In vivo ratiometric optical mapping enables high-resolution cardiac electrophysiology in pig models

Abstract

Aims: Cardiac optical mapping is the gold standard for measuring complex electrophysiology in ex vivo heart preparations. However, new methods for optical mapping in vivo have been elusive. We aimed at developing and validating an experimental method for performing in vivo cardiac optical mapping in pig models.

Methods and results: First, we characterized ex vivo the excitation-ratiometric properties during pacing and ventricular fibrillation (VF) of two near-infrared voltage-sensitive dyes (di-4-ANBDQBS/di-4-ANEQ(F)PTEA) optimized for imaging blood-perfused tissue (n = 7). Then, optical-fibre recordings in Langendorff-perfused hearts demonstrated that ratiometry permits the recording of optical action potentials (APs) with minimal motion artefacts during contraction (n = 7). Ratiometric optical mapping ex vivo also showed that optical AP duration (APD) and conduction velocity (CV) measurements can be accurately obtained to test drug effects. Secondly, we developed a percutaneous dye-loading protocol in vivo to perform high-resolution ratiometric optical mapping of VF dynamics (motion minimal) using a high-speed camera system positioned above the epicardial surface of the exposed heart (n = 11). During pacing (motion substantial) we recorded ratiometric optical signals and activation via a 2D fibre array in contact with the epicardial surface (n = 7). Optical APs in vivo under general anaesthesia showed significantly faster CV [120 (63-138) cm/s vs. 51 (41-64) cm/s; P = 0.032] and a statistical trend to longer APD90 [242 (217-254) ms vs. 192 (182-233) ms; P = 0.095] compared with ex vivo measurements in the contracting heart. The average rate of signal-to-noise ratio (SNR) decay of di-4-ANEQ(F)PTEA in vivo was 0.0671 ± 0.0090 min-1. However, reloading with di-4-ANEQ(F)PTEA fully recovered the initial SNR. Finally, toxicity studies (n = 12) showed that coronary dye injection did not generate systemic nor cardiac damage, although di-4-ANBDQBS injection induced transient hypotension, which was not observed with di-4-ANEQ(F)PTEA.

Conclusions: In vivo optical mapping using voltage ratiometry of near-infrared dyes enables high-resolution cardiac electrophysiology in translational pig models.

Keywords: In vivo imaging; Cardiac fibrillation; Cardiotoxicity; Optical mapping; Voltage-sensitive dyes.

Graphical Abstract

Figure 1

In vivo optical mapping systems. (A) Schematic representation of the system built to optically map the exposed heart in the open-chest pig during dye loading and ventricular fibrillation (dye-loading region highlighted purple). (B) Schematic representation of the modified system using an optical-fibre array to measure action potentials during regular contraction. (C) 3D representation of the system in (A) during an in vivo experiment. (D) 3D representation of the system in (B) during an in vivo experiment.

Figure 2

Excitation ratiometry of di-4-ANBDQBS and di-4-ANEQ(F)PTEA in the isolated pig heart with excitation-contraction uncoupled. (A) Flow-chart of optical mapping experiments represented in the figure. (B) Action potential (AP) signals at sites ‘a’, ‘b’ (di-4-ANBDQBS), ‘c,’ and ‘d’ (di-4-ANEQ(F)PTEA) on the epicardial surface of the heart obtained by exciting the tissue at ∼480 nm (numerator; blue) and ∼640 nm (denominator; red). The ratio of the numerator to denominator signal is shown in black (stimulation site: red square pulse). (C) Time series of normalized transmembrane voltage fluorescence intensity maps (using ratio signals) during point electrical pacing (500 ms CL) (di-4-ANBDQBS) and sinus rhythm (di-4-ANEQ(F)PTEA). (D) DF maps during VF using di-4-ANBDQBS (left panels) and di-4-ANEQ(F)PTEA (right panels). Sample AP signals and DF values are shown at sites ‘a’ (di-4-ANBDQBS) and ‘b’ (di-4-ANEQ(F)PTEA). AP signals illustrate irregular electrical activity and alterations in emission intensity of opposite polarity. Signals are in arbitrary fluorescence units. DF, dominant frequency; LV, left ventricle; RV, right ventricle; SR, sinus rhythm.

Figure 3

Ex vivo optical fibre-based excitation ratiometry of di-4-ANBDQBS and di-4-ANEQ(F)PTEA in contracting tissue. (A) Flow-chart of optical mapping experiments represented in the figure. (B) 2D optical-fibre array pressed against the surface of a Langendorff-perfused pig heart (left panel). Numerator (blue), denominator (red) and ratio (black) action potential signals from two sample fibres using di-4-ANBDQBS (middle panel), and before and after blebbistatin using di-4-ANEQ(F)PTEA (right panel). (C) Sample activation maps illustrating propagation of the wavefront (left panel) and APD maps (right panel) before and after blebbistatin in the perfusate. Stimulation sites (black square pulses) are shown relative to the fibre array. (D, E) APD (D) and CV (E) comparisons (Wilcoxon Signed Rank test) before and after blebbistatin in the perfusate. Five hearts and four sample fibres per heart were included in the analysis. (F) AP signals from a 500 µm fibre, demonstrating motion artefact cancellation. Signals are in arbitrary fluorescence units. APD, action potential duration; CV, conduction velocity; LAT, local activation time.

Figure 4

In vivo dye loading. (A) Left panel: Brightfield image of a sample heart loaded using balloon occlusion of the LAD coronary artery. Right panel: Fluorescence image of the same heart using red LED excitation (∼640 nm). (B) Left panel: Brightfield image of another sample heart with the dye-loading region delineated by the black dashed line. Right panels: Snapshots of a fluorescence coronary angiogram recorded at 300 fps with a high-resolution CMOS camera during dye loading in the presence of regular coronary blood flow.

Figure 5

In vivo optical mapping of paced rhythms and ventricular fibrillation (VF). (A) Flow-chart of optical mapping experiments represented in the figure. (B) Action potential (AP) signals at sites ‘a’, ‘b’ (di-4-ANBDQBS), ‘c,’ and ‘d’ (di-4-ANEQ(F)PTEA) on the epicardial surface of the heart obtained by exciting the tissue at ∼480 nm (numerator; blue) and ∼640 nm (denominator; red). The ratio of the numerator to denominator signal is shown in black (stimulation sites: red square pulses). Despite the substantial motion, ratiometric optical mapping could detect the AP upstroke. (C, D) Time series of normalized transmembrane voltage fluorescence intensity maps and activation map (using ratio signals) during point electrical pacing (300 ms CL). (E) DF maps during VF using di-4-ANBDQBS (left panels) and di-4-ANEQ(F)PTEA (right panels). Sample AP signals and DF values are shown at sites ‘a’ (di-4-ANBDQBS) and ‘b’ (di-4-ANEQ(F)PTEA). AP signals illustrate irregular electrical activity during VF. Signals are in arbitrary fluorescence units. (F) DF and SPD comparisons (Mann–Whitney U test) between in vivo (n = 4) and ex vivo (n = 5) VF episodes. DF, dominant frequency; LV, left ventricle; RV, right ventricle; SPD, singularity point density; VF, ventricular fibrillation.

Figure 6

In vivo optical-fibre fluorescence recordings. (A) Flow-chart of optical mapping experiments represented in the figure. (B) Positioning of the optical-fibre array during an experimental procedure (left panel). Sample numerator (blue), denominator (red) and ratio (black) AP signals at 500 and 250 ms BDCLs using di-4-ANBDQBS (middle panel) and di-4-ANEQ(F)PTEA (right panel). (C) Sample activation maps illustrating propagation of the wavefront (right panels) and APD maps (left panels) at 500 and 250 ms BDCLs using di-4-ANEQ(F)PTEA. Stimulation sites (black square pulses) are shown relative to the fibre array. (D) APD and CV comparisons (Mann–Whitney U test) between in vivo (n = 5) and ex vivo (n = 5) hearts (four sample fibres per heart). (E, F) Two sample SNR decays (E, F) and reloading (F) with di-4-ANEQ(F)PTEA. Signals are in arbitrary fluorescence units. APD, action potential duration; BDCL, basic drive cycle length; CV, conduction velocity; LAT, local activation time; SNR, signal-to-noise ratio.

Figure 7

Dye toxicity assessment. (A) Flow-chart of the toxicity protocol. (B) Sample ECG tracings (lead V2) during the monitoring period in the three subgroups. (C–F) QRS complex duration (C), corrected QT interval (D), mean blood pressure (E), and Troponin I (cTnI) (F) comparisons among subgroups. The infarct threshold criterion after percutaneous coronary interventions is represented with a brown dashed line (F). (G, H) Comparisons of T2 GraSE (G) and post-contrast T1 mapping (H) sequences among subgroups. Data are represented as mean and standard deviation, but in (E), standard errors of the means are shown because of the large variability in blood pressure measurements. Physiological ranges are represented with green dashed lines. Blue circles: solvent (n = 4). Red squares: di-4-ANBDQBS+solvent (n = 4). Purple diamonds: di-4-ANEQ(F)PTEA+solvent (n = 4). ^# and ^† indicate P-values for two-way repeated measures ANOVA of solvent vs. di-4-ANBDQBS (^#), and solvent vs. di-4-ANEQ(F)PTEA (^†).