Real-time optical manipulation of cardiac conduction in intact hearts

Abstract

Key points: Although optogenetics has clearly demonstrated the feasibility of cardiac manipulation, current optical stimulation strategies lack the capability to react acutely to ongoing cardiac wave dynamics. Here, we developed an all-optical platform to monitor and control electrical activity in real-time. The methodology was applied to restore normal electrical activity after atrioventricular block and to manipulate the intraventricular propagation of the electrical wavefront. The closed-loop approach was also applied to simulate a re-entrant circuit across the ventricle. The development of this innovative optical methodology provides the first proof-of-concept that a real-time all-optical stimulation can control cardiac rhythm in normal and abnormal conditions.

Abstract: Optogenetics has provided new insights in cardiovascular research, leading to new methods for cardiac pacing, resynchronization therapy and cardioversion. Although these interventions have clearly demonstrated the feasibility of cardiac manipulation, current optical stimulation strategies do not take into account cardiac wave dynamics in real time. Here, we developed an all-optical platform complemented by integrated, newly developed software to monitor and control electrical activity in intact mouse hearts. The system combined a wide-field mesoscope with a digital projector for optogenetic activation. Cardiac functionality could be manipulated either in free-run mode with submillisecond temporal resolution or in a closed-loop fashion: a tailored hardware and software platform allowed real-time intervention capable of reacting within 2 ms. The methodology was applied to restore normal electrical activity after atrioventricular block, by triggering the ventricle in response to optically mapped atrial activity with appropriate timing. Real-time intraventricular manipulation of the propagating electrical wavefront was also demonstrated, opening the prospect for real-time resynchronization therapy and cardiac defibrillation. Furthermore, the closed-loop approach was applied to simulate a re-entrant circuit across the ventricle demonstrating the capability of our system to manipulate heart conduction with high versatility even in arrhythmogenic conditions. The development of this innovative optical methodology provides the first proof-of-concept that a real-time optically based stimulation can control cardiac rhythm in normal and abnormal conditions, promising a new approach for the investigation of the (patho)physiology of the heart.

Keywords: Cardiac electrophysiology; Digital Micromirror Device; Optical mapping; Optogenetics.

Figure 1. Targeted optogenetic manipulation of cardiac conduction

A, scheme of the wide‐field fluorescence mesoscope. A red LED followed by a band‐pass filter (640/40 nm) excites through a ×2 objective the whole mouse heart, stained with a red‐shifted electro‐chromic voltage‐sensitive dye (Di‐4‐ANBDQPQ). A dichroic beam splitter followed by a band‐pass filter (775/140 nm) is used for collecting the emitted fluorescence signal. A 4f system is adopted to collimate the beam onto a ×20 objective. The signal is then focused into a central portion (128 × 128 pixels) of a sCMOS sensor operating at a frame rate of 1.6 kHz (630 μs actual exposure time). A commercial light steering solution based on a digital micro‐mirror device (DMD) was coupled with the mesoscope using a high numerical aperture relay system and a dichroic beam splitter. B, optical mapping during four patterns of optogenetic stimulation: single‐point, whole ventricle, vertical line and horizontal line, designed as reported by the blue traits on the fluorescence image (F ₀) of the heart. Six representative frames of optical mapping (∆F/F ₀) showing the electrical activation in red and the baseline in cyan. All stimulations were performed with a light intensity on the sample of 4 mW mm⁻² and 2 ms exposure time. Scale bar: 2 mm. C, on the left, a representative trace of membrane potential recorded in cardiomyocytes isolated from ChR2 heart using patch clamp. At the beginning of the recording (OFF1), the optogenetic excitability of the cell was confirmed inducing sixteen action potentials using blue light pulses (blue lines). In the remainder of the recording, the red LED used for VSD imaging was turned on with two different power intensities (2 mW mm⁻², thin red line and 40 mW mm⁻², thick red line) and turned off again (OFF2) showing no variation in resting membrane potential. On the right, graph shows resting membrane potential during different states of red LED illumination. Each data point (black circle) represents the average of membrane resting potential of a different cell. Also indicated is the mean and the standard error of the mean of each illumination state. No statistically significant differences (ANOVA; number of mice, 2; number of cells, 6) were found between categories.

Figure 2. Real‐time optical intervention

A, schematic workflow of the hardware and software architecture. A sCMOS camera, connected through two camera links to the workstation, saves the data in a RAID array of solid‐state disks (SSD). The camera can run either in high‐speed (blue cycle) or time‐lapse modality for closed‐loop operation (green cycle). During the latter, custom‐written LabVIEW software performs a real‐time analysis on images as they are saved on the SSD (green cycle): a previously acquired image of the heart is used as reference to select two ROIs, whose mean values are compared every 2 ms. When the ratio between the two ROIs exceeds the absolute value of a set threshold (Thr), optogenetic stimulation is activated, with user‐defined temporal delay and intensity. B, optical mapping during sinus rhythm with the two modalities: in loop‐off at a frame rate of 1.6 kHz (16‐bit images) and in loop‐on modality operating at 0.5 kHz (8‐bit images). The fluorescence signal (∆F/F ₀) of two ROIs selected on atrium and ventricle (green and red circle respectively, reported on the fluorescence baseline image) are shown for both acquisition modalities. Scale bar: 2 mm.

Figure 3. Intra‐ventricular manipulation

A, a slowing down of the ventricular action potential propagation was induced by applying an electrical stimulation on the apex of the ventricle (yellow lightning). Two reference ROIs were selected (red and green rectangles) and the ROI ratio was obtained, with a 1% threshold and with no delay in activation. ChR2 stimulation was achieved with a light intensity of 4 mW mm⁻² and 2 ms exposure time (blue arrowhead). B, the wave‐front propagation without the real‐time intervention (OFF) and with the optogenetic activation (ON). Frames acquired after photo‐stimulation (blue arrowhead) clearly show the differences in wave‐front propagation, highlighting the acceleration of ventricle activation. Scale bar: 2 mm.

Figure 4. Optical manipulation of atrioventricular delay

A, optical mapping of spontaneous heart activity, with typical atrioventricular (AV) temporal delay around 70 ms. B, the optical system was used to reduce AV delay by triggering the optical stimulation of the ventricle according to optically mapped atrial activity. The two reference ROIs (red and green circles) are positioned on atrium and ventricle respectively (1% threshold). As first stimulation pattern, a single point (blue area) was applied on the ventricle with three different temporal delays: 0, 10 and 20 ms after atrium activation. The underlined time frames highlight different delays. In the last row, whole‐ventricle activation was performed with a temporal delay of 0 ms. ChR2 stimulation was achieved with a light intensity of 4 mW mm⁻² and 2 ms exposure time. White arrowheads indicate atrial activation. Scale bar: 2 mm. C, a stationary AV block was established by adding ethanol to the perfusion solution. ECG clearly demonstrates the presence of an AV block, since the P wave is not followed by a proper QRS complex. D, after inducing an AV block, the OFF modality shows no ventricular activation after atrial triggering. A real‐time intervention (ON) was performed using single‐point and wide‐area illumination, with 50 ms delay (1% threshold, light intensity of 4 mW mm⁻² and 2 ms exposure time). Photo‐activation results in AV resynchronization, restoring normal cardiac conduction patterns during sinus rhythm. White arrowheads indicate atrial activation. Scale bar: 2 mm.

Figure 5. Optically induced re‐entrant circuit

Left, fluorescence image of the heart. Re‐entrant circuit was started by detection of spontaneous ventricular activity at the base of the heart (green ROI). After detection, ChR2 stimulation at ventricle apex (blue circle) was applied after a delay of 200 ms (A) or 150 ms (B) concluding one loop‐of the cycle. Scale bar: 2 mm. Centre, corresponding colour‐scaled isochronal map reporting the activation time per pixel. Right, fluorescence signal (∆F/F ₀) extracted from the green ROI. The stable re‐entrant circuit at a stimulus delay of 200 ms (A) becomes unstable at a delay of 150 ms (B) showing discontinuity in the action potential time interval. Spontaneous action potentials (red asterisks) detected in the green ROI are needed in this case to re‐start the re‐entrant cycle.

Figure A1. Custom mesoscope

C, collimation lens; DC, dichroic mirror; DMD, digital micro‐mirror device; F, filter; L, lens; LED, light emitting diode; M, mirror; O, objective; sCMOS, scientific complementary metal‐oxide semiconductor camera; WD, working distance.

Figure A2. Main considerations for the mesoscope's temporal and spatial resolution

BFP, back focal plane diameter; NA, numerical aperture; O, objective; WD, working distance.

Figure A3. Specifications of the digital micro‐mirror device (DMD) and its relay optics after removing the projection lens

Figure A4. The lightcrafter as optogenetic stimulation light source

A, remove the projection lens (yellow arrow) by gently wedging it out of its mount with a screwdriver. To bypass the native LED driver, populate Jumper 30 (white arrow). Unplug the connectors (purple arrows) for the blue and green LEDs (cyan arrows). B–D, using spacers (orange arrows, B) mount an aluminium plate with adequately spaced and threaded through holes (C) to mount the external LED driver (D). E, on the LED driver, mount connectors for the blue and green LED and connect them to the LED+ and LED− terminals (white arrows) in a series configuration and solder a pull‐up resistor (blue arrowhead). F, the lightcrafter is mounted on a manual xyz tip stage which is approximately inclined by 10 degrees. Without the projection lens, the lightcrafter projects a primary image (white arrow) approximately 10 mm from the front housing.

Figure A5. Emission spectra of the red green and blue LEDs in the light engine

Spectrum is indicative only. Also shown is the transmission curve of the dichroic filter (black) used for injecting the optogenetic excitation light into the mesoscope beam path and the excitation spectrum of ChR2 (cyan).

Figure A6. Connectivity scheme of the mesoscope

DAQ, data acquisition device; ECG, electrocardiogram; ext, external; HDMI, high‐definition multimedia interface; I/O, input/output; LED, light emitting diode; USB, universial serial bus; PC, personal computer.