Optogenetic control of cardiomyocytes via viral delivery

Abstract

Optogenetics is an emerging technology for the manipulation and control of excitable tissues, such as the brain and heart. As this technique requires the genetic modification of cells in order to inscribe light sensitivity, for cardiac applications, here we describe the process through which neonatal rat ventricular myocytes are virally infected in vitro with channelrhodopsin-2 (ChR2). We also describe in detail the procedure for quantitatively determining the optimal viral dosage, including instructions for patterning gene expression in multicellular cardiomyocyte preparations (cardiac syncytia) to simulate potential in vivo transgene distributions. Finally, we address optical actuation of ChR2-transduced cells and means to measure their functional response to light.

Fig. 1

ChR2 expression in cardiomyocytes: Representative fluorescent images of NRVMs infected with Ad-ChR2(H134R)-eYFP at (a) MOI 0, control; (b) MOI 25; and (c) MOI 100. Blue stain is DAPI. Scale bar is 100 μm

Fig. 2

Cell viability upon viral dosing: Representative fluorescent images of NRVMs infected with Ad-ChR2(H134R)-eYFP and stained with PI at (a) MOI 0, control; (b) MOI 25; and (c) MOI 100. Scale bar is 100 μm. (d) Quantification of PI staining presented as average number of pixels with PI stain above threshold normalized to area (mm²)

Fig. 3

Patterning stencils: Example schematic of silicone elastomer stencil used to pattern an island of ChR2-expressing NRVMs surrounded by normal, uninfected NRVMs. Not drawn to scale

Fig. 4

Spatial patterning of ChR2 expression: Representative binarized distributions of ChR2-expressing NRVMs (eYFP, white) in four configurations: (a) global expression of ChR2; (b) mixture of 35 % ChR2-expressing NRVMs and 65 % non-infected NRVMs; (c) mixture of 10 % ChR2-expressing NRVMs and 90 % non-infected NRVMs; (d) island of ChR2-expressing NRVMs

Fig. 5

Combining optical actuation with optical imaging: (a) Experimental setup for high-resolution, high-speed optical imaging and optical control of cardiac syncytia. (b) Activation maps in cardiac syncytia by electrical and optical pacing at 0.5 Hz. Normalized calcium transients (acquired with Rhod 4 staining) are shown from two locations—A and B