Simultaneous Optical Pacing and Membrane Voltage Mapping in ChR2-Expressing Neonatal Rat Ventricular Myocyte Cultures

Abstract

Optogenetics is an emerging technology that combines optics and genetics to remotely and precisely control individual cells in a living tissue. Due to the high spatial and temporal resolution, this innovative approach has been widely employed in the field of neuroscience to manipulate the electrical activity of various types of neuron cells. However, its application in the study and therapy of cardiovascular diseases is still in the infancy. In this work, primary cultures of neonatal rat ventricular myocytes (NRVMs) were infected with lentivirus containing the channelrhodopsin (ChR2) gene (teto-FUW-ChR2-eYFP) and paced with short pulses of LED light (wavelength 475 nm) delivered via optical fibers of various diameters (from 0.3 to 1mm). Wave propagation induced by optical pacing was monitored using a Vm-sensitive dye and a photodiode optical mapping system. Parameters of excitation waves induced by optical pulses were compared with those ones induced by electrical pacing. Our data showed that ChR2 protein can be stably and homogeneously expressed on the cell membrane of NRVMs without significantly affecting cell shape and monolayer confluence. The ChR2-expressing cultures could be paced by light pulses at varying frequencies (from 0.25 to 4Hz), using even the smallest 0.3-mm optical fibers, suggesting the high light sensitivity of ChR2 in NRVM cells. In addition, optical Vm mapping showed that key parameters of excitation waves (e.g., shape of optical action potentials and conduction velocity) induced by light pulses were similar to that ones induced by electrical stimulation. These results demonstrate the feasibility of optical pacing and simultaneous optical Vm mapping in ChR2-expressing NVRM monolayers, and lay a foundation for the development of novel optical-based pacing devices.