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The complex and highly organized structural arrangement of some five billion cardiomyocytes directs the coordinated electrical activity and mechanical contraction of the human heart. The characteristic transmural change in cardiomyocyte orientation underlies base-to-apex shortening, circumferential shortening, and left ventricular torsion during contraction. Individual cardiomyocytes shorten approximately 15% and increase in diameter approximately 8%. Remarkably, however, the left ventricular wall thickens by up to 30-40%. To accommodate this, the myocardium must undergo significant structural rearrangement during contraction. At the mesoscale, collections of cardiomyocytes are organized into sheetlets, and sheetlet shear is the fundamental mechanism of rearrangement that produces wall thickening. Herein we review the histological and physiological studies of myocardial mesostructure that have established the sheetlet shear model of wall thickening. Recent developments in tissue clearing techniques allow for imaging of whole hearts at the cellular scale, while magnetic resonance imaging (MRI) and computed tomography (CT) can image the myocardium at the mesoscale (100µm to 1mm) to resolve cardiomyocyte orientation and organization. Through histology, cardiac diffusion tensor imaging (DTI) and other modalities, mesostructural sheetlets have been confirmed in both animal and human hearts. Recent in vivo cardiac DTI methods have measured reorientation of sheetlets during the cardiac cycle. We also examine the role of pathological cardiac remodeling on sheetlet organization and reorientation, and the impact this has on ventricular function and dysfunction. We also review the unresolved mesostructural questions and challenges that may direct future work in the field.

Background: Cardiac remodeling underpinning progressive hypertensive heart disease alters the three-dimensional structure of the extracellular matrix, as well as the laminar organization of myocardial sheetlets. The effect of pharmacological intervention on microstructural remodeling is not known. Objective: To examine the changes in myocardial microstructure that occur as a result of hypertensive heart disease, and the effect of pharmacological treatment. Methods: The spontaneously hypertensive rat (SHR) model of hypertensive heart disease was studied, and compared with the normotensive Wistar Kyoto (WKY) rat, and SHRs treated with angiotensin converting enzyme inhibitors (TSHR). Cardiac magnetic resonance was used to assess cardiac function and ventricular geometry. Excised myocardium was stained (picrosirius red) for collagen and extended volume confocal microscopy used to produce high-resolution image stacks. Results: At 24 months, TSHRs had significantly greater ejection fraction (P

Heart failure (HF) is one of the leading causes of death worldwide. HF is associated with substantial microstructural remodeling, which is linked to changes in left ventricular geometry and impaired cardiac function. The role of myocardial remodeling in altering the mechanics of failing hearts remains unclear. Structurally based constitutive modeling provides an approach to improve understanding of the relationship between biomechanical function and tissue organization in cardiac muscle during HF. In this study, we used cardiac magnetic resonance imaging and extended-volume confocal microscopy to quantify the remodeling of left ventricular geometry and myocardial microstructure of healthy and spontaneously hypertensive rat hearts at the ages of 12 and 24 months. Passive cardiac mechanical function was characterized using left ventricular pressure-volume compliance measurements. We have developed a, to our knowledge, new structurally based biomechanical constitutive equation built on parameters quantified directly from collagen distributions observed in confocal images of the myocardium. Three-dimensional left ventricular finite element models were constructed from subject-specific in vivo magnetic resonance imaging data. The structurally based constitutive equation was integrated into geometrically subject-specific finite element models of the hearts and used to investigate the underlying mechanisms of ventricular dysfunction during HF. Using a single pair of material parameters for all hearts, we were able to produce compliance curves that reproduced all of the experimental compliance measurements. The value of this study is not limited to reproducing the mechanical behavior of healthy and diseased hearts, but it also provides important insights into the structure-function relationship of diseased myocardium that will help pave the way toward more effective treatments for HF.

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.

4A three-dimensional finite element model of the mechanical and electrical behavior of the heart is being developed in a collaboration among Auckland University, New Zealand; the University of California at San Diego, U.S.; and McGill University, Canada. The equations of continuum mechanics from the theory of finite deformation elasticity are formulated in a prolate spheroidal coordinate system and solved using a combination of Galerkin and collocation techniques. The finite element basis functions used for the dependent and independent variables range from linear Lagrange to cubic Hermite, depending on the degree of spatial variation and continuity required for each variable. Orthotropic constitutive equations derived from biaxial testing of myocardial sheets are defined with respect to the microstructural axes of the tissue at the Gaussian quadrature points of the model. In particular, we define the muscle fiber orientation and the newly identified myocardial sheet axis orientation throughout the myocardium using finite element fields with nodal parameters fitted by least-squares to comprehensive measurements of these variables. Electrical activation of the model is achieved by solving the FitzHugh–Nagumo equations with collocation at fixed material points of the anatomical finite element model. Electrical propagation relies on an orthotropic conductivity tensor defined with respect to the local material axes. The mechanical constitutive laws for the Galerkin continuum mechanics model are (1) an orthotropic “pole–zero” law for the passive mechanical properties of myocardium and (2) a Wiener cascade model of the active mechanical properties of the muscle fibers. This chapter concentrates on two aspects of the model: first, grid generation, including both the generation of nodal coordinates for the finite element mesh and the generation of orthotropic material axes at each computational point, and, second, the formulation of constitutive laws suitable for numerically intensive finite element computations. Extensions to this model and applications to the mechanical and electrical function of the heart are described in Chapter 2 by McCulloch and co-workers.

Orthotropic Intramural Spread of LV Electrical PotentialsIntroduction 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.

Testing new therapies in heart failure (HF) requires a chronic stable model of HF in large animals. Microembolization of the coronary arteries has been used to model HF previously; however, neural control has not been previously explored in this model. Thus the aim of this study was to further characterize neural control in this model of HF. HF was induced by infusion of microspheres (45 micron; 1.3 ml) into the proximal left coronary artery or left descending coronary arteries, with three sequential embolizations over 3 weeks. Twelve to 14 weeks after the final embolization, and when ejection fraction had decreased below 45%, animals were instrumented to record blood pressure and heart rate. Baroreflex control of heart rate was investigated in conscious animals. Additionally, pressure-volume loops were constructed under anesthesia. Embolization-induced HF was associated with a decrease in mean arterial pressure (67 ± 2 vs. 85 ± 4 mmHg, p < 0.05), an increase in heart rate (108 ± 4 vs. 94 ± 4 bpm, p < 0.05), and a significant increase in left ventricular end-diastolic pressure (11.4 ± 2 vs. 6.2 ± 1 mmHg, p < 0.01). Under conscious conditions, there was a significant decrease in the gain (−8.2 ± 2 vs. −4.1 ± 1 beats/min/mmHg, p < 0.05) as well as the lower plateau of the baroreflex in HF compared to control animals. HF was also associated with significantly increased respiratory rate (107 ± 4 vs. 87 ± 4 breaths/min, p < 0.01) and incidence of apneas (520 ± 24 vs. 191 ± 8 apnea periods >4 s, p < 0.05), compared to control sheep. The microembolization model of heart failure is associated with an increase in left ventricular end-diastolic pressure, impaired cardiac function, and altered baroreflex control of the heart. These findings suggest this chronic model of HF is appropriate to use for investigating interventions aimed at improving neural control in HF.

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 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

Key points Vagal reflexes slow heart rate and can change where the heartbeat originates within the sinoatrial node (SAN). The mechanisms responsible for this process - termed leading pacemaker (LP) shift - have not been investigated fully. We used optical mapping to measure the effects of baroreflex, chemoreflex and carbachol on pacemaker entrainment and electrical conduction across the SAN. All methods of stimulation triggered shifts in LP site from the central SAN to one or two caudal pacemaker regions. These shifts were associated with reduced current generation capacity centrally and increased electrical load caudally. Previous studies suggest LP shift is a rate-dependent phenomenon whereby acetylcholine slows central pacemaker rate disproportionately, enabling caudal cells that are less acetylcholine sensitive to assume control. However, our findings indicate the LP region is defined by both pacemaker rate and capacity to drive activation. Shifts in LP site provide an important homeostatic mechanism for rapid switches in heart rate. Abstract Reflex vagal activity causes abrupt heart rate slowing with concomitant caudal shifts of the leading pacemaker (LP) site within the sinoatrial node (SAN). However, neither the mechanisms responsible nor their dynamics have been investigated fully. Therefore, the objective of this study was to elucidate the mechanisms driving cholinergic LP shift. Optical maps of right atrial activation were acquired in a rat working heart-brainstem preparation during baroreflex and chemoreflex stimulation or with carbachol. All methods of stimulation triggered shifts in LP site from the central SAN to caudal pacemaker regions, which were positive for HCN4 and received uniform cholinergic innervation. During baroreflex onset, the capacity of the central region to drive activation declined with a decrease in amplitude and gradient of optical action potentials (OAPs) in the surrounding myocardium. Accompanying this decline, there was altered entrainment in the caudal SAN as shown by decreased conduction velocity, OAP amplitude, gradient and activation time. Atropine abolished these responses. Chemoreflex stimulation produced similar effects but central capacity to drive activation was preserved before the LP shift. In contrast, carbachol produced a prolonged period of reduced capacity to drive and altered entrainment. Previous studies suggest LP shift is a rate-dependent phenomenon whereby acetylcholine slows central pacemaker rate disproportionately, enabling caudal cells that are less acetylcholine sensitive to assume control. Our findings indicate that cholinergic LP shifts are also determined by altered electrical source-to-sink balance in the SAN. We conclude that the LP region is defined by both rate and capacity to drive atrial activation.

Myocardial fibrosis is a pathological process that occurs during heart failure (HF). It involves microstructural remodeling of normal myocardial tissue, and consequent changes in both cardiac geometry and function. The role of myocardial structural remodeling in the progression of HF remains poorly understood. We propose a constitutive modeling framework, informed by high-resolution images of cardiac tissue structure, to model the mechanical response of normal and fibrotic myocardium. This image-driven constitutive modeling approach allows us to better reproduce and understand the relationship between structural and functional remodeling of ventricular myocardium during HF.

Building anatomically accurate models of the coronary vascular system enables potentially deeper understandings of coronary circulation. To achieve this, (a) images at different levels of vascular network-arteries, arterioles, capillaries, venules, and veins-need to be obtained through suitable imaging modalities; and (b) from images, morphological and topological information needs to be extracted using image processing techniques. While there are several modalities that enable the imaging of large vessels, microcirculation imaging-capturing vessels having diameter lesser than 100 μm-has to date been typically confined to small regions of the heart. This spatially limited microcirculatory information has often been used within cardiac models, with the potentially erroneous assumption that it is representative of the whole organ. However, with the recent advancements in imaging and image processing, it is rapidly becoming feasible to acquire, process, and quantify microcirculation data at the scale of whole organ. In this review, we summarize the progress toward this goal followed through a presentation of the current state-of-the-art imaging and image processing techniques in the context of coronary microcirculation extraction, prominently but not exclusively, from small animals.

Background— The relationship between epicardial and body surface potentials defines the forward problem of electrocardiography. A robust formulation of the forward problem is instrumental to solving the inverse problem, in which epicardial potentials are computed from known body surface potentials. Here, the accuracy of different forward models has been evaluated experimentally. Methods and Results— Body surface and epicardial potentials were recorded simultaneously in anesthetized closed-chest pigs (n=5) during sinus rhythm, and epicardial and endocardial ventricular pacing (65 records in total). Body surface potentials were simulated from epicardial recordings using experiment-specific volume conductor models constructed from magnetic resonance imaging. Results for homogeneous (isotropic electric properties) and inhomogeneous (incorporating lungs, anisotropic skeletal muscle, and subcutaneous fat) forward models were compared with measured body surface potentials. Correlation coefficients were 0.85±0.08 across all animals and activation sequences with no significant difference between homogeneous and inhomogeneous solutions ( P =0.85). Despite this, there was considerable variance between simulated and measured body surface potential distributions. Differences between the body surface potential extrema predicted with homogeneous forward models were 55% to 78% greater than observed ( P P Conclusions— These results demonstrate that homogeneous volume conductor models introduce substantial spatial inaccuracies in forward problem solutions. This probably affects the precision of inverse reconstructions of cardiac potentials, in which this assumption is made.

Background: Inverse electrocardiographic mapping reconstructs cardiac electrical activity from recorded body surface potentials. This noninvasive technique has been used to identify potential ablation targets. Despite this, there has been little systematic evaluation of its reliability. Methods: Torso and ventricular epicardial potentials were recorded simultaneously in anesthetized, closed-chest pigs (n=5), during sinus rhythm, epicardial, and endocardial ventricular pacing (70 records in total). Body surface and cardiac electrode positions were determined and registered using magnetic resonance imaging. Epicardial potentials were reconstructed during ventricular activation using experiment-specific magnetic resonance imaging–based thorax models, with homogeneous or inhomogeneous (lungs, skeletal muscle, fat) electrical properties. Coupled finite/boundary element methods and a meshless approach based on the method of fundamental solutions were compared. Inverse mapping underestimated epicardial potentials >2-fold ( P RESULTS: Mean correlation coefficients for reconstructed epicardial potential distributions ranged from 0.60±0.08 to 0.64±0.07 across all methods. Epicardial electrograms were recovered with reasonable fidelity at ≈50% of sites (median correlation coefficient, 0.69–0.72), but variation was substantial. General activation spread was reproduced (median correlation coefficient, 0.72–0.78 for activation time maps after spatio-temporal smoothing). Epicardial foci were identified with a median location error ≈16 mm (interquartile range, 9–29 mm). Inverse mapping with meshless method of fundamental solutions was better than with finite/boundary element methods, and the latter were not improved by inclusion of inhomogeneous torso electrical properties. Conclusions: Inverse potential mapping provides useful information on the origin and spread of epicardial activation. However the spatio-temporal variability of recovered electrograms limit resolution and must constrain the accuracy with which arrhythmia circuits can be identified independently using this approach.

High resolution optical mapping of cardiac activation spread is a powerful tool for investigating the mechanisms of arrhythmia. However, due to the complex geometry of the atria, mapping global electrical activity is challenging. In this study, we present a high resolution optical mapping set-up and a data processing pipeline for characterising bi-atrial arrhythmia. Our system enables recording of bi-atrial electrical activity with high spatial and temporal resolution and provides a flexible tool for programming pacing protocols. Preliminary results indicate that this imaging system and data processing pipeline provide a reliable platform for experimental characterisation of bi-atrial arrhythmia.

Transmural variations in the relationship between structural and fluid transport properties of myocardial capillary networks are determined via continuum modelling approaches using recent three-dimensional (3D) data on the microvascular structure. Specifically, the permeability tensor, which quantifies the inverse of the blood flow resistivity of the capillary network, is computed by volume-averaging flow solutions in synthetic networks with geometrical and topological properties derived from an anatomically-detailed microvascular data set extracted from the rat myocardium. Results show that the permeability is approximately ten times higher in the principal direction of capillary alignment (the ‘longitudinal’ direction) than perpendicular to this direction, reflecting the strong anisotropy of the microvascular network. Additionally, a 30% increase in capillary diameter from subepicardium to subendocardium is shown to translate to a 130% transmural rise in permeability in the longitudinal capillary direction. This result supports the hypothesis that perfusion is preferentially facilitated during diastole in the subendocardial microvasculature to compensate for the severely-reduced systolic perfusion in the subendocardium.

Introduction: More effective methods for characterizing 3D electrical activity in human left atrium (LA) are needed to identify substrates/triggers and microreentrant circuit for paroxysmal atrial fibrillation (PAF). We describe a novel wavelet-based approach and wave-front centroid tracking that have been used to reconstruct regional activation frequency and electrical activation pathways from non-contact multi-electrode array. Methods: Data from 13 patients acquired prior to ablation for PAF with a 64 electrode noncontact catheter positioned in the LA were analysed. Unipolar electrograms were reconstructed at 2048 locations across each LA endocardial surface. Weighted fine- and coarse-scale electrograms were constructed by wavelet decomposition and combined with peak detection to identify atrial fibrillation (AF) activation frequency and fractionated activity at each site. LA regions with upper quartile AF frequencies were identified for each patient. On the other hand, a wave-front centroid tracking approach was introduced for this first time to detect macro-reentrant circuit during PAF. Results: The results employing wavelet-based analysis on atrial unipolar electrograms are validated by the signals recorded simultaneously via the contacted ablation catheter and visually tracking the 3D spread of activation through the interest region. Multiple connected regions of high frequency electrical activity were seen; most often in left superior pulmonary vein (10/12), septum (9/12) and atrial roof (9/12), as well as the ridge (8/12). The wave-front centroid tracking approach detects a major macro circuit involving LPVs, PLA, atrial floor, MV, septum, atrial roof and ridge. The regions with high frequency by wave-front tracking are consistent with the results using wavelet approach and our clinical observations. Conclusions: The wavelet-based technique and wave-front centroid tracking approach provide a robust means of extracting spatio-temporal characteristics of PAF. The approach could facilitate accurate identification of pro-arrhythmic substrate and triggers, and therefore, to improve success rate of catheter ablation for AF.

The spontaneously hypertensive rat (SHR) is an established model of human hypertensive heart disease transitioning into heart failure. The study of the progression to heart failure in these animals has been limited by the lack of longitudinal data. We used MRI to quantify left ventricular mass, volume, and cardiac work in SHRs at age 3 to 21 month and compared these indices to data from Wistar‐Kyoto (WKY) controls. SHR had lower ejection fraction compared with WKY at all ages, but there was no difference in cardiac output at any age. At 21 month the SHR had significantly elevated stroke work (51 ± 3 mL.mmHg SHR vs. 24 ± 2 mL.mmHg WKY; n = 8, 4; P

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.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.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.

Understanding of the microvasculature has previously been limited by the lack of methods capable of capturing and modelling complete vascular networks. We used novel imaging and computational techniques to establish the topology of the entire blood vessel network of a murine lymph node, combining 63706 confocal images at 2 μm pixel resolution to cover a volume of 3.88 mm3. Detailed measurements including the distribution of vessel diameters, branch counts, and identification of voids were subsequently re-visualised in 3D revealing regional specialisation within the network. By focussing on critical immune microenvironments we quantified differences in their vascular topology. We further developed a morphology-based approach to identify High Endothelial Venules, key sites for lymphocyte extravasation. These data represent a comprehensive and continuous blood vessel network of an entire organ and provide benchmark measurements that will inform modelling of blood vessel networks as well as enable comparison of vascular topology in different organs.

Background— Computer models that capture key features of the heterogeneous myofiber architecture of right and left atria and interatrial septum provide a means of investigating the mechanisms responsible for atrial arrhythmia. The data necessary to implement such models have not previously been available. The aims of this study were to characterize surface geometry and myofiber architecture throughout the atrial chambers and to investigate the effects of this structure on atrial activation. Methods and Results— Atrial surface geometry and myofiber orientations were reconstructed in 3D at 50×50×50-μm 3 resolution from serial images acquired throughout the sheep atrial chambers. Myofiber orientations were determined by Eigen-analysis of the structure tensor. These data have been incorporated into an anatomic model that provides the first quantitative representation of myofiber architecture throughout the atrial chambers. By simulating activation on this 3D structure, we have confirmed the roles of specialized myofiber tracts such as the crista terminalis, pectinate muscles, and the Bachman bundle on the spread of activation from the sinus node. We also demonstrate how the complex myocyte arrangement in the posterior left atrium contributes to activation time dispersion adjacent to the pulmonary veins and increased vulnerability to rhythm disturbance generated by ectopic stimuli originating in the pulmonary vein sleeves. Conclusions— We have developed a structurally detailed, image-based model of atrial anatomy that provides deeper understanding of the role that myocyte architecture plays in normal and abnormal atrial electric function.

The cardiac extracellular matrix (ECM) is the three-dimensional scaffold that defines the geometry and muscular architecture of the cardiac chambers and transmits forces produced during the cardiac cycle throughout the heart wall. The cardiac ECM is an active system that responds to the stresses to which it is exposed and in the normal heart is adapted to facilitate efficient mechanical function. There are marked differences in the short- and medium-term changes in ventricular geometry and cardiac ECM that occur as a result of volume overload, hypertension, and ischemic cardiomyopathy. Despite this, there is a widespread view that a common remodeling “phenotype” governs the final progression to end-stage heart failure in different forms of heart disease. In this review article, we make the case that this interpretation is not consistent with the clinical and experimental data on the topic. We argue that there is a need for new theoretical and experimental models that will enable stresses acting on the ECM and resultant deformations to be estimated more accurately and provide better spatial resolution of local signaling mechanisms that are activated as a result. These developments are necessary to link the effects of structural remodeling with altered cardiac mechanical function.

Cardiac ventricular trabeculae are widely used in the study of cardiac muscle function, primarily because their myocytes are axially-aligned. However, their collagen content has not been rigorously determined. In particular, it is unknown whether the content of collagen differs between specimens originating from the left (LV) and right (RV) ventricles and whether, indeed, either corresponds to the collagen content of the ventricular walls themselves. In order to redress this deficit of knowledge, we have used the techniques of fluorescence confocal microscopy and environmental scanning electron microscopy to quantify the proportion of perimysial collagen comprising the cross-sectional area of trabeculae carneae. In trabeculae from both the RV and LV of adult rat hearts, collagen may occupy as little as 1% or as much as 100% of the cross-section. For specimens of dimensions typically used experimentally, there was no difference in average collagen content (6.03 ± 5.14%, n = 33) of preparations from the two ventricles.

There is a significant gap in our knowledge of engineered heart valve tissue (EHVT) development regarding detailed three-dimensional (3D) tissue formation and remodeling from the point of in vitro culturing to full in vivo function. As a step toward understanding the complexities of EHVT formation and remodeling, a novel serial confocal microscopy technique was employed to obtain 3D micro-structural information of pre-implant (PRI) and post-implant for 12 weeks (POI) EHVT fabricated from PGA: PLLA scaffolds and seeded with ovine bone-marrow-derived mesenchymal stem cells. Custom scaffold fiber tracking software was developed to quantify scaffold fiber architectural features such as length, tortuosity, and minimum scaffold fiber–fiber separation distance and scaffold fiber orientation was quantified utilizing a 3D fabric tensor. In addition, collagen and cellular density of ovine pulmonary valve leaflet tissue were also analyzed for baseline comparisons. Results indicated that in the unseeded state, scaffold fibers formed a continuous, oriented network. In the PRI state, the scaffold showed some fragmentation with a scaffold volume fraction of 7.79%. In the POI specimen, the scaffold became highly fragmented, forming a randomly distributed short fibrous network (volume fraction of 2.03%) within a contiguous, dense collagenous matrix. Both PGA and PLLA scaffold fibers were observed in the PRI and POI specimens. Collagen density remained similar in both PRI and POI specimens (74.2 and 71.5%, respectively), though the distributions in the transmural direction appeared slightly more homogenous in the POI specimen. Finally, to guide future 2D histological studies for large-scale studies (since acquisition of high-resolution volumetric data is not practical for all specimens), we investigated changes in relevant collagen and scaffold metrics (collagen density and scaffold fiber orientation) with varying section spacing. It was found that a sectioning spacing up to 25 μm (for scaffold morphology) and 50 μm (for collagen density) in both PRI and POI tissues did not result in loss of information fidelity, and that sectioning in the circumferential or radial direction provides the greatest preservation of information. This is the first known work to investigate EHVT microstructure over a large volume with high resolution and to investigate time evolving in vivo EHVT morphology. The important scaffold fiber structural changes observed provide morphological information crucial for guiding future structurally based constitutive modeling efforts focused on better understanding EHVT tissue formation and remodeling.

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.

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 ≈4�4�1-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.

Introduction: Atrial fibrillation is prevalent in the elderly and contributes to mortality in congestive heart failure. Development of computer models of atrial electrical activation that incorporate realistic structures provides a means of investigating the mechanisms that initiate and maintain reentrant atrial arrhythmia. As a step toward this, we have developed a model of the right atrial appendage (RAA) including detailed geometry of the pectinate muscles (PM) and crista terminalis (CT) with high spatial resolution, as well as complete fiber architecture. Methods and Results: Detailed structural images of a pig RAA were acquired using a semiautomated extended-volume imaging system. The generally accepted anisotropic ratio of 10:1 was adopted in the computer model. To deal with the regional action potential duration heterogeneity in the RAA, a Courtemanche cell model and a Luo-Rudy cell model were used for the CT and PM, respectively. Activation through the CT and PM network was adequately reproduced with acceptable accuracy using reduced-order computer models. Using a train of reducing cycle length stimuli applied to a CT/PM junction, we observed functional block both parallel with and perpendicular to the axis of the CT. Conclusion: With stimulation from the CT at the junction of a PM, we conclude: (a) that conduction block within the CT is due to a reduced safety factor; and (b) that unidirectional block and reentry within the CT is due to its high anisotropy. Regional differences in effective refractive period do not explain the observed conduction block.

Trabeculae carneae are the smallest naturally arising collections of linearly arranged myocytes in the heart. They are the preparation of choice for studies of function of intact myocardium in vitro. In vivo, trabeculae are unique in receiving oxygen from two independent sources: the coronary circulation and the surrounding ventricular blood. Because oxygen partial pressure (PO2) in the coronary arterioles is identical in specimens from both ventricles, whereas that of ventricular blood is 2.5-fold higher in the left ventricle than in the right ventricle, trabeculae represent a “natural laboratory” in which to examine the influence of “extravascular” PO2 on the extent of capillarization of myocardial tissue. We exploit this advantage to test four hypotheses. (1) In trabeculae from either ventricle, a peripheral annulus of cells is devoid of capillaries. (2) Hence, sufficiently small trabeculae from either ventricle are totally devoid of capillaries. (3) The capillary-to-myocyte ratios in specimens from either ventricle are identical to those of their respective walls. (4) Capillary-to-myocyte ratios are comparable in specimens from either ventricle, reflecting equivalent energy demands in vivo, driven by identical contractile frequencies and comparable wall stresses. We applied confocal fluorescent imaging to trabeculae in cross section, subsequently using semi-automated segmentation techniques to distinguish capillaries from myocytes. We quantified the capillary-to-myocyte ratios of trabeculae from both ventricles and compared them to those determined for the ventricular free walls and septum. Quantitative interpretation was furthered by mathematical modeling, using both the classical solution to the diffusion equation for elliptical cross sections, and a novel approach applicable to cross sections of arbitrary shape containing arbitrary disposition of capillaries and non-respiring collagen cords.

The passive material properties of myocardium play a major role in diastolic performance of the heart. In particular, the shear behaviour is thought to play an important mechanical role due to the laminar architecture of myocardium. We have previously compared a number of myocardial constitutive relations with the aim to extract their suitability for inverse material parameter estimation. The previous study assumed a homogeneous deformation. In the present study we relaxed the homogeneous assumption by implementing these laws into a finite element environment in order to obtain more realistic measures for the suitability of these laws in both their ability to fit a given set of experimental data, as well as their stability in the finite element environment. In particular, we examined five constitutive laws and compare them on the basis of (i) “goodness of fit”: how well they fit a set of six shear deformation tests, (ii) “determinability”: how well determined the objective function is at the optimal parameter fit, and (iii) “variability”: how well determined the material parameters are over the range of experiments. Furthermore, we compared the FE results with those from the previous study. It was found that the same material law as in the previous study, the orthotropic Fung-type “Costa-Law”, was the most suitable for inverse material parameter estimation for myocardium in simple shear.

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.

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.

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.

1. Detailed information about three-dimensional structure is key to understanding biological function. 2. Confocal laser microscopy has made it possible to reconstruct three-dimensional organization with exquisite resolution at cellular and subcellular levels. 3. There have been few attempts to acquire large image volumes using the confocal laser scanning microscope. 4. Previously, we have used manual techniques to construct extended volumes (several mm in extent, at 1.5 microm voxel size) of myocardial tissue. 5. We are now developing equipment and efficient automated methods for acquiring extended morphometric databases using confocal laser scanning microscopy.

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.