Multiscale computational models for optogenetic control of cardiac function

Abstract

The ability to stimulate mammalian cells with light has significantly changed our understanding of electrically excitable tissues in health and disease, paving the way toward various novel therapeutic applications. Here, we demonstrate the potential of optogenetic control in cardiac cells using a hybrid experimental/computational technique. Experimentally, we introduced channelrhodopsin-2 into undifferentiated human embryonic stem cells via a lentiviral vector, and sorted and expanded the genetically engineered cells. Via directed differentiation, we created channelrhodopsin-expressing cardiomyocytes, which we subjected to optical stimulation. To quantify the impact of photostimulation, we assessed electrical, biochemical, and mechanical signals using patch-clamping, multielectrode array recordings, and video microscopy. Computationally, we introduced channelrhodopsin-2 into a classic autorhythmic cardiac cell model via an additional photocurrent governed by a light-sensitive gating variable. Upon optical stimulation, the channel opens and allows sodium ions to enter the cell, inducing a fast upstroke of the transmembrane potential. We calibrated the channelrhodopsin-expressing cell model using single action potential readings for different photostimulation amplitudes, pulse widths, and frequencies. To illustrate the potential of the proposed approach, we virtually injected channelrhodopsin-expressing cells into different locations of a human heart, and explored its activation sequences upon optical stimulation. Our experimentally calibrated computational toolbox allows us to virtually probe landscapes of process parameters, and identify optimal photostimulation sequences toward pacing hearts with light.

Figure 1

Channelrhodopsin-2 (ChR2) is a light-gated cation channel native to the green alga C. reinhardtii. It consists of seven transmembrane proteins and absorbs light through its interaction with retinal. Here, we induce channelrhodopsin coupled to enhanced yellow fluorescent protein (eYFP) into undifferentiated human embryonic stem cells via a lentiviral vector and differentiate these cells into cardiomyocytes.

Figure 2

Channelrhodopsin-2 (ChR2) is activated by photoisomerization of all-trans retinal to 13-cis retinal at wavelengths of 470 nm. After photoisomerization, the covalently bound retinal spontaneously relaxes to all-trans in the dark, providing closure of the ion channel and regeneration of the chromophore.

Figure 3

Three-state model for the channelrhodopsin photocycle. Upon photoabsorption, molecules in the closed state g_c undergo a fast transition into the open state g_ChR2. After for some time, molecules spontaneously turn into the recovering state g_r where the ion channels are closed, but the molecules are not yet ready to photoswitch again. After a recovery period, the molecules finally return to the closed state g_c, ready to undergo a new photocycle when subjected to light.

Figure 4

Ionic model of genetically engineered light sensitive cardiac cell. The electrochemical state of the cell is characterized in terms of n_ion = 8 ion concentrations, c_ion = [c^e_Na, c^e_K, c^e_Ca, cⁱ_Na, cⁱ_K, cⁱ_Ca, c^up_Ca, c^rel_Ca], the extracellular and intracellular sodium, potassium, and calcium concentrations, and the sarcoplasmic reticulum calcium uptake and release. Ion concentrations are controlled through n_crt = 12 ionic currents, I_crt = [I_Na, I_bNa, I_f, I_NaCa, I_NaK, I_ChR2, I_K, I_bK, I_CaL, I_CaT, I_up, I_rel], where the baseline autorhythmic cell model (26) has been enhanced with the channelrhodopsin current I_ChR2 (shown in blue). The channels are governed by n_gate = 10 gating variables g_gate = [g_m, g_h, g_ChR2, g_y, g_x, g_dL, g_fL, g_fCa, g_dT, g_fT], which may be functions of the current membrane potential ϕ.

Figure 5

Undifferentiated human embryonic stem cells (hESC) stably transduced with a ChR2-eYFP lentiviral vector (hESC^ChR2+) remain pluripotent and can differentiate into cardiomyocytes (hESC^ChR2+-CM). (a) PCR shows that hESC ^ChR2+ express the pluripotent Oct-4 gene (169 bp, lane 4) and Nanog gene (154 bp, lane 5) (blue box). In addition, amplification within the ChR2 gene (174 bp, lane 6), across the ChR2-eYFP gene (197 bp, lane 7), and within the eYFP gene (187 bp, lane 8), confirms stable transduction of the ChR2-eYFP lentivirus in undifferentiated hESC^ChR2+ (yellow box). A ladder (100 bp, lane 1) confirms the predicted sizes of PCR products. Nontemplate control (lane 2) and GAPDH (152 bp, lane 3) serve as negative and positive controls, respectively. (b) Pluripotent hESC^ChR2+ stain is positive for alkaline phosphatase (red). (c) Fluorescence microscopy shows hESC^ChR2+ has a positive eYFP signal (green). (d) hESC^ChR2+-CM have positive TnI signals (red), consistent with a CM phenotype. DAPI staining (blue) demonstrates the position of nuclei. (e) Transmission electron microscopy shows sarcomeres with associated z-lines (z) and mitochondria (m) in hESC^ChR2+-CM. (f) Light microscopy shows three hESC^ChR2+-CM colonies (dashed white circles) on a multielectrode array.

Figure 6

Experimental and computational sensitivity of hESC^ChR2-CM with respect to light intensity. With light on, the photocurrent I_ChR2 increases rapidly, peaks, and decays toward a characteristic plateau value. With light off, the photocurrent I_ChR2 drops rapidly and decays to zero. Light intensity is varied from 12.5% to 25%, 50%, and 100% (top). Whole-cell voltage-clamp reveals an increased photocurrent I_ChR2 as the light intensity increases (middle). The computational hESC^ChR2-CM model captures the light sensitivity and displays increased photocurrents I_ChR2 with increased light intensity (bottom).

Figure 7

Experimental and computational sensitivity of hESC^ChR2-CM with respect to stimulation frequency. Light stimulation (blue) evokes field potentials (black) that translate into mechanical contractions (red). Light stimulation at 100% intensity is performed at 0.5 Hz (top), 1.0 Hz (middle), and 1.5 Hz (bottom). Evoked signals during light stimulation (center) are markedly different from pre- and poststimulation signals at all frequencies (left and right). The computational hESC^ChR2-CM model (green) captures the electrical signal at all frequencies, both during light stimulation (center), and pre- and poststimulation (left and right).

Figure 8

Virtual activation sequences of light-paced hearts. Atrioventricular node (top) and biventricular (bottom) photosimulations are initiated through hESC^ChR2-CM, virtually injected into the basal septum and into both lateral walls, respectively. All other regions are modeled as standard ventricular CM. The color code indicates the magnitude of the transmembrane potential ϕ varying from −90 mV (blue) to +20 mV (red).