Abstract-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. (Circ Res. 2007;101:e103-e112.)

There is strong support for the view that the ventricular myocardium has a laminar organization in which myocytes are grouped into branching layers separated by cleavage planes. However, understanding of the extent and functional implications of this architecture has been limited by the lack of a systematic three-dimensional description of the organization of myocytes and associated perimysial collagen. We imaged myocytes and collagen across the left ventricular wall at high resolution in seven normal rat hearts using extended volume confocal microscopy. We developed novel reconstruction and segmentation techniques necessary for the quantitative analysis of three-dimensional myocyte and perimysial collagen organization. The results confirm that perimysial collagen has an ordered arrangement and that it defines a laminar organization. Perimysial collagen is composed of three distinct forms: extensive meshwork on laminar surfaces, convoluted fibers connecting adjacent layers, and longitudinal cords. While myolaminae are the principal form of structural organization throughout most of the wall, they are not seen in the subepicardium, where perimysial collagen is present only as longitudinal cords.

Confocal microscopy enables constitutive elements of cells and tissues to be viewed at high resolution and reconstructed in three dimensions, but is constrained by the limited extent of the volumes that can be imaged. We have developed an automated technique that enables serial confocal images to be acquired over large tissue areas and volumes. The computer-controlled system, which integrates a confocal microscope and an ultramill using a high-precision translation stage, inherently preserves specimen registration, and the user control interface enables flexible specification of imaging protocols over a wide range of scales and resolutions. With this system it is possible to reconstruct specified morphological features in three dimensions and locate them accurately throughout a tissue sample. We have successfully imaged various samples at 1-mum voxel resolution on volumes up to 4 mm3 and on areas up to 75 mm2. Used in conjunction with appropriate embedding media and immuno-histochemical probes, the techniques described in this paper make it possible to routinely map the distributions of key intracellular structures over much larger tissue domains than has been easily achievable in the past.

Rationale: Slow nonuniform electric propagation in the border zone (BZ) of a healed myocardial infarct (MI) cangive rise to reentrant arrhythmia. The extent to which this is influenced by structural rather than cellular electric remodeling is unclear.Objective: To determine whether structural remodeling alone in the infarct BZ could provide a substrate for re-entry by (i) characterizing the 3-dimensional (3D) structure of the myocardium surrounding a healed MI at high spatial resolution and (ii) modeling electric activation on this structure. Methods and Results: Anterior left ventricular (LV) infarcts were induced in 2 rats by coronary artery ligation.Three-dimensional BZ volume (4.1 mm 3 and 5.6 mm 3 ) were imaged at 14 days using confocal microscopy. Viable myocytes were identified, and their connectivity and orientation were quantified. Preserved cell networks were observed in the subendocardium and subepicardium of the infarct. Myocyte tracts traversed the BZ, and there was heavy infiltration of collagen into the adjacent myocardium. Myocyte connectivity decreased by Ϸ65% over 250 m across the BZ. This structure was incorporated into 3D network models on which activation was simulated using Luo-Rudy membrane dynamics assuming normal cellular electric properties. Repetitive stimulation was imposed at selected BZ sites. Stimulus site-specific unidirectional propagation occurred in the BZ with rate-dependent slowing and conduction block, and reentry was demonstrated in one substrate. Activation times were prolonged because of tract path length and local slowing. Conclusions:

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 Ϸ4ϫ4ϫ1-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 0.67Ϯ0.019 ms Ϫ1 was aligned with the myofiber axis. However, transverse to this, the maximum conduction velocity was 0.30Ϯ0.010 ms Ϫ1, parallel to the myocyte layers and 0.17Ϯ0.004 ms Ϫ1 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. (Circ Arrhythmia Electrophysiol. 2009;2:433-440.)Key Words: anisotropy Ⅲ mapping Ⅲ structure Ⅲ computer modeling Ⅲ intramural pacing A ccurate information about the electric properties of cardiac tissue is central to understanding the biophysical basis of normal and aberrant heart rhythm. Electric anisotropy influences the spread of activation in the heart, plays a critical role both in the initiation and maintenance of reentrant arrhythmia, and is an important determinant of the effectiveness of cardioversion. Knowledge of the nature and extent of electric anisotropy is required for computer models of heart activation that provide a means of probing intramural electric behavior that cannot be accessed from clinical and experimental measurements made on the surfaces of the heart. Clinical Perspective on p 440Normal ventricular myocardium is generally thought to function as a syncytium in which side-to-side electric coupling between adjacent myocytes is uniform. 1,2 The electric properties of ventricular myocardium are assumed to be axially anisotropic with respect to the local myofiber axis, 1,2 with activation propagating most rapidly in the myofiber direction and at uniform velocity in planes transverse to this.This view is not consistent with the laminar model of ventricular myocardium t...