Optical Mapping of Membrane Potential and Epicardial Deformation in Beating Hearts

Abstract

Cardiac optical mapping uses potentiometric fluorescent dyes to image membrane potential (Vm). An important limitation of conventional optical mapping is that contraction is usually arrested pharmacologically to prevent motion artifacts from obscuring Vm signals. However, these agents may alter electrophysiology, and by abolishing contraction, also prevent optical mapping from being used to study coupling between electrical and mechanical function. Here, we present a method to simultaneously map Vm and epicardial contraction in the beating heart. Isolated perfused swine hearts were stained with di-4-ANEPPS and fiducial markers were glued to the epicardium for motion tracking. The heart was imaged at 750 Hz with a video camera. Fluorescence was excited with cyan or blue LEDs on alternating camera frames, thus providing a 375-Hz effective sampling rate. Marker tracking enabled the pixel(s) imaging any epicardial site within the marked region to be identified in each camera frame. Cyan- and blue-elicited fluorescence have different sensitivities to Vm, but other signal features, primarily motion artifacts, are common. Thus, taking the ratio of fluorescence emitted by a motion-tracked epicardial site in adjacent frames removes artifacts, leaving Vm (excitation ratiometry). Reconstructed Vm signals were validated by comparison to monophasic action potentials and to conventional optical mapping signals. Binocular imaging with additional video cameras enabled marker motion to be tracked in three dimensions. From these data, epicardial deformation during the cardiac cycle was quantified by computing finite strain fields. We show that the method can simultaneously map Vm and strain in a left-sided working heart preparation and can image changes in both electrical and mechanical function 5 min after the induction of regional ischemia. By allowing high-resolution optical mapping in the absence of electromechanical uncoupling agents, the method relieves a long-standing limitation of optical mapping and has potential to enhance new studies in coupled cardiac electromechanics.

Figure 1

Selection of emission band. (A and B) Emission spectra recorded during action potential plateau and during rest for blue (450 nm) and cyan (505 nm) excitation, respectively. (C and D) Difference spectra obtained by subtracting the resting from plateau spectra from (A) and (B), respectively. Horizontal bars in (C) and (D) indicate the passband of the emission filter (635 ± 27.5 nm). Plots in (C) and (D) have the same vertical scale. The emission spectra are averages; the gray bands show the 95% confidence intervals for the spectra.

Figure 2

Optical mapping system with two geometry cameras. MC, mapping camera; GC1 and GC2, geometry cameras; F1 and F3, emission filter (590-nm long-pass filter); F2, emission filter (635 ± 27.5-nm band-pass filter). EX, excitation light source units (see Figs. S1 and S2). “Fire” is a digital pulse marking the start of each MC frame.

Figure 3

Evaluation of motion tracking with different marker spacings. (A) Pattern of marker placement. (B) Histograms of distance error with different marker spacings. Distance error is defined as the distance (in pixels) between the estimated image coordinates of site R and the actual image coordinates of site R.

Figure 4

V_m reconstruction on a Langendorff-perfused beating heart. The heart was paced at 500 ms BCL from the LV subepicardium near the apex. (A) Triangulation of trackable markers and location of epicardial sites. The signals in (B) are from epicardial site 1. Blue/cyan: deinterlaced blue- and cyan-elicited signals from the pixel where site 1 is located at the first frame, after spatial filtering. Blue tracked/cyan tracked: deinterlaced blue- and cyan-elicited signals, respectively, after motion tracking and spatial filtering. The bars are CCD signal counts (proportional to fluorescence intensity). Ratio: the motion-corrected V_m signal is the ratio of the blue- to cyan-elicited signals. The bar is dF/F. (C) Reconstructed V_m signals and dF/F from sites 2–5, respectively.

Figure 5

APDs of monophasic action potential (APD_MAP) and reconstructed optical V_m (APD_OAP). In three Langendorff-perfused hearts, MAP and V_m signals were simultaneously recorded at different spontaneous or paced activation rates. One-hundred-thirty activations from a total of 23 MAP-V_m site-pairs were analyzed. MAP-V_m site separation was <2 cm in all cases.

Figure 6

Reconstructed V_m signals in the setting of manually induced motion in a heart treated with BDM. Residual motion artifacts are apparent as shifts of V_m baseline. Angular displacement indicates the angle between the local surface normal vector and the optical axis of the mapping camera. Linear displacement indicates 3D displacement of the recording site. (Column A) The heart was rotated about its vertical axis. (Column B) The heart was swung from left-to-right relative to the camera. (Column C) The heart was swung forward and backward relative to the camera. (Column D) The heart was translated forward and backward in the direction parallel to optical axis of the camera. The calibration bars represent 2.5°, 5 mm, and 5%, respectively.

Figure 7

Simultaneously recorded V_m and mechanical contraction from an LV working heart. (A) Action potential propagation (upper rows) and the resulting mechanical contraction (lower rows) from a single beat. Regions in which V_m is unavailable are colored gray. (B) Mechanical contraction (fine line) and the simultaneously recorded V_m signal (bold line) from sites 1 and 2 in (A). The spatial average of shortening is reported at the bottom of each shortening map. Shortening is defined as the most negative eigenvalue of the stretch tensor, U. The heart was paced from the LV apex at 700 ms BCL, and LV preload and afterload pressures were set to 15 and 30 mmHg, respectively.

Figure 8

Altered electromechanical function after coronary artery occlusion. (A) Shortening (negative deformation) or stretch (positive deformation) in the major deformation direction (thin lines) and V_m (thick lines) before and 5 min after coronary artery occlusion. Site 1 is in a perfused region adjacent to the ischemic zone and site 2 is in the ischemic zone. Numbers under V_m show the APDs in milliseconds. (B) Spatial maps of strains before and after occlusion. This is an anterior view of the LV; the base is at the top and the LAD runs along the top-left edge of the mapped region. The bold circle indicates the LAD occlusion site. The strain maps were computed for the times (end-systole) indicated by the arrows in (A). White bars indicate positive strains (stretch); black bars indicate negative strains (shortening). Bar length is proportional to stretch or shortening relative to end-diastole. The bars are oriented in the major/minor directions. End-diastole was taken as the time immediately before action potential upstroke. The heart was paced at 600 ms BCL, and LV preload and afterload pressure values were set to 15 and 30 mmHg, respectively.