Mechanism of the Spontaneous Beating of Skeletal-Based Precursor of Cardiomyocytes (SPOC)

Abstract

Here we demonstrate a method that allows light-induced activation of voltagegated ion channels and the concurrent imaging of membrane potential changes using voltage-sensitive dyes. This light-induced voltage clamp (LIVC) method uses photostimulation through channelrhodopsin-2 (ChR2) to activate voltagegated ion channels. ChR2 allows light to be immediately transduced into a depolarizing ionic current, which in turn causes voltage-gated ion channels to open. In our system we coexpressed ChR2 either with the voltage-gated potassium channels hERG or hKv1.5 in cell lines and in Xenopus oocytes. In electrophysiological experiments we show that light-induced depolarization through ChR2 sufficed to activate hERG as well as hKv1.5 channels. We were further able to optically monitor the light-induced membrane de-and hyperpolarizations on a millisecond timescale with the voltage-sensitive RH421 and Annine6. The fluorescence readout reflected the dose-response relationships of the hERG blocker Terfenadine and the hKv1.5 inhibitor DPO-1 obtained from patch-clamp measurements. LIVC represents a solely optical technology with remote activation of the target voltage-gated ion channels by the delivery of a flash of blue light and simultaneous detection of their activity employing voltage-sensitive dyes. It combines the high-throughput of optical methods with the high-content of patch clamp concerning high temporal resolution, membrane potential control and repetitive stimulation.