Cardiac intramural electrical mapping reveals focal delays but no conduction velocity slowing in the peri-infarct region.

Altered electrical behavior alongside healed myocardial infarcts (MIs) is associated with increased risk of sudden cardiac death. However, the multidimensional mechanisms are poorly understood and described. This study characterizes, for the first time, the intramural spread of electrical activation in the peri-infarct region of chronic reperfusion MIs. Four sheep were studied 13 wk after antero-apical reperfusion infarction. Extracellular potentials (ECPs) were recorded in a ~20 × 20-mm2 region adjacent to the infarct boundary (25 plunge needles <0.5-mm diameter with 15 electrodes at 1-mm centers) during multisite stimulation. Infarct geometry and electrode locations were reconstructed from magnetic resonance images. Three-dimensional activation spread was characterized by local activation times and interpolated ECP fields (n = 191 records). Control data were acquired in 4 non-infarcted sheep (n = 96 records). Electrodes were distributed uniformly around 15 ± 5% of the intramural infarct boundary. There were marked changes in pacing success and ECP morphology across a functional border zone (BZ) ±2 mm from the boundary. Stimulation adjacent to the infarct boundary was associated with low-amplitude electrical activity within the BZ and delayed activation of surrounding myocardium. Bulk tissue depolarization occurred 3.5-14.6 mm from the pacing site for 39% of stimuli with delays of 4-37 ms, both significantly greater than control (P < 0.0001). Conduction velocity (CV) adjacent to the infarct was not reduced compared with control, consistent with structure-only computer model results. Insignificant CV slowing, irregular stimulus-site specific activation delays, and obvious indirect activation pathways strongly suggest that the substrate for conduction abnormalities in chronic MI is predominantly structural in nature.NEW & NOTEWORTHY Intramural in vivo measurements of peri-infarct electrical activity were not available before this study. We use pace-mapping in a three-dimensional electrode array to show that a subset of stimuli in the peri-infarct region initiates coordinated myocardial activation some distance from the stimulus site with substantial associated time delays. This is site dependent and heterogeneous and occurs for <50% of ectopic stimuli in the border zone. Furthermore, once coordinated activation is initiated, conduction velocity adjacent to the infarct boundary is not significantly different from control. These results give new insights to peri-infarct electrical activity and do not support the widespread view of uniform electrical remodeling in the border zone of chronic myocardial infarcts, with depressed conduction velocity throughout.

Quantitative descriptions of extended volume cardiac tissue architecture from multiple large hearts.

The arrangement of cardiac cells into strand and sheet-like structures within the heart wall, confers important electrical properties onto heart tissue. Unraveling cardiomyocyte architecture in both healthy and diseased hearts is fundamental to understanding the mechanisms generating normal rhythm and arrhythmia. We analyzed five extended volume serial image stacks of normal pig left ventricular tissue. Analysis included: (1) reconstruction of original tissue volume and shape with non-linear correction maps; (2) segmentation and higher-order descriptions, areas and orientations of laminar structures through the heart wall; (3)computation of fiber directions; (4) computation of tissue connectivity using a shell filter. These measures contributed to a deeper and more objective understanding of cardiac tissue structures and their spatial variation than previously possible.

Development of 3-D Intramural and Surface Potentials in the LV: Microstructural Basis of Preferential Transmural Conduction.

INTRODUCTION: Extracellular potentials measured on the heart surfaces are used to infer events that originate deep within the heart wall. We have reconstructed intramural potentials in three dimensions for the first time, and compare these with epicardial and endocardial surface potentials and cardiac microstructure. METHODS AND RESULTS: Extracellular potentials from intramural point stimulation were measured from a high density 3-D electrode array in the in vivo pig LV. MR and extended volume imaging were used to register electrode locations and characterize fiber and laminar orientations throughout the recording volume. Measured potentials were compared with predictions of tissue-specific bidomain computer activation models. Positive potentials recorded in the LV wall preceded the depolarization wavefront as it spread in the fiber direction. Transverse to this, passive and active potentials spread preferentially in the laminar direction (anisotropy ratio ∼1.6:1). Epicardial surface potentials reflect initial intramural propagation at the stimulus location, but endocardial potentials do not, particularly adjacent to papillary muscles. Measured 3-D potentials were consistently better captured by computer models that incorporate three distinct conductivities aligned with local microstructural axes, but the preferential spread of potentials in the laminar direction was not fully predicted. CONCLUSIONS: This study provides evidence for preferential transmural conduction and raises questions about the extent to which intramural electrical events can be inferred from endocardial potentials.

Cardiac response to low-energy field pacing challenges the standard theory of defibrillation.

BACKGROUND: The electric response of myocardial tissue to periodic field stimuli has attracted significant attention as the basis for low-energy antifibrillation pacing, potentially more effective than traditional single high-energy shocks. In conventional models, an electric field produces a highly nonuniform response of the myocardial wall, with discrete excitations, or hot spots (HS), occurring at cathodal tissue surfaces or large coronary vessels. We test this prediction using novel 3-dimensional tomographic optical imaging. METHODS AND RESULTS: Experiments were performed in isolated coronary perfused pig ventricular wall preparations stained with near-infrared voltage-sensitive fluorescent dye DI-4-ANBDQBS. The 3-dimensional coordinates of HS were determined using alternating transillumination. To relate HS formation with myocardial structures, we used ultradeep confocal imaging (interrogation depths, >4 mm). The peak HS distribution is located deep inside the heart wall, and the depth is not significantly affected by field polarity. We did not observe the strong colocalization of HS with major coronary vessels anticipated from theory. Yet, we observed considerable lateral displacement of HS with field polarity reversal. Models that de-emphasized lateral intracellular coupling and accounted for resistive heterogeneity in the extracellular space showed similar HS distributions to the experimental observations. CONCLUSIONS: The HS distributions within the myocardial wall and the significant lateral displacements with field polarity reversal are inconsistent with standard theories of defibrillation. Extended theories based on enhanced descriptions of cellular scale electric mechanisms may be necessary. The considerable lateral displacement of HS with field polarity reversal supports the hypothesis of biphasic stimuli in low-energy antifibrillation pacing being advantageous.

Imaging electrical excitation inside the myocardial wall.

Cardiac arrhythmias are often triggered by ectopic membrane depolarization originating deep inside the myocardial wall. Here we propose a new method utilizing a novel near-infrared voltage-sensitive fluorescent dye DI-4-ANBDQBS to determine the three-dimensional (3D) coordinates of the sources of such depolarization. We tested the method in live preparations of pig left and right ventricular myocardium (thickness 8-18 mm) and phantoms imitating the optical properties of myocardial tissue. The method utilizes an alternating transillumination approach that involves comparing pairs of simultaneously recorded broad-field epifluorescence and transillumination images produced at two alternating directions of illumination. Recordings were taken simultaneously by two CCD cameras facing the endocardial and epicardial surfaces of the heart at a frame rate up to 3 KHz. In live preparations, we were able to localize the origin of the depolarization wave with a precision of ±1.3mm in the transmural direction and 3 mm in the image plane. The accuracy of detection was independent of the depth of the source inside ventricular wall.

Three-dimensional cardiac tissue image registration for analysis of in vivo electrical mapping.

A method is presented for registering 3D cardiac tissue images to reference data, for the purpose of analyzing recorded electrical activity. Following left-ventricular in vivo electrical mapping studies in pig hearts, MRI is used to define a reference geometry in the tissue segment around the recording electrodes. The segment is then imaged in 3D using a high-resolution serial imaging microscopy technique. The tissue processing required for this introduces segment-wide distortion. Piecewise-smooth maps are used to correct the tissue distortion and register the 3D images with the reference MRI data. The methods are validated and techniques for identifying the preferred maps are proposed. Recorded electrical activation is shown to map reliably onto cardiac tissue structure using this registration method.

Probing field-induced tissue polarization using transillumination fluorescent imaging.

Despite major successes of biophysical theories in predicting the effects of electrical shocks within the heart, recent optical mapping studies have revealed two major discrepancies between theory and experiment: 1), the presence of negative bulk polarization recorded during strong shocks; and 2), the unexpectedly small surface polarization under shock electrodes. There is little consensus as to whether these differences result from deficiencies of experimental techniques, artifacts of tissue damage, or deficiencies of existing theories. Here, we take advantage of recently developed near-infrared voltage-sensitive dyes and transillumination optical imaging to perform, for the first time that we know of, noninvasive probing of field effects deep inside the intact ventricular wall. This technique removes some of the limitations encountered in previous experimental studies. We explicitly demonstrate that deep inside intact myocardial tissue preparations, strong electrical shocks do produce considerable negative bulk polarization previously inferred from surface recordings. We also demonstrate that near-threshold diastolic field stimulation produces activation of deep myocardial layers 2-6 mm away from the cathodal surface, contrary to theory. Using bidomain simulations we explore factors that may improve the agreement between theory and experiment. We show that the inclusion of negative asymmetric current can qualitatively explain negative bulk polarization in a discontinuous bidomain model.

Shock induced electrical activation in structurally detailed models of pig left-ventricular tissue.

Detailed models of sample specific structures in pig left-ventricular tissue have been constructed. These models include epicardial and endocardial surfaces, fiber and sheet orientations, vessels and cleavage planes with significant dimensions. This work shows that it is possible to extract from 3D tissue images reduced dimension descriptions of cleavage planes in the heart wall. These descriptions are used to analyze the response of tissue to electrical shocks of varying strengths. The presence of explicit discontinuities in the heart significantly reduces the time required for transmural activation and provides a basis for understanding successful defibrillation.

Three distinct directions of intramural activation reveal nonuniform side-to-side electrical coupling of ventricular myocytes.

BACKGROUND: The anisotropy of cardiac tissue is a key determinant of 3D electric propagation and the stability of activation wave fronts in the heart. The electric properties of ventricular myocardium are widely assumed to be axially anisotropic, with activation propagating most rapidly in the myofiber direction and at uniform velocity transverse to this. We present new experimental evidence that contradicts this view. METHODS AND RESULTS: For the first time, high-density intramural electric mapping (325 electrodes at approximately 4x4x1-mm spacing) from pig left ventricular tissue was used to reconstruct 3D paced activation surfaces projected directly onto 3D tissue structure imaged throughout the same left ventricular volume. These data from 5 hearts demonstrate that ventricular tissue is electrically orthotropic with 3 distinct propagation directions that coincide with local microstructural axes defined by the laminar arrangement of ventricular myocytes. The maximum conduction velocity of 0.67+/-0.019 ms(-1) was aligned with the myofiber axis. However, transverse to this, the maximum conduction velocity was 0.30+/-0.010 ms(-1), parallel to the myocyte layers and 0.17+/-0.004 ms(-1) normal to them. These orthotropic conduction velocities give rise to preferential activation pathways across the left ventricular free wall that are not captured by structurally detailed computer models, which incorporate axially anisotropic electric properties. CONCLUSIONS: Our findings suggest that current views on uniform side-to-side electric coupling in the heart need to be revised. In particular, nonuniform laminar myocardial architecture and associated electric orthotropy should be included in future models of initiation and maintenance of ventricular arrhythmia.

Experiment-specific models of ventricular electrical activation: construction and application.

Experimental intramural recordings of electrical activity at high resolution have been made in the in-vivo pig LV free wall. To analyze features of these recordings experiment-specific 3D computer models of tissue structures and electrical behavior around the recording sites were constructed. The construction of the models used novel tissue image registration, correction and feature extraction methods. Appropriate model conductivity parameters were deduced from measurements and used to replicate features of experimental recordings.

Laminar arrangement of ventricular myocytes influences electrical behavior of the heart.

The response of the heart to electrical shock, electrical propagation in sinus rhythm, and the spatiotemporal dynamics of ventricular fibrillation all depend critically on the electrical anisotropy of cardiac tissue. A long-held view of cardiac electrical anisotropy is that electrical conductivity is greatest along the myocyte axis allowing most rapid propagation of electrical activation in this direction, and that conductivity is isotropic transverse to the myocyte axis supporting a slower uniform spread of activation in this plane. In this context, knowledge of conductivity in two directions, parallel and transverse to the myofiber axis, is sufficient to characterize the electrical action of the heart. Here we present new experimental data that challenge this view. We have used a novel combination of intramural electrical mapping, and experiment-specific computer modeling, to demonstrate that left ventricular myocardium has unique bulk conductivities associated with three microstructurally-defined axes. We show that voltage fields induced by intramural current injection are influenced by not only myofiber direction, but also the transmural arrangement of muscle layers or myolaminae. Computer models of these experiments, in which measured 3D tissue structure was reconstructed in-silico, best matched recorded voltages with conductivities in the myofiber direction, and parallel and normal to myolaminae, set in the ratio 4:2:1, respectively. These findings redefine cardiac tissue as an electrically orthotropic substrate and enhance our understanding of how external shocks may act to successfully reset the fibrillating heart into a uniform electrical state. More generally, the mechanisms governing the destabilization of coordinated electrical propagation into ventricular arrhythmia need to be evaluated in the light of this discovery.

Surface imaging microscopy using an ultramiller for large volume 3D reconstruction of wax- and resin-embedded tissues.

Three-dimensional reconstruction of large tissue volumes using histological thin sections poses difficulties because of registration of sections, section distortion, and the possibility of incomplete data set collection due to section loss. We have constructed an integrated surface imaging system that successfully addresses these problems. Embedded tissue is mounted on a high precision XYZ stage and the upper surface is iteratively: (i) stained to provide an effective optical section, (ii) imaged using a digital camera, and (iii) removed with an ultramiller. This approach provides for the reconstruction of high-quality 3D images by inherently preserving image registration, eliminates section distortion, thus removing the need for complex realignment and correction, and also ensures full capture of all image planes. The system has the capacity to acquire images of tissue structure with voxel sizes from 0.5 to 50 mum over dimensions ranging from micrometers to tens of millimeters. The ultramiller enables large samples to be imaged by reliably removing tissue over their full extent. The ability to visualize key features of 3D tissue structure across such a range of scale and resolution will facilitate the development of a greater understanding of the relationship between structure and function. This understanding is essential for better analyses of the structural changes associated with different disease states, and the development of structure-based computer models of biological function.

Cardiac electrophysiology and tissue structure: bridging the scale gap with a joint measurement and modelling paradigm.

Significant tissue structures exist in cardiac ventricular tissue that are of supracellular dimension. It is hypothesized that these tissue structures contribute to the discontinuous spread of electrical activation, may contribute to arrhymogenesis and also provide a substrate for effective cardioversion. However, the influences of these mesoscale tissue structures in intact ventricular tissue are difficult to understand solely on the basis of experimental measurement. Current measurement technology is able to record at both the macroscale tissue level and the microscale cellular or subcellular level, but to date it has not been possible to obtain large volume, direct measurements at the mesoscales. To bridge this scale gap in experimental measurements, we use tissue-specific structure and mathematical modelling. Our models have enabled us to consider key hypotheses regarding discontinuous activation. We also consider the future developments of our intact tissue experimental programme.

Intramural measurement of transmembrane potential in the isolated pig heart: validation of a novel technique.

INTRODUCTION: Transmembrane potentials can be recorded at multiple intramural sites in the intact heart using fiber optic probes or optrodes. The technique has considerable potential utility for studies of arrhythmia and defibrillation, but has not been validated in large mammalian hearts. METHODS AND RESULTS: An optrode was used to acquire intramural transmembrane potentials in six isolated Langendorff-perfused pig hearts. Mechanical activity was suppressed with 2,3-butanedione monoxime (BDM). Excitation light (488 nm) was delivered to and fluorescence collected from six sites, each spaced 1.4 mm apart across the left ventricle (LV) free wall that was stained with di-4 ANEPPS. Intramural membrane potentials were compared with extracellular potentials recorded at adjacent locations in sinus rhythm, and during atrial and subepicardial ventricular pacing (1-3 Hz). In three hearts, epicardial intracellular potentials were also measured close to the optrode. Optical action potentials were reproducible, with no significant transmural variation in morphology. There was close correspondence between subepicardial optical and intracellular potentials (R2 = 0.948, n = 23). The onset of activation at and its progression across adjacent optical and extracellular recording sites were consistent, as was the variation of action potential duration (APD) with cycle length. However, there was greater variability in absolute APD estimated from optical and extracellular records (R2 = 0.773, n = 258). Comparison of extracellular potentials at the same intramural sites in vivo confirms that heart isolation and BDM slow electrical propagation and depress restitution relationships, but otherwise preserve intramural patterns of electrical activation. CONCLUSIONS: We have demonstrated that our optrode provides reliable intramural transmembrane potential recordings in the isolated pig heart preparation.

Correction of motion artifact in transmembrane voltage-sensitive fluorescent dye emission in hearts.

Fast voltage-sensitive dyes are widely used to image cardiac electrical activity. Typically, the emission spectrum of these fluorochromes is wavelength shifted with altered membrane potential, but the optical signals obtained also decay with time and are affected by contraction. Ratiometry reduces, but may not fully remove, these artifacts. An alternate approach has been developed in which the time decay in simultaneously acquired short- and long-wavelength signals is characterized nonparametrically and removed. Motion artifact is then identified as the time-varying signal component common to both decay-corrected signals and subtracted. Performance of this subtraction technique was compared with ratiometry for intramural optical signals acquired with a fiber-optic probe in an isolated, Langendorff-perfused pig heart preparation (n = 4) stained with di-4-ANEPPS. Perfusate concentration of 2,3-butanedione monoxime was adjusted (7.5-12.5 mM) to alter contractile activity. Short-wavelength (520-600 nm) and long-wavelength (>600 nm) signals were recorded over 8-16 cardiac cycles at 6 sites across the left ventricular free wall in sinus rhythm and during pacing. A total of 451 such data sets were acquired. Appreciable wall motion was observed in 225 cases, with motion artifact classed as moderate (less than modulation due to action potential) in 187 and substantial (more than modulation due to action potential) in 38. In all cases, subtraction performed as well as, or better than, ratiometry in removing motion artifact and decay. Action potential morphology was recovered more faithfully by subtraction than by ratiometry in 58 of 187 and 31 of 38 cases with moderate and substantial motion artifact, respectively. This novel subtraction approach may therefore provide a means of reducing the concentration of uncoupling agents used in cardiac optical mapping studies.

Cardiac structure and electrical activation: models and measurement.

1. Our group has developed finite element models of ventricular anatomy that incorporate detailed structural information. These have been used to study normal electrical activation and re-entrant arrhythmia. 2. A model based on the actual three-dimensional microstructure of a transmural left ventricular (LV) segment predicts that cleavage planes between muscle layers may give rise to non-uniform, anisotropic electrical propagation and also provide a substrate for bulk resetting of the myocardium during defibrillation. 3. The model predictions are consistent with the results of preliminary experiments in which a novel fibre-optic probe is used to record transmembrane potentials at multiple intramural sites in the intact heart. Extracellular potentials are recorded at adjacent LV sites in these studies. 4. We conclude that structural discontinuities in ventricular myocardium may play a role in the initiation of re-entrant arrhythmia and discuss future studies that address this hypothesis.