR of Archon1 are compared to traditional electrophysiology and Di-8-ANEPPS measurements. Additionally, E-4031 (K+ Channel Blocker) and Nifedipine (Ca2+ Channel Blocker) were used to demonstrate the sensitivity of this sensor in a drug dosage study. To study the APs of single cells within a 3D engineered microtissue, cardiomyocytes expressing Archon1 were seeded into a force transducing micro-pillar device and the APs for optically isolated cells were recorded. Demonstration of this new genetically encoded voltage sensor in cardiac cells enables the monitoring of single and multi-cell APs in 2D and 3D applications and can be extended to in vivo. This tool, newly applied to cardiac biology and tissue engineering will allow for better and more accurate observation of cardiac electrical activity to probe human cardiovascular disease.