Stimulating cardiac muscle by light: cardiac optogenetics by cell delivery

Abstract

Background: After the recent cloning of light-sensitive ion channels and their expression in mammalian cells, a new field, optogenetics, emerged in neuroscience, allowing for precise perturbations of neural circuits by light. However, functionality of optogenetic tools has not been fully explored outside neuroscience, and a nonviral, nonembryogenesis-based strategy for optogenetics has not been shown before.

Methods and results: We demonstrate the utility of optogenetics to cardiac muscle by a tandem cell unit (TCU) strategy, in which nonexcitable cells carry exogenous light-sensitive ion channels, and, when electrically coupled to cardiomyocytes, produce optically excitable heart tissue. A stable channelrhodopsin2 (ChR2)-expressing cell line was developed, characterized, and used as a cell delivery system. The TCU strategy was validated in vitro in cell pairs with adult canine myocytes (for a wide range of coupling strengths) and in cardiac syncytium with neonatal rat cardiomyocytes. For the first time, we combined optical excitation and optical imaging to capture light-triggered muscle contractions and high-resolution propagation maps of light-triggered electric waves, found to be quantitatively indistinguishable from electrically triggered waves.

Conclusions: Our results demonstrate feasibility to control excitation and contraction in cardiac muscle by light, using the TCU approach. Optical pacing in this case uses less energy, offers superior spatiotemporal control and remote access and can serve not only as an elegant tool in arrhythmia research but may form the basis for a new generation of light-driven cardiac pacemakers and muscle actuators. The TCU strategy is extendable to (nonviral) stem cell therapy and is directly relevant to in vivo applications.

Figure 1

The functional “tandem cell unit” (TCU) concept of donor-host cells. Non-excitable cells (e.g. HEK cells here) are transfected to express a light-sensitive ion channel (ChR2). When coupled via gap junctions to excitable cardiomyocytes (CM) they form an optically controllable functional TCU, i.e. the CM will generate an action potential upon light-triggered opening of the depolarizing ChR2 in the HEK cell.

Figure 2

Development and functional characterization of a cell delivery system for ChR2. a: Stable HEK−ChR2 cell line – shown is EYFP-reported ChR2 expression in the 10^th passage after transfection and purification; scale bar is 50μm. b: Voltage-clamp test protocol and example traces for quantification of the steady-state ChR2 current in single HEK−ChR2 cells with 500ms voltage pulses in the range (−80 to +50mV) with and without excitation light for ChR2 on (470nm, 0.24 mW/mm²). c: Example curves for the light-sensitive component after subtraction of current in dark, and the resultant average current-voltage (I–V) relationship for n=12 cells, cell capacitance 43.3±7.5pF, data are presented as mean±SD. d: Magnitude of the light-triggered current does not depend on the duration of rest (Δt rest) or activation (Δt act), thus indicating relatively fast deactivation in the examined range. Holding potential is −80mV. e: Kinetics of activation (on) and deactivation (off), quantified by a τ_sl parameter in the sigmoid curve fits to the light-controlled current transitions (see inset); bar graphs represent mean±SEM.

Figure 3

Implementation and validation of the TCU concept for neonatal rat CM and adult canine CM coupled to HEK+ChR2 cells. a: Phase and fluorescence images of neonatal rat CM and HEK−ChR2 co-culture. Immunostaining in red forα-actinin (CMs), green is EYFP-ChR2-expressing HEK cells, typically forming small clusters as shown. Scale bar is 20μm. b: Western blot for Cx43 andα-tubulin (at 55kD) in the cell delivery system (HEK−ChR2), column 2; column 1 shows a positive control of stably transfected HeLa-Cx43 cells; column 3 shows parental HEK cells without ChR2; column 4 shows the ladder – MagicMark^™ bands in kDa; Normalized (Cx43/tubulin) expression is provided for four gels (mean±95%CI). c: Histogram of measured coupling conductances in TCUs of canine CMs and HEK−ChR2 cells, n=31, median value of 4nS and IQR (2 – 11nS); red arrow indicates coupling levels allowing optical excitability of the TCUs. d: Dual whole-cell voltage clamp of a TCU - adult canine ventricular CM (1) and HEK−ChR2 cell (2). Voltage steps (V1=10mV, 0.4s), applied to the canine CM (cell 1), induced junctional currents (I2) in this cell pair (estimated g.j. conductance of 11nS). e: Action potentials in a cell pair (canine CM and HEK−ChR2 cell, phase image on the left) in response to optical pacing (0.13 mW/mm², 10ms pulses). Due to coupling, the HEK cell exhibits a low-pass filtered version of the CM-generated action potentials. f: Action potentials in a cell pair (canine CM and HEK−ChR2 cell) in response to continuous optical pacing before, during and after washout of uncoupler carbenoxolone (CBX).

Figure 4

Optical control of cardiac tissue function over space-time: light-triggered excitation waves and light-triggered contractions. a: Experimental setup for ultra-high resolution high-speed optical imaging and optical control of cardiac excitation: 1) experimental chamber with tangential light illumination for calcium imaging, focused LED illumination on a moveable stage for ChR2 excitation (see inset on the right); 2) high-NA optics for high-resolution macroscopic imaging; 3) Gen III MCP intensifier; 4) pco 1200hs CMOS camera; 5) light source, excitation filter and optical light guides for tangential excitation; 6) computer system and software for data acquisition and control of electrical and optical stimulation; 7) interface for stimulation control; 8) controllable stimulator for electrical pacing (analog output); 9) controllable stimulator for optical pacing (TTL output). 10) LED for ChR2 excitation, driven by the TTL stimulator output. b: Activation maps in a cardiac monolayer by electrical and optical pacing at 0.5Hz. Color represents time of activation; isochrones are shown in black at 0.15s. Calcium transient traces in response to electrical or optical stimulation are shown from 2 locations (A and B), normalized fluorescence. Blue marks indicate time of stimulation (electrical pulses were 10ms, optical −20ms each). c: Normalized Ca²⁺ transients from CM monolayer (red), CM:HEK (black) and CM:HEK+ChR2 co-culture 100:1 (blue) at 1Hz pacing. d: Quantification of calcium transient duration (CTD) – CTD25, CTD50 and CTD80 for pure CM monolayer, 45:1 and 100:1 CM:HEK, as well as 100:1 CM:(HEK+ChR2) co-culture under electrical and optical pacing at 1Hz. e: Comparison of conduction velocity (CV) among the same 5 groups as in (d); for d and e optical pacing was at irradiance of 0.01– 0.04mW/mm², 50ms pulses; data are shown as mean±95%CI; listed number of samples applies to both; f: Strength-duration curve (along with the equation for the fitted curve) obtained for optical pacing in co-cultures (100:1 CM:HEK ratio) at 30°C, n=8, mean±SEM. g: Example contractility recording from optically-driven CM+HEK+ChR2 – displacement normalized to cell length. Scale bar is 1s.