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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.

Recent developments in clearing and microscopy enable 3D imaging with cellular resolution up to the whole organ level. These methods have been used extensively in neurobiology, but their uptake in other fields has been much more limited. Application of this approach to the human heart and effective use of the data acquired present challenges of scale and complexity. Four interlinked issues need to be addressed: 1) efficient clearing and labelling of heart tissue, 2) fast microscopic imaging of human-scale samples, 3) handling and processing of multi-terabyte 3D images, and 4) extraction of structural information in computationally tractable structure-based models of cardiac function. Preliminary studies show that each of these requirements can be achieved with the appropriate application and development of existing technologies.

Ablation has recently emerged as a tool for modulating gastric electrical activation and may hold interventional potential for disorders of gastric function. However, previous studies have been limited to the acute intraoperative setting. This study now presents the safety of gastric ablation after postsurgical recovery and healing. Localized electrical conduction blocks created by ablation remained after 2 wk of healing, and no perforation or other complications were observed over the postsurgical period.

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

Plasticity is a fundamental property of neurons in both the central and peripheral nervous systems, enabling rapid changes in neural network function. The intracardiac nervous system (ICNS) is an extensive network of neurons clustered into ganglionated plexi (GP) on the surface of the heart. GP neurons are the final site of neuronal control of heart rhythm, and pathophysiological remodeling of the ICNS is proposed to feature in multiple cardiovascular diseases, including heart failure and atrial fibrillation. To examine the potential role of GP neuron plasticity in atrial arrhythmia and hypertension, we developed whole cell patch clamp recording techniques from GP neurons in isolated ICNS preparations from aged control (Wistar-Kyoto) and spontaneously hypertensive rats (SHRs). Anesthetized SHRs showed frequent premature ventricular contractions and episodes of atrial arrhythmia following carbachol injection, and isolated SHR atrial preparations were susceptible to pacing induced atrial arrhythmia. Whole cell recordings revealed elevated spontaneous postsynaptic current frequency in SHR GP neurons, as well as remodeled electrophysiology, with significant decreases in action potential amplitude and half-width. SHRs also showed a parallel increase in the number of cholinergic neurons and adrenergic glomus cells in cardiac ganglia, a higher proportion of synaptic α7-subunit but not β2-containing nicotinic receptors, and an elevation in the number of synaptic terminals onto GP neurons. Our data show that significant structural and functional plasticity occurs in the intracardiac nervous system and suggest that enhanced excitability through synaptic plasticity, together with remodeling of cardiac neuron electrophysiology, contributes to the substrate for atrial arrhythmia in hypertensive heart disease.NEW & NOTEWORTHY We have developed intracardiac neuron whole cell recording techniques in atrial preparations from control and spontaneous hypertensive rats. This has enabled the identification of significant synaptic plasticity in the intracardiac nervous system, including enhanced postsynaptic current frequency, increased synaptic terminal density, and altered postsynaptic receptors. This increased synaptic drive together with altered cardiac neuron electrophysiology could increase intracardiac nervous system excitability and contribute to the substrate for atrial arrhythmia in hypertensive heart disease.

Introduction: Myocardial collagen structure is a complex three dimensional (3D) hierarchy, organizing cardiomyocytes and providing structural integrity. Changes in collagen organization have a crucial role in cardiac remodeling. However, quantitative methods for the analysis of collagen structures are still lacking. Herein, we apply our collagen shape analysis framework to compare within-sheetlet collagen (both endomysial collagen surrounding myocytes and perimysial cords) against sheetlet-surface collagen (perimysial meshwork collagen deposited between sheetlets) in spontaneously hypertensive rats undergoing quinapril treatment. Hypothesis: The different functional properties of endomysial and perimysial meshwork collagen are linked to their structure and will have markedly different features quantifiable by 3D shape analysis. Methods: Rat myocardium was stained for collagen (picrosirius red), and the left ventricular free wall imaged using extended volume confocal microscopy (0.4 μm 3 voxel size). The 3D image volumes were segmented into within-sheetlet and sheetlet-surface regions. Shape analysis was performed using eigenanalysis of the inertia matrix to derive 3D morphological (elongation and flatness) parameters, characterizing the collagen shape in the two regions. Results: Shape analysis revealed marked differences between within-sheetlet and sheetlet-surface collagen organization. Long-term quinapril treatment resulted in notable differences in within-sheetlet collagen shape in rats at 14 months of age (mo) compared with 24 mo. At 14 mo, the within-sheetlet collagen is a mix of sheet-, ribbon-, and rod-like structures (elongation: 0.43, flatness: 0.05), but by 24 mo this has remodeled to predominately thin rod-like structures (elongation: 0.03, flatness: 0.35). The sheetlet-surface collagen consisted of thick sheet-like structures in both 14 and 24 mo rats (elongation: 0.83 vs 0.79, flatness: 0.13 vs 0.09). Conclusion: Shape analysis in 3D showed marked differences between within-sheetlet and sheetlet-surface collagen structure, as well as differences between early treatment and late treatment of diseased hearts.

Underlying electrical propagation in the heart and potentially fatal arrhythmia is the cardiac microstructure. Despite the critical role of muscle architecture, a non-destructive approach to examine not only myocyte orientation, but cellular arrangement in to laminar organization is lacking in hearts from translational animal models and humans. X-ray micro computed tomography using contrast enhancing agents achieves three-dimensional images at near-histological resolutions. However, imaging large mammalian hearts presents challenges including X-ray over-attenuation and loss of image contrast. The goal of this study was to rethink tissue pre-treatment to optimize, and benefit from micro computed tomography imaging resolution in large tissues. Whole pig and human hearts were dehydrated and perfused with a tissue reinforcing agent, hexamethyldisilazane, and slowly air-dried. Heart morphology was conserved and temporally stable. This enabled direct air-mounting for micro computed tomography imaging. Moreover, the desiccated tissue density was significantly reduced compared to the initial hydrated state (P=0.04). Three-dimensional image reconstructions of air-dried hearts segmented using a single intensity threshold revealed detailed microstructural architecture of myolaminae. Conversely, one-step segmentation of hearts loaded with contrast agents poorly estimated the gross anatomical morphology of the heart and lacked identification of tissue microarchitecture. Air-drying large mammalian hearts optimizes X-ray imaging of cardiac microstructure.

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.

Cardiomyocytes branch and interconnect with one another, providing important redundancy for propagation of electro-chemical signals. Despite this, cardiomyocyte branching structure remains poorly understood. Herein, myocardium from spontaneously hypertensive rats (SHR) was imaged using extended volume confocal microscopy. Samples from untreated SHRs (\(n = 2\)) were compared with SHRs undergoing ACE inhibitor treatment (\(n = 2\)). From these image-stacks the cardiomyocyte network (center-lines and branches) were manually tracked. The frequency of cardiomyocyte branching was calculated and these branching frequencies were compared according to spatial position within a myocardial sheetlet. ACE inhibitor treatment resulted in significantly reduced total cardiomyocyte branching compared with untreated SHR myocardium at 24 mo (0.49 ± 0.04 vs 1.07 ± 0.15 branches per 100 \(\upmu \)m, \(P = 0.020\)). Cardiomyocytes on the sheetlet-surface branched more frequently within their respective cell-layer (0.59 ± 0.07 branches per 100 \(\upmu \)m) compared with cardiomyocytes in the sheetlet interior (0.29 ± 0.12 branches per 100 \(\upmu \)m). The cardiomyocytes in the sheetlet interior exhibited more frequent between-layer branching (0.56 ± 0.06 branches per 100 \(\upmu \)m) compared to sheetlet-surface cardiomyocytes (0.17 ± 0.03 branches per 100 \(\upmu \)m). The ratio of within-layer to between-layer branching was significantly greater at the surface layer compared with the interior layer (3.93 ± 1.06 vs 0.47 ± 0.16, \(P=0.018\)). This proof-of-concept study demonstrates an approach to measuring branching cardiomyocyte networks and shows the spatial heterogeneity of cardiomyocyte branching.

Introduction: Cardiomyocytes form a continuously branching syncytium that also form sheetlets four to six cells thick. Myocytes and sheetlets have essential structural and functional roles, but descriptions of their complex branching structure are limited. Herein, we characterize the branching structure of cardiomyocytes within a myocardial sheetlet segment. Hypothesis: Due to topological constraints, cardiomyocytes on the sheetlet surfaces branch within the fiber-sheetlet plane more than myocytes on the interior of the sheetlet. Methods: Rodent myocardium was stained for collagen and the left ventricular free wall was imaged using extended volume confocal microscopy (0.4 μm 3 voxel size), a and sheetlet portion segmented (146 х 214 х 339 μm 3 , approx. 5 by 10 by 3 cells). The centerline of each myocyte was tracked, with branch-points identified where the cell bodies of adjacent cardiomyocytes merge. This produced a connected network (Figure) with five cell-layers along the normal axis: lower surface, lower interior, middle interior, upper interior, and upper surface. Results: The cardiomyocyte branching frequency varied between cell-layers (Figure); the sheetlet-surface cell-layers branched within their own cell-layer more frequently than myocytes in the sheetlet interior (0.28 vs 0.11 branches per 100 μm). Cardiomyocytes on the sheetlet surface also branched less frequently with cardiomyocytes of a different cell-layer, compared with cardiomyocytes in the sheetlet interior (0.31 vs 0.67 branches per 100 μm). Cardiomyocytes in the middle interior cell-layer had the highest number of branches between cell-layers (0.86 per 100 μm), and the lowest number of branches within their own cell-layer (0.03 per 100 μm). Conclusion: Analysis of confocal images revealed differences in cardiomyocyte branching between cell-layers of a sheetlet segment, and confirmed that sheetlet boundaries provide a topological constraint for cardiomyocyte branching.

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.

Multi-electrode basket catheters are used for 3D mapping of complex focal arrhythmias in real-time, but full contact is hard to achieve for all electrodes in the atria. We have developed a novel inverse mapping approach, which enables endocardial potentials to be recovered from noncontact recordings and report on its efficacy. Intra-atrial electrograms were acquired with 64 channel Constellation™ catheters in 4 anesthetized sheep in sinus rhythm (SR), atrial pacing (AP) and atrial fibrillation (AF), with catheters of different dimension at various locations in right and/or left atria (RA, LA). Atrial endocardial potentials were recovered from noncontact recordings using finite-element and meshless inverse methods. Inverse mapping reproduced contact electrograms, recorded separately at distributed sites, with excellent accuracy in SR and AP [CC 0.8+0.18, time delay 1+3 ms, relative RMS error 0.53+0.17, n=96]. Moreover, repeatable activation time maps were obtained in SR and AP. Focal activity was identified in AF and precise noncontact region-of-interest mapping was also demonstrated.

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.

The mechanical properties of myocardial tissue are primarily determined by the organisation of the collagen network. Quantitative measurements of collagen morphology can help to understand the structure-function relationship in cardiac tissue. In this study, we segmented collagen from high-resolution three-dimensional (3D) images of the left ventricle (LV) mid-wall myocardium obtained using extended-volume confocal microscopy. 3D shape analysis was used to compute the morphological parameters elongation (e), flatness (f), and anisotropy (a). We applied this analysis to both control and hypertensive rat hearts and showed distinct differences between the control and remodelled hearts, particularly in collagen elongation. The predominant form of collagen in the control rat is elongated with a value of e = 0.846 ± 0.041, whereas in the hypertensive rat collagen, is arranged mostly in a sheet-like form with e = 0.301 ± 0.023. Such quantitative information can be used to develop microstructural models of the myocardium that link the observed changes in cardiac microstructure to changes in mechanical function during the progression of heart diseases, which will help to elucidate the underlying pathological mechanisms.

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.

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

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.

This study presents a novel method for building parametric representations of myocardial microstructure of the left ventricle from multi-directional diffusion weighted magnetic resonance images DWI. The direction of maximal diffusion is directly estimated from the DWI signal intensities using finite element field fitting. This framework avoids the need to compute diffusion tensors, which introduces errors due to least squares fitting that are generally neglected when building microstructural models of the heart from DWI. Nodal parameters describing cardiac myocyte orientations throughout a finite element model of the left ventricle were fitted to a series of raw diffusion signals using non-linear least squares optimisation to determine the direction of maximum diffusion. An ex vivo DWI data set from a Wystar-Kyoto rat was processed using the proposed method. The fitted myocyte orientations were compared against conventional diffusion tensor/eigenvector analysis and the degree of correlation was measured using a normalised dot product nDP. Good agreement nDPi¾?=i¾?0.979 between the new method and the traditional tensor analysis approach was observed for regions of high fractional anisotropy FA. In regions of low FA, the errors were much more variable, but the proposed method maintains a smoothly varying myocyte angle distribution as is generally used in tissue and organ scale heart models.

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.

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.

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.

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.

Changes in heart geometry and microstructural remodelling of myocardium are pathological processes that occur in heart failure (HF). The combination of effects often leads to compromised mechanical function of the left ventricle (LV). We have developed an image-driven constitutive modelling framework, which integrates subject-specific geometric data and microstructural organisation of the myocardial tissue, in order to investigate the mechanisms of heart function in health and disease. In this study, we investigated the effect of myocardial microstructural remodelling on passive mechanical function of the LV during the progression of hypertension-induced HF. We constructed finite element models of the LV using geometric data derived from in vivo magnetic resonance images of healthy and failing rat hearts at both 14-months and 24-months of age. By incorporating subject-specific LV geometries and parameters that directly reflect the tissue microstructure, we demonstrated that a single set of fitted constitutive parameters could reproduce the observed LV chamber compliance curves in all four cases (healthy and diseased hearts at 14-months and 24-months). This study highlights the important role that remodelling of myocardial microstructure plays in the mechanics of the failing heart.