Optogenetic Control of Vascular Tone with High Temporal Resolution

Abstract

Blood pressure is directly controlled by the contractile state of vascular smooth muscle cells (VSMCs) which is regulated by membrane potential-dependent Ca2+-entry through Ca2+-channels and Ca2+-release from intracellular stores. Modulation of membrane potential in VSMCs in intact vessels can be performed by electrical field stimulation or elevation of extracellular potassium concentration, but both methods are not cell-specific and have low spatio-temporal resolution. To overcome these limitations, we have used the light-gated cation channel channelrhodopsin2 for optogenetical control of VSMCs membrane potential. ChR2 was expressed in a7r5 VSMCs and patch clamp experiments showed light-induced inward currents. Brief light pulses with increasing light intensities led to graded depolarization and eventually initiation of action potentials. Prolonged illumination resulted in constant depolarization and elevated intracellular Ca2+-concentrations. Optogenetic stimulation of VSMCs in intact vessels was performed using transgenic mice that express ChR2 (Nat Methods. 2010;7:897-900) in VSMCs but not endothelial cells. Isometric force was analyzed in aortic rings with a wire-myograph. Illumination with blue light (475nm, 2.7mW/mm2) reliably induced contractions with very fast on- (< 800ms) and off-kinetics (< 2.5s) and contraction could be maintained up to 10 min. The amplitude of contraction could be graded by variation of light intensity and reached a maximum comparable to stimulation with noradrenalin (10μM). Control vessels from wild-type mice or transgenic mice expressing EGFP did not show light-induced contractions. Taken together ChR2 enables light-induced contraction of VSMCs with high temporal resolution and represents a powerful technique to analyze membrane-potential dependent mechanisms in VSMCs. Optogenetic stimulation allows cell-specific and localized stimulation down to single cell level and therefore can be used in the future to investigate the interplay of endothelial cells and VSMCs as well as the electrical coupling between cells within intact vessels.