A Palette of Near-Infrared Voltage Sensors for Cardiac Research and Safety Pharmacology

PROJECT SUMMARY/ABSTRACT POTENTIOMETRIC PROBES The overall goal of this Phase I SBIR project is to expand the capabilities of tools, invented by the founders of Potentiometric Probe, LLC, for high-fidelity optical recording of electrical activity in cardiac cells, tissues, and whole hearts. Potentiometric Probes develops organic voltage-sensitive dyes (VSDs) that convert the changes in voltage across cell membranes to visible changes in fluorescence. Instead of individual electrodes, this technology enables the use of sensitive high-speed cameras, where each pixel can be considered an electrode, for massively parallel high throughput screening, or for high-resolution spatiotemporal maps of electrical signal propagation. Importantly, our VSDs are unique in enabling dual-wavelength ratiometric recording of electrical activity; this is particularly important for cardiac studies because it eliminates contraction-induced motion artifacts, permitting high-fidelity records of the action potential waveform. Human stem cell-derived cardiomyocytes (hiPSC-CMs) are human skin or other cells that are “reprogrammed” to be beating heart cells in a dish. These human cells and the tissues derived from them are increasingly being used for research and development purposes, including screening new drugs for cardiotoxicity, and in the future may be used for “personalized medicine” and the analysis of patient-derived cells. We believe high throughput recording of accurate action potential waveforms from hiPSC-CMs and tissues will be a major market for the proposed technology. While these cells beat spontaneously, pacing or stimulating these cells to beat at specific frequencies is a critical part of screening protocols because electrical activity and the contribution of specific ion channels is highly dependent on beat frequency. This external stimulation can be applied using light and researchers have incorporated light-dependent channel rhodopsins to conveniently achieve the desired pacing. However, it is crucial to separate the spectra of light used for optical voltage imaging and stimulation. Preliminary work shows that a new chromophore developed at our company can extend the wavelength of our VSDs to the near-infrared and allow them to be used to their full potential, including ratiometric imaging, in concert with channel rhodopsins for pacing. In this proposal, Potentiometric Probes will synthesize a set of near-infrared VSDs and will characterize their performance in cells including hiPSC-CMs. Ultimately, the technology may allow much greater flexibility to researchers, enable multiplexed imaging experiments not previously possible, and in the future may also be applied clinically for high-resolution imaging of action potential propagation in the heart.