Optogenetic manipulation of cardiac electrical dynamics using sub-threshold illumination: dissecting the role of cardiac alternans in terminating rapid rhythms

Abstract

Cardiac action potential (AP) shape and propagation are regulated by several key dynamic factors such as ion channel recovery and intracellular Ca²⁺ cycling. Experimental methods for manipulating AP electrical dynamics commonly use ion channel inhibitors that lack spatial and temporal specificity. In this work, we propose an approach based on optogenetics to manipulate cardiac electrical activity employing a light-modulated depolarizing current with intensities that are too low to elicit APs (sub-threshold illumination), but are sufficient to fine-tune AP electrical dynamics. We investigated the effects of sub-threshold illumination in isolated cardiomyocytes and whole hearts by using transgenic mice constitutively expressing a light-gated ion channel (channelrhodopsin-2, ChR2). We find that ChR2-mediated depolarizing current prolongs APs and reduces conduction velocity (CV) in a space-selective and reversible manner. Sub-threshold manipulation also affects the dynamics of cardiac electrical activity, increasing the magnitude of cardiac alternans. We used an optical system that uses real-time feedback control to generate re-entrant circuits with user-defined cycle lengths to explore the role of cardiac alternans in spontaneous termination of ventricular tachycardias (VTs). We demonstrate that VT stability significantly decreases during sub-threshold illumination primarily due to an increase in the amplitude of electrical oscillations, which implies that cardiac alternans may be beneficial in the context of self-termination of VT.

Keywords: Cardiac alternans; Optogenetics; Ventricular tachycardias; Voltage imaging.

Fig. 1

Sub-threshold illumination in single cardiomyocytes isolated from CTRL and ChR2 mouse hearts. A Representative traces of V_rest recorded in CTRL (black) and ChR2 cardiomyocytes (blue) during global and constant sub-threshold illumination of cardiomyocytes at increasing light intensities (LIs). The bottom bar shows the LIs used. Light was incremented of 2 µW/mm² every 5 s in the range 0–11 µW/mm². Blue light was turned off at the end of the illumination protocol to assess reversibility. B) Absolute values (left), percentage variation and absolute variation (right) of V_rest in CTRL and ChR2 cardiomyocytes. C Representative traces of APs recorded in CTRL (black) and ChR2 cardiomyocytes (blue) during global and constant sub-threshold illumination of the cell at several increasing LIs. APs were induced by current injection at a frequency of 1 Hz. The bottom bar shows the LIs used. Light was incremented in 2 µW/mm² steps every 2 s in the range 0–11 µW/mm² and then turned off at the end of the illumination protocol to assess reversibility. The top three panels show two successive APs recorded before (left dashed red frame), during (middle solid red frame) and after (right dashed red frame) sub-threshold illumination (zoom of traces in the bottom). D–F Absolute values (left), percentage variation and absolute variation (right) of APA, APRS and APD90 in CTRL and ChR2 cardiomyocytes. Empty diamonds represent values when the light was turned off at the end of the illumination protocol to assess reversibility. G Absolute values of membrane resistance (R_m) in CTRL and ChR2 cardiomyocytes measured at diastolic potential. H Absolute values of current intensity normalized to cell capacitance required to achieve AP firing in CTRL and ChR2 cardiomyocytes. Data was collected from 6 CTRL mice (36 cardiomyocytes) and 10 ChR2 mice (30 cardiomyocytes). Data are reported as mean ± standard error of the mean (SEM) and a linear fit on experimental data was superimposed. Regression analysis results (REG; ANOVA test) are shown for both CTRL and ChR2 cardiomyocytes. No significant difference was found between CTRL and ChR2 cardiomyocytes in V_rest, APA, APRS and APD in absence of sub-threshold illumination (two-way RM ANOVA test with Tukey’s post hoc test)

Fig. 2

Sub-threshold illumination in intact hearts isolated from CTRL and ChR2 mice. A Representative fluorescence images of a mouse heart showing the illumination protocol. Mouse hearts were electrically paced at the apex using an electrode with a burst of 15 stimuli at 5 Hz (the location of electrical stimuli are shown using a yellow bolt symbol). At the same time the entire surface of the heart was constantly sub-threshold illuminated with increasing LIs. Electrical activity was optically recorded before (left panel), during (middle panel) and at the end (right panel) of sub-threshold illumination of the whole heart (filled blue circle). B Fluorescent signals (ΔF/F) extracted from the red ROIs in CTRL (black trace) and ChR2 (blue trace) mouse hearts. The blue bar shows the timing of the sub-threshold illumination with LI = 0.153 mW/mm². C APD90 map (top) and activation map (bottom) of ChR2 mouse heart shown in A (LI = 0.153 mW/mm²). Black arrows highlight the light-mediated delay in AP wavefront propagation. D–F Absolute values (left), percentage variation and absolute variation (right) of APRS, APD90 and CV in CTRL and ChR2 hearts. Empty diamonds represent values when the light was turned off at the end of the illumination protocol to assess reversibility. Data were collected from 7 CTRL and 7 ChR2 hearts. Data is reported as mean ± SEM and exponential (in D and E) and linear (in F) fit on experimental data was superimposed. Regression analysis results (REG; ANOVA test) are shown for both CTRL and ChR2 hearts. No significant difference was found between CTRL and ChR2 hearts in APRS, APD and CV in the absence of sub-threshold illumination (two-way RM ANOVA test with Tukey’s post hoc test)

Fig. 3

Patterned sub-threshold illumination in ChR2 intact mouse hearts. A, F Left panels: representative fluorescence images of a mouse heart (the same reported in Fig. 2C) showing the illumination protocols. ChR2 mouse hearts were electrically paced at the apex with an electrode at 5 Hz (yellow bolt symbol) and at the same time the left (A) and the right (F) half of the heart was illuminated with increasing sub-threshold LIs. Right panels: fluorescent signal (ΔF/F) extracted from the red ROIs in the illuminated region in blue (LI 0.153 mW/mm²) and in the unilluminated region in gray. B, G APD90 (left) and activation (right) maps corresponding to experimental condition in A,F. Black dashed line indicates the border between the illuminated and unilluminated region. Percentage variation and absolute variation of APRS C, H, APD90 (D,I), and CV (E,L) measured in the illuminated region and in the unilluminated region. Data were collected from 6 ChR2 hearts. Data is reported as mean ± SEM and exponential or linear fits on experimental data was superimposed. A two-way RM ANOVA with Tukey’s post hoc test was applied. M) Scheme showing the illumination protocol tested in a simulation of an optogenetically-modified mouse ventricular monolayer. The simulation domain (2.5 × 2.5 cm) consists of a mouse ventricular monolayer expressing ChR2 in which APs propagate as plane waves from the bottom (yellow arrows) to the top side of the domain. APs were electrically elicited with a stimulation frequency of 5 Hz. At the same time half of the domain was continuously sub-threshold illuminated (LI 0.02 mW/mm²) with the illumination edge perpendicular to the AP wave-front. N) Representative frames of the simulation movie showing a clear delay of the propagating AP wave in the illuminated region compared to the unilluminated region

Fig. 4

Pacing rate dependency of sub-threshold illumination in ChR2 intact mouse hearts. A, B) Representative traces optically recorded at the pacing rate of 5 Hz (A) and 13 Hz (B) during light-off (LI 0 mW/mm², in gray) and light-on (LI = 0.153 mW/mm², in blue) condition. C–E Graphic representation of APRS (C), APD50 (D) and APD70 (E) oscillations regarding the 13 APs showed in B. F–H APRS, APD50 and APD70 during odd (downward pointing triangles) and even (upward pointing triangles) beats. APRS, APD50 and APD70 mid-values are also shown (solid line) by averaging the even and odd beats. I-M) APRS, APD50, and APD70 alternans magnitude (Alt) measured during light-off and light-on condition. Data was collected from 8 ChR2 hearts. Data is reported as mean ± SEM and exponential or linear fits on experimental data was superimposed. A two-way RM ANOVA with Tukey’s post hoc test was applied to mid-values, while a two-way RM ANOVA with Tukey’s post hoc test was applied to alternans

Fig. 5

Sub-threshold illumination in ChR2 intact mouse hearts during electrically stimulated VT. A Scheme showing electrically stimulated VT in intact mouse heart expressing ChR2 in the presence (bottom) and absence (top) of sub-threshold illumination of the whole heart with LI = 0.153 mW/mm². An electrical pulse (yellow bolt symbol) was applied to the apex of the ventricle to induce an AP which propagated towards the base of the heart (red dashed lines). Once the propagating wave was optically detected in the user-defined ROI (green rectangle), the system reinjects the electrical stimulus at the apex of the heart with a pre-defined fixed DT (clock). B, C Fluorescent signals (ΔF/F) extracted from the cyan ROIs showing VTs stimulated with a DT of 60 ms and 50 ms in the absence (traces in gray) and presence (traces in blue) of sub-threshold illumination. Spontaneous APs (red arrowhead) detected in the green ROI in A are needed to start or re-start the re-entrant cycle. D Number of interruptions per second of VT as a function of the DT, measured under light-off (LI = 0 mW/mm², in gray) and light-on (LI = 0.153 mW/mm², in blue) conditions. E) APD70 (left) and CT (right) oscillations in VTs showed in B and C (dotted magenta frame). The last beat before VT block has a long AP and a short CT. F–H APD50, APD70 and CT during odd (downward pointing triangles) and even (upward pointing triangles) beats. APD50, APD70 and CT mid-values are also shown (solid line) by averaging the even and odd beats. I-M) APD50, APD70 and CT alternans magnitude (Alt) measured under light-off and light-on conditions. A linear or an exponential fit was superimposed on experimental data. N) Probability that the last beat before VT block displays both a long AP and short CT calculated under light-off and light-on conditions. O APD70 Alt in several VT terminations measured under light-off and light-on conditions as a function of the last beat before VT termination with a short AP/long CT or a long AP/short AP. Data were collected from eight ChR2 hearts. Data are reported as mean ± SEM. Two-way ANOVA with Tukey test means comparison (F–M) and Student’s t test (N, O) were applied