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Background: Recent clinical studies have indicated the presence of localized electrical abnormalities in idiopathic ventricular fibrillation and J-wave syndrome patients., Objectives: This study aims to characterize the specific electrical signatures of localized repolarization and conduction heterogeneities and their respective role in vulnerability to arrhythmias., Methods: Optical mapping was performed in porcine right ventricles with local: 1) repolarization shortening; 2) conduction slowing; or 3) structural heterogeneity induced by locally perfusing: 1) pinacidil (20 μmol/L, n = 13); or 2) flecainide (2 μmol/L, n = 13) via an epicardial catheter; or 3) by local epicardial tissue destruction (9 radiofrequency lesions n = 12). Electrograms were recorded (n = 5 in each group) and spontaneous and induced arrhythmias were quantified and optically mapped., Results: Electrograms were normal in (1) but showed local fragmentation in 40% of preparations in (2) with greater effects observed at high pacing frequencies dependent on the wavefront direction. In (3), the structural substrate alone increased the width and number of peaks in the electrograms, and addition of flecainide induced pronounced fragmentation (≥3 peaks and ≥70 ms) in all cases. Occurrence of spontaneous arrhythmias was significantly increased in (1) and (2) (P < 0.0001 and 0.05, respectively, vs baseline) and were triggered by ectopies. Vulnerability to arrhythmias at high pacing frequencies (≥2 Hz) was the lowest in (1) and greatest in (2)., Conclusions: Microstructural substrates have the most pronounced impact on electrograms, especially when combined with sodium channel blockers, whereas local action potential duration shortening does not lead to electrogram fragmentation even though it is associated with the highest prevalence of spontaneous arrhythmias., Competing Interests: Funding Support and Author Disclosures This study received financial support from the French Government as part of the “Investments of the Future” program managed by the National Research Agency (ANR-10-IAHU04-LIRYC). It was funded by the Leducq-Foundation (RHYTHM network, 16CVD02), the Fondation Coeur et Artères (FC17T2), and the European Research Council (ERC-2021-ADG 101054717). All authors have reported that they have no relationships relevant to the contents of this paper to disclose., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background Dispersion and gradients in repolarization have been associated with life-threatening arrhythmias, but are difficult to quantify precisely from surface electrocardiography. The objective of this study was to evaluate electrocardiographic imaging (ECGI) to noninvasively detect repolarization-based abnormalities. Methods and Results Ex vivo data were obtained from Langendorff-perfused pig hearts (n=8) and a human donor heart. Unipolar electrograms were recorded simultaneously during sinus rhythm from an epicardial sock and the torso-shaped tank within which the heart was suspended. Regional repolarization heterogeneities were introduced through perfusion of dofetilide and pinacidil into separate perfusion beds. In vivo data included torso and epicardial potentials recorded simultaneously in anesthetized, closed-chest pigs (n=5), during sinus rhythm, and ventricular pacing. For both data sets, ECGI accurately reconstructed T-wave electrogram morphologies when compared with those recorded by the sock (ex vivo: correlation coefficient, 0.85 [0.52-0.96], in vivo: correlation coefficient, 0.86 [0.52-0.96]) and repolarization time maps (ex-vivo: correlation coefficient, 0.73 [0.63-0.83], in vivo: correlation coefficient, 0.76 [0.67-0.82]). ECGI-reconstructed repolarization time distributions were strongly correlated to those measured by the sock (both data sets, R 2 ≥0.92). Although the position of the gradient was slightly shifted by 8.3 (0-13.9) mm, the mean, max, and SD between ECGI and recorded gradient values were highly correlated ( R 2 =0.87, 0.75, and 0.86 respectively). There was no significant difference in ECGI accuracy between ex vivo and in vivo data. Conclusions ECGI reliably and accurately maps potentially critical repolarization abnormalities. This noninvasive approach allows imaging and quantifying individual parameters of abnormal repolarization-based substrates in patients with arrhythmogenesis, to improve diagnosis and risk stratification.

The epicardial and endocardial surfaces of the heart are attractive targets to administer antiarrhythmic electrotherapies. Electrically stimulating wide areas of the surfaces of small mammalian ventricles is straightforward given the relatively small scale of their myocardial dimensions compared to the tissue space constant and electrical field. However, it has yet to be proven for larger mammalian hearts with tissue properties and ventricular dimensions closer to humans. Our goal was to address the feasibility and impact of wide-area electrical stimulation on the ventricular surfaces of large mammalian hearts at different stimulus strengths. This was accomplished by placing long line electrodes on the ventricular surfaces of pig hearts that span wide areas, and activating them individually. Stimulus efficacy was assessed and compared between surfaces, and tissue viability was evaluated. Activation time was dependent on stimulation strength and location, achieving uniform linear stimulation at 9x threshold strength. Endocardial stimulation activated more tissue transmurally than epicardial stimulation, which could be considered a potential target for future cardiac electrotherapies. Overall, our results indicate that electrically stimulating wide areas of the ventricular surfaces of large mammals is achievable with line electrodes, minimal tissue damage, and energies under the human pain threshold (100 mJ).

Objective: The growing adult population with surgically corrected tetralogy of Fallot (TOF) is at risk of arrhythmias and sudden cardiac death. We sought to investigate the contribution of right ventricular (RV) structural and electrophysiological remodelling to arrhythmia generation in a preclinical animal model of repaired TOF (rTOF)., Methods and Results: Pigs mimicking rTOF underwent cardiac MRI functional characterisation and presented with pulmonary regurgitation, RV hypertrophy, dilatation and dysfunction compared with Sham-operated animals (Sham). Optical mapping of rTOF RV-perfused wedges revealed a significant prolongation of RV activation time with slower conduction velocities and regions of conduction slowing well beyond the surgical scar. A reduced protein expression and lateralisation of Connexin-43 were identified in rTOF RVs. A remodelling of extracellular matrix-related gene expression and an increase in collagen content that correlated with prolonged RV activation time were also found in these animals. RV action potential duration (APD) was prolonged in the epicardial anterior region at early and late repolarisation level, thus contributing to a greater APD heterogeneity and to altered transmural and anteroposterior APD gradients in rTOF RVs. APD remodelling involved changes in Kv4.3 and MiRP1 expression. Spontaneous arrhythmias were more frequent in rTOF wedges and more complex in the anterior than in the posterior RV., Conclusion: Significant remodelling of RV conduction and repolarisation properties was found in pigs with rTOF. This remodelling generates a proarrhythmic substrate likely to facilitate re-entries and to contribute to sudden cardiac death in patients with rTOF., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.)

Myocardial conduction velocity is important for the genesis of arrhythmias. In the normal heart, conduction is primarily dependent on fiber direction (anisotropy) and may be discontinuous at sites with tissue heterogeneities (trabeculated or fibrotic tissue). We present a semi-automated method for the accurate measurement of conduction velocity based on high-resolution activation mapping following central stimulation. The method was applied to activation maps created from myocardium from man, sheep and mouse with anisotropic and discontinuous conduction. Advantages of the presented method over existing methods are discussed., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The spatiotemporal dynamics of arrhythmias are likely to be complex three-dimensional phenomena. Yet, the lack of high-resolution three-dimensional imaging techniques, both in the clinic and the experimental lab, limits our ability to better understand the mechanisms of such arrhythmias. Optical mapping using voltage-sensitive dyes is a widely used tool in experimental electrophysiology. It has been known for decades that even in its most basic application, epi-fluorescence, the optical signal contains information from within a certain intramural volume. Understanding of this fundamental property of optical signals has paved the way towards novel three-dimensional optical imaging techniques. Here, we review our current understanding of the three-dimensional nature of optical signals; how penetration depths of cardiac optical imaging can be improved by using novel imaging modalities and finally, we highlight new techniques inspired from optical tomography and aiming at full depth-resolved optical mapping of cardiac electrical activity.

We demonstrate the synergistic benefits of using multiple technologies to investigate complex multi-scale biological responses. The combination of reductionist and integrative methodologies can reveal novel insights into mechanisms of action by tracking changes of in vivo phenomena to alterations in protein activity (or vice versa). We have applied this approach to electrical and mechanical remodelling in right ventricular failure caused by monocrotaline-induced pulmonary artery hypertension in rats. We show arrhythmogenic T-wave alternans in the ECG of conscious heart failure animals. Optical mapping of isolated hearts revealed discordant action potential duration (APD) alternans. Potential causes of the arrhythmic substrate; structural remodelling and/or steep APD restitution and dispersion were observed, with specific remodelling of the Right Ventricular Outflow Tract. At the myocyte level, [Ca(2+)]i transient alternans were observed together with decreased activity, gene and protein expression of the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA). Computer simulations of the electrical and structural remodelling suggest both contribute to a less stable substrate. Echocardiography was used to estimate increased wall stress in failure, in vivo. Stretch of intact and skinned single myocytes revealed no effect on the Frank-Starling mechanism in failing myocytes. In isolated hearts acute stretch-induced arrhythmias occurred in all preparations. Significant shortening of the early APD was seen in control but not failing hearts. These observations may be linked to changes in the gene expression of candidate mechanosensitive ion channels (MSCs) TREK-1 and TRPC1/6. Computer simulations incorporating MSCs and changes in ion channels with failure, based on altered gene expression, largely reproduced experimental observations., (Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Optical mapping has become an indispensible tool for studying cardiac electrical activity. However, due to the three-dimensional nature of the optical signal, the optical upstroke is significantly longer than the electrical upstroke. This raises the issue of how to accurately determine the activation time on the epicardial surface. The purpose of this study was to establish a link between the optical upstroke and exact surface activation time using computer simulations, with subsequent validation by a combination of microelectrode recordings and optical mapping experiments. To simulate wave propagation and associated optical signals, we used a hybrid electro-optical model. We found that the time of the surface electrical activation (t(E)) within the accuracy of our simulations coincided with the maximal slope of the optical upstroke (t(F)*) for a broad range of optical attenuation lengths. This was not the case when the activation time was determined at 50% amplitude (t(F50)) of the optical upstroke. The validation experiments were conducted in isolated Langendorff-perfused rat hearts and coronary-perfused pig left ventricles stained with either di-4-ANEPPS or the near-infrared dye di-4-ANBDQBS. We found that t(F)* was a more accurate measure of t(E) than was t(F50) in all experimental settings tested (P = 0.0002). Using t(F)* instead of t(F50) produced the most significant improvement in measurements of the conduction anisotropy and the transmural conduction time in pig ventricles., (Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Transmural myocardial activation is influenced by myocardial structure, including structural differences between the compacta (Cta) and the trabeculata (Tta), although this has not been fully explained. Hearts from rats were Langendorff perfused, stained with DI-4-ANEPPS, the apex was cut off and fluorescence acquired from the exposed short-axis surface. The hearts were stimulated at 160 ms cycle length at the anterior, lateral, posterior left ventricle (LV) and septal sub-epicardial sites. Conduction velocity perpendicular to the wave front orientation was measured in each pixel using a gradient-based approach. After optical mapping the cut surface was imaged using a light microscope and the extent of the Cta and Tta mapped and validated against 50 u, m isotropic MRI images. We used a 3D rat ventricle computational model, with architecture obtained from 200 u, m isotropic diffusion tensor MRI and kinetics from the modified Pandit model to determine the relative roles of fibers and sheets on propagation. We show in the experimental study that circumferential propagation around the LV cavity is fast in the Cta: 63.2±19.5 and is slower in the Tta: 32.7±11.0(∗) (mean ± s.d cms-1, ∗ p < 0.01 by two sample t test). In the simulation study the pattern and velocity are not replicated in an isotropic model (I), are partially replicated in a simulation study including fiber anisotropy (A) and is more fully replicated in orthotropic (O) ventricles (fiber and sheet anisotropy), where the circumferential propagation velocity is, I: Cta: 54.2±3.9; Tta:54.3±3.9; A: Cta:43.6±3.2; Tta: 40.6±6.6; O: Cta: 63.2±19.5; Tta: 32.7±11.9(∗). We show that sheet orientation is important in understanding activation differences between Cta and Tta.

Current techniques to map intramural activation patterns in ex vivo cardiac tissue have limited spatial resolution. Here, we report on the experimental validation of a novel optical technique that was recently proposed to resolve the size and depth of intramural wave fronts using alternating transillumination (AT). AT was achieved by simultaneously mapping the epi- and endocardial surfaces with two synchronized CCD cameras and rapidly alternating LED illumination between both surfaces. Optical phantoms were made based on tissue optical properties measured using a hybrid optical spectrometer. Spherical fluorescent sources (Scarlet microspheres, Invitrogen, UK) of varying sizes were embedded at known depths in the phantoms. Coronary-perfused procine left ventricular slab preparations were stained with DI-4-ANBDQBS (n = 3) and paced at known intramural depths. In phantoms we were able to reliably estimate the depth of the center of fluorescent sources (9.6 ± 5.4% error), as well as their transmural extent (15.7 ± 11.5% error). In ventricular slabs we were able to localize the sites of origin of intramural excitation waves with a precision of ± 1.6 mm. Transmural conduction velocities were, for the first time, measured optically from the surfaces and found to be 21.0 ± 12.4 cm/s. In conclusion, alternating transillumination is a promising technique for reliable reconstruction of depth and expansion rate of intramural activation wave fronts in cardiac tissue.

The development of voltage-sensitive dyes has revolutionized cardiac electrophysiology and made optical imaging of cardiac electrical activity possible. Photon diffusion models coupled to electrical excitation models have been successful in qualitatively predicting the shape of the optical action potential and its dependence on subsurface electrical wave orientation. However, the accuracy of the diffusion equation in the visible range, especially for thin tissue preparations, remains unclear. Here, we compare diffusion and Monte Carlo (MC) based models and we investigate the role of tissue thickness. All computational results are compared to experimental data obtained from intact guinea pig hearts. We show that the subsurface volume contributing to the epi-fluorescence signal extends deeper in the tissue when using MC models, resulting in longer optical upstroke durations which are in better agreement with experiments. The optical upstroke morphology, however, strongly correlates to the subsurface propagation direction independent of the model and is consistent with our experimental observations.

Information about intramural propagation of electrical excitation is crucial to understanding arrhythmia mechanisms in thick ventricular muscle. There is currently a controversy over whether it is possible to extract such information from the shape of the upstroke in optical mapping recordings. We show that even in the complex geometry of a whole guinea pig heart, optical upstroke morphology reveals the 3D wavefront orientation near the surface. To characterize the upstroke morphology, we use V(F)(*), the fractional level at which voltage-sensitive fluorescence, V(F), has maximal time derivative. Low values of V(F)(*)( approximately 0.2) indicate a wavefront moving away from the surface, high values of V(F)(*) ( approximately 0.6) a wavefront moving toward the surface, and intermediate values of V(F)(*) ( approximately 0.4) a wavefront moving parallel to the surface. We further performed computer simulations using Luo-Rudy II electrophysiology and a simplified 3D geometry. The simulated V(F)(*) maps for free wall and apical stimulations as well as for sinus rhythm are in good quantitative agreement with the averaged experimental results. Furthermore, computer simulations show that the effect of the curvature of the heart on wave propagation is negligible.

We present a study of the 3-dimensional (3D) propagation of electrical waves in the heart wall using Laminar Optical Tomography (LOT). Optical imaging contrast is provided by a voltage sensitive dye whose fluorescence reports changes in membrane potential. We examined the transmural propagation dynamics of electrical waves in the right ventricle of Langendorf perfused rat hearts, initiated either by endo-cardial or epi-cardial pacing. 3D images were acquired at an effective frame rate of 667Hz. We compare our experimental results to a mathematical model of electrical transmural propagation. We demonstrate that LOT can clearly resolve the direction of propagation of electrical waves within the cardiac wall, and that the dynamics observed agree well with the model of electrical propagation in rat ventricular tissue.

Optical imaging using voltage-sensitive dyes has become an important tool for studying vortex-like electrical waves in the heart. Such waves, known as spiral or scroll waves, can spontaneously form in pathological ventricular myocardium, causing ventricular fibrillation and sudden death. Until recently, observations of scroll waves were limited to their surface manifestations, thus providing little information about the shape and location of their organizing center, the filament. We use computer modeling to assess the feasibility of visualizing filaments using dynamic transillumination imaging in conjunction with near-IR voltage-sensitive absorptive dyes (absorptive transillumination). We simulate transillumination signals produced by the intramural scroll waves in a realistic slab of ventricular tissue with trabeculated endocardial surface. The computations use a detailed ionic model of electrical excitation (LRd) coupled to a photon transport model for cardiac tissue. Our simulations show that dynamic absorptive transillumination data, with subsequent processing involving either amplitude maps, time-space plots, or power-of-the-dominant-frequency maps, can be used to reliably detect intramural scroll waves through the whole thickness (approximately 10 mm) of the ventricular wall. Neither variations in the thickness of the myocardial wall nor noise impeded the detection of intramural filaments.

Optical mapping using voltage-sensitive fluorescent dyes has become a major tool for studying excitation propagation in the heart. Computational and experimental studies have indicated that the optical upstroke morphology reflects the orientation of the subsurface excitation front. In a recent whole heart computational study performed by Bishop et al. (Bishop, M. J., B. Rodriguez, J. Eason, J. P. Whiteley, N. Trayanova, and D. J. Gavaghan. 2006. Synthesis of voltage-sensitive optical signals: application to panoramic optical mapping. Biophys. J. 90:2938-2945), an example was provided of two different directions of propagation having nevertheless very similar epicardial optical upstrokes. The goal of this comment is to clarify the interpretation of optical upstroke morphologies and reconcile the results obtained by Bishop et al. with previous computational and experimental studies.

Cardiac electrical activity can be mapped today through the response of voltage-sensitive dyes; but poor transparency of muscle tissue has enforced shallow-depth imaging. We present a three-dimensional (3D) reconstruction method for electrical activity deep inside the myocardial wall. Our approach is nonlinear and differs substantially from standard diffusive optical tomography. It does not require matrix inversion, data regularization or a priori information concerning the original object. Opposite sides of a slab-shaped preparation are scanned in parallel by detection and illumination points with a constant vector offset between illumination and detection axes (biaxial scanning). Scanning is performed in two perpendicular directions. In each direction, a pair of 2D images is obtained under offsets of opposite signs. These two pairs are the input for a multiplicative reconstruction algorithm, whose output is a 3D image. The overall procedure was successfully tested on computer-generated sources that include points, lines and hemispheres, patterned after actual electrophysiological excitations. The algorithm is computationally efficient and stable with respect to varying noise levels in the raw data.

This study explores the possibility of localizing the excitation centers of electrical waves inside the heart wall using voltage-sensitive dyes (fluorescent or absorptive). In the present study, we propose a method for the 3-D localization of excitation centers from pairs of 2-D images obtained in two modes of observation: reflection and transillumination. Such images can be obtained using high-speed charge-coupled device (CCD) cameras and photodiode arrays with time resolution up to 0.5 ms. To test the method, we simulate optical signals produced by point sources and propagating ellipsoidal waves in 1-cm-thick slabs of myocardial tissue. Solutions of the optical diffusion equation are constructed by employing the method of images with Robin boundary conditions. The coordinates of point sources as well as of the centers of expanding waves can be accurately determined using the proposed algorithm. The method can be extended to depth estimations of the outer boundaries of the expanding wave. The depth estimates are based on ratios of spatially integrated images. The method shows high tolerance to noise and can give accurate results even at relatively low signal-to-noise ratios. In conclusion, we propose a novel and efficient algorithm for the localization of excitation centers in 3-D cardiac tissue.

The rotating fiber orientation within the cardiac wall substantially affects the electrical propagation and can cause intra-myocardial cusp waves. Numerical simulations have shown that the cusps form in layers where propagation is perpendicular to the fiber orientation and lead to complex wave front morphologies. They can travel across layers and break through at the epi- or endocardial surfaces where they cause apparent accelerations of propagation. The validation of these results remains a major experimental challenge. In the present study, we investigate both computationally and experimentally how intramural cusp waves can be detected using optical imaging. Our simulations show that cusps alter the optical upstroke morphology and can be detected well before they reach the surface (up to 1 mm deep). Experiments in Langendorff-perfused guinea pig hearts are consistent with our numerical findings.

Aims: Here, we investigate the mechanisms underlying the onset of conduction-related arrhythmias in a three-dimensional (3D) computational model of acute regional ischaemia., Methods: Ischaemia was introduced by realistic gradients of potassium, pH, oxygen and electrical coupling in a 3D slab of ventricular tissue using the LRd model. We focused on a specific stage (10-15 min after occlusion) at which an intramural non-conductive ischaemic core (IC) surrounded by a border zone (BZ) has formed., Results: At pacing frequencies greater than 4.5 Hz, we observed narrow areas (0.5 mm wide) of 2:1 conduction blocks at the periphery of the IC. As the pacing frequency increased, the area of block widened to 9 mm and gave rise to reentry at the periphery of the BZ. Alternating conduction blocks produced discordant action potential duration (APD) alternans throughout the slab and T-wave alternans in pseudo-ECG. Slowing the recovery of the calcium current broadened the range of pacing frequencies at which blocks were observed. Hyperkalaemia alone was sufficient to induce the alternating blocks., Conclusion: Computer modelling predicts that ischaemia-related arrhythmias are triggered by calcium-mediated alternating conduction blocks in the ischaemic border zone. Alternating conduction blocks lead to intramural reentry and APD alternans.

Voltage-sensitive dyes are an important tool in visualizing electrical activity in cardiac tissue. Until today, they have mainly been applied in cardiac electrophysiology to subsurface imaging. In the present study, we assess different imaging methods used in optical tomography with respect to their effectiveness in visualizing 3D cardiac activity. To achieve this goal, we simulate optical signals produced by excitation fronts initiated at different depths inside the myocardial wall and compare their properties for various imaging modes. Specifically, we consider scanning and broad-field illumination, including trans- and epi-illumination. We focus on the lateral optical resolution and signal intensity, as a function of the source depth. Optical diffusion theory is applied to derive a computationally efficient approximation of the point-spread function and to predict voltage-sensitive signals. Computations were performed both for fluorescent and absorptive voltage-sensitive dyes. Among all the above-mentioned methods, fluorescent coaxial scanning yields the best resolution (<2.5 mm) and gives the most information about the intramural cardiac activity.

The cardiac muscle is well known to conduct electric impulses anisotropically, showing a larger conduction velocity along than across fibers. Fiber orientation is not uniform within the cardiac wall, but rotates by as much as 180 degrees throughout the wall thickness. Numerical simulations and experiments have indicated that this rotational anisotropy considerably affects the spread of excitation in cardiac tissue: the wave front shows a complex intramural shape with trailing cusps. The cusps can travel across layers and reach the epicardial and endocardial surfaces where they cause apparent accelerations of propagation. In the present study we provide an analytical description of the asymptotic wave front, as well as of cusp waves. We investigate the motion of cusp waves, based on the assumption that they occur at the intersection of asymptotic solutions, and we show that our theoretical analysis is in close agreement with numerical simulations. The asymptotic solutions are found to be determined purely by the fiber organization within the cardiac wall, independent of the excitable properties of cardiac tissue.