Electrophysiology and 3D-imaging reveal properties of human intracardiac neurons and increased excitability with atrial fibrillation.

Altered autonomic input to the heart plays a major role in atrial fibrillation (AF). Autonomic neurons termed ganglionated plexi (GP) are clustered on the heart surface to provide the last point of neural control of cardiac function. To date the properties of GP neurons in humans are unknown. Here we have addressed this knowledge gap in human GP neuron structure and physiology in patients with and without AF. Human right atrial GP neurons embedded in epicardial adipose tissue were excised during open heart surgery performed on both non-AF and AF patients and then characterised physiologically by whole cell patch clamp techniques. Structural analysis was also performed after fixation at both the single cell and at the entire GP levels via three-dimensional confocal imaging. Human GP neurons were found to exhibit unique properties and structural complexity with branched neurite outgrowth. Significant differences in excitability were revealed between AF and non-AF GP neurons as measured by lower current to induce action potential firing, a reduced occurrence of low action potential firing rates, decreased accommodation and increased synaptic density. Visualisation of entire GPs showed almost all neurons are cholinergic with a small proportion of noradrenergic and dual phenotype neurons. Phenotypic distribution differences occurred with AF including decreased cholinergic and dual phenotype neurons, and increased noradrenergic neurons. These data show both functional and structural differences occur between GP neurons from patients with and without AF, highlighting that cellular plasticity occurs in neural input to the heart that could alter autonomic influence on atrial function. KEY POINTS: The autonomic nervous system plays a critical role in regulating heart rhythm and the initiation of AF; however, the structural and functional properties of human autonomic neurons in the autonomic ganglionated plexi (GP) remain unknown. Here we perform the first whole cell patch clamp electrophysiological and large tissue confocal imaging analysis of these neurons from patients with and without AF. Our data show human GP neurons are functionally and structurally complex. Measurements of action potential kinetics show higher excitability in GP neurons from AF patients as measured by lower current to induce action potential firing, reduced low firing action potential rates, and decreased action potential accommodation. Confocal imaging shows increased synaptic density and noradrenergic phenotypes in patients with AF. Both functional and structural differences occur in GP neurons from patients with AF that could alter autonomic influence on atrial rhythm.

Successful induction of oestrus, ovulation and pregnancy in adult ewes and ewe lambs out of the breeding season using a GnRH+progesterone oestrus synchronisation protocol.

A series of experiments was designed to assess the effect of a treatment protocol (U-synch) for inducing oestrus and ovulation out of the breeding season in adult ewes and ewe lambs. The protocol consisted of a 7-day treatment with an intravaginal progesterone-releasing device (IPRD), administration of GnRH at IPRD insertion on Day 0, and equine chorionic gonadotropin (eCG) and prostaglandin F2α at IPRD removal on Day 7. In Experiment 1, 50 or 100 µg GnRH were sufficient to induce ovulation at the beginning of the protocol in 3/9 and 4/9 ewes, respectively; while the resulting proportion of sheep ovulating after the treatment protocol was 88.9% and 77.8% in ewes initially treated with 50 or 100 µg GnRH, respectively. In Experiment 2, the proportion of Romney-cross ewe lambs ovulating was greater (P<0.0001) in the U-synch group (95.4%) than in the untreated Control group (3.2%). In Experiment 3, pregnancy rates of Dorset-cross sheep in the U-synch (60.7%) and Standard (12-day IPRD and eCG treatment; 56.5%) groups were greater (P=0.01) than in the untreated Control group (43.4%). The incidence of twin pregnancies was greater (P=0.005) in the U-synch group than in the Control group. A 7-day IPRD treatment including GnRH treatment at device insertion and eCG treatment at device removal induced oestrus and ovulation during the non-breeding season in a high proportion of mature ewes and ewe lambs. High pregnancy rates to natural mating, with a low rate of triplet pregnancies, were also observed.

Intravaginal progesterone devices in synchronization protocols for artificial insemination in beef heifers.

Two experiments were designed to investigate the administration of intravaginal progesterone in protocols for oestrus and ovulation synchronization in beef heifers. In Experiment 1, cyclic Black Angus heifers (n = 20) received an Ovsynch protocol and were randomly assigned to receive (CIDR-Ovsynch) or not (Ovsynch) a progesterone device between Days 0 and 7. Treatment with a controlled internal drug release (CIDR) device significantly increased the size of the dominant follicle prior to ovulation (12.8 ± 0.4 CIDR-Ovsynch vs 11.4 ± 0.4 Ovsynch) (p < 0.02). Plasma progesterone concentrations throughout the experiment were affected by the interaction between group and day effects (p < 0.004). In Experiment 2, cyclic Polled Hereford heifers (n = 382) were randomly assigned to one of the six treatment groups (3 × 2 factorial design) to receive a CIDR, a used bovine intravaginal device (DIB), or a medroxiprogesterone acetate (MAP) sponge and GnRH analogues (lecirelin or buserelin). All heifers received oestradiol benzoate plus one of the devices on Day 0 and PGF on Day 7 pm (device withdrawal). Heifers were detected in oestrus 36 h after PGF and inseminated 8-12 h later, while the remainder received GnRH 48 h after PGF and were inseminated on Day 10 (60 h). The number of heifers detected in oestrus on Day 8 and conception rate to AI on Day 9 were higher (p < 0.01) in the used-DIB than in the CIDR or MAP groups, while the opposite occurred with the pregnancy rate to FTAI on Day 10 (p < 0.01). There was no effect of progesterone source, GnRH analogue or their interaction on overall pregnancy rates (64.9%). Progesterone treatment of heifers during an Ovsynch protocol resulted in a larger pre-ovulatory follicle in beef heifers. Progesterone content of intravaginal devices in synchronization protocols is important for the timing of AI, as the use of low-progesterone devices can shorten the interval to oestrus.

Heterozygous Inverdale ewes show increased ovulation rate sensitivity to pre-mating nutrition.

This study aimed to determine whether ewes heterozygous (I+) for the Inverdale mutation of the bone morphogenetic protein-15 (BMP15) gene with high natural ovulation rate (OR) show similar sensitivity to nutritional manipulation as non-carriers (++). Increasing pre-mating nutrition results in OR increases in sheep, but whether this effect occurs in ewes with naturally high OR is unknown. Over 2 years, I+ or ++ ewes were given high (ad libitum) or control (maintenance) pasture allowances for 6 weeks prior to mating at a synchronised oestrus, with OR measured 8 days later. The high group increased in weight compared with controls (+5.84kg; P<0.01), accompanied by increased OR (+19%; P<0.01). As well as having higher OR (+45%; P<0.01), I+ ewes responded to increased feed with a larger proportional increase in OR (+27%; P<0.01) compared with the response in ++ ewes (+11%; P<0.05), suggesting an interaction between BMP15 levels and nutritional signals in the follicle to control OR. Although litter size increases only tended to significance (+12%; P=0.06), extra feed resulted in over 50% of I+ ewes giving birth to more than three lambs, compared with 20-31% of I+ ewes on maintenance rations. This information can guide feed management of prolific Inverdale ewes prior to breeding.

Myocardial material parameter estimation: a non-homogeneous finite element study from simple shear tests.

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.

Gene expression of stretch-activated channels and mechanoelectric feedback in the heart.

1. Mechanoelectric feedback (MEF) in the heart is the process by which mechanical forces on the myocardium can change its electrical properties. Mechanoelectric feedback has been demonstrated in many animal models, ranging from isolated cells, through isolated hearts to whole animals. In humans, MEF has been demonstrated directly in both the atria and the ventricles. It seems likely that MEF provides either the trigger or the substrate for some types of clinically important arrhythmias. 2. Mechanoelectric feedback may arise because of the presence of stretch-sensitive (or mechano-sensitive) ion channels in the cell membrane of the cardiac myocytes. Two types have been demonstrated: (i) a non-specific cation channel (stretch-activated channel (SAC); conductance of approximately 25 pS); and (ii) a potassium channel with a conductance of approximately 100 pS. The gene coding for the SAC has not yet been identified. The gene for the potassium channel is likely to be TREK, a member of the tandem pore potassium channel gene family. We have recorded stretch-sensitive potassium channels in rat isolated myocytes that have the properties of TREK channels expressed in heterologous systems. 3. It has been shown that TREK mRNA is expressed heterogeneously in the rat ventricular wall, with 17-fold more expression in endocardial compared with epicardial cells. This difference is reflected in the TREK currents recorded from endocardial and epicardial cells using whole-cell patch-clamp techniques, although the difference in current density was less pronounced (approximately threefold). Consistent with this, we show here that when the ventricle is stretched by inflation of an intraventricular balloon in a Langendorff perfused rat isolated heart, action potential shortening was more pronounced in the endocardium (30% shortening at 40 mmHg) compared with that in the epicardium (10% shortening at the same pressure). 4. Computer models of the mechanics of the (pig) heart show pronounced spatial variations in strain in the myocardium with large transmural differences (in the left ventricle in particular) and also large differences between the base and apex of the ventricle. 5. The importance of MEF and the non-homogeneous gene expression and strain distribution for arrhythmias is discussed.

Automated extended volume imaging of tissue using confocal and optical microscopy.

Conventional histologic techniques cannot readily be used for 3D reconstruction of large tissue volumes. We have developed an imaging rig which supports both confocal and light microscopy, and utilizes a surface imaging approach to serially image embedded tissue blocks while maintaining alignment and registration of the image series.

Constructing a tissue-specific model of ventricular microstructure.

Simulations of cardiac electrical activity are generally computed in idealized or generic domains. We have developed a semi-automated technique for imaging an extended volume of cardiac ventricular tissue at a resolution of approximately 1 microm, and constructing from those images a geometric and structural model with 10 microm resolution suitable for solving the bidomain equations. This technique enables experimental modeling and computer simulation to be integrated by constructing a tissue-specific structural model in less than one week. We demonstrate the use of this procedure applied to a sample of rat ventricle.

Intramural multisite recording of transmembrane potential in the heart.

Heart surface optical mapping of transmembrane potentials has been widely used in studies of normal and pathological heart rhythms and defibrillation. In these studies, three-dimensional spatio-temporal events can only be inferred from two-dimensional surface potential maps. We present a novel optical system that enables high fidelity transmural recording of transmembrane potentials. A probe constructed from optical fibers is used to deliver excitation light and collect fluorescence from seven positions, each 1 mm apart, through the left ventricle wall of the rabbit heart. Excitation is provided by the 488-nm line of a water-cooled argon-ion laser. The fluorescence of the voltage-sensitive dye di-4-ANEPPS from each tissue site is split at 600 nm and imaged onto separate photodiodes for later signal ratioing. The optics and electronics are easily expandable to accommodate multiple optical probes. The system is used to record the first simultaneous measurements of transmembrane potential at a number of sites through the intact heart wall.

A triaxial-measurement shear-test device for soft biological tissues.

A novel shear-test device for soft biological tissue, capable of applying simple shear deformations simultaneously in two orthogonal directions while measuring the resulting forces generated in three axes, is described. We validated the device using a synthetic gel, the properties of which were ascertained from independent tensile and rotational shear tests. Material parameters for the gel were fitted using neo-Hookean analytical solutions to the independent test data, and these matched the results from the device. Preliminary results obtained with rat septal myocardium are also presented to demonstrate the feasibility of the apparatus in determining the shear characteristics of living tissue.

The effect of synthetic patch repair of coarctation on regional deformation of the aortic wall.

BACKGROUND: A long-term complication of synthetic patch repair of coarctation is true aneurysm formation. AIM: An in vitro study was undertaken to determine the effects of patch angioplasty on aortic geometry and strain adjacent to the patch. METHODS: Segments of human descending thoracic aorta were subject to 10 pressure loading cycles (10-120 mm Hg; 1.36-16.32 kPa) before and after simulated coarctation repair with a synthetic patch. Local curvature and strain were estimated by fitting a geometric model to reconstructed three-dimensional surface marker points. RESULTS: In the control aortas, when pressure increased from 11 +/- 1.0 to 124 +/- 4.0 mm Hg (1.5 +/- 0.14 to 16.86 +/- 0.54 kPa), average circumferential curvature decreased from 0.1543 +/- 0.03 to 0.1065 +/- 0.03 mm(-1). The average major extension reached a maximum of 1.43 +/- 0.08. After patch implantation, the average circumferential curvature was reduced relative to control at all pressures. Average major extensions were significantly greater than paired control values and reached a maximum of 1.55 +/- 0.08 at 122 +/- 4.0 mm Hg (16.59 +/- 0. 54 kPa). Substantial strain inhomogeneity was observed and major extensions were greatest immediately adjacent to the patch. INFERENCE: Synthetic patch repair of coarctation of the aorta increases wall strain and produces significant regional gradients in strain. With control aortic material properties there may be a substantial increase in wall stress immediately adjacent to the aorta, which could lead to true aneurysm formation.

Extended confocal microscopy of myocardial laminae and collagen network.

Ventricular myocardium has a complex three-dimensional structure which has previously been inferred from two-dimensional images. We describe a technique for imaging the 3D organization of myocytes in conjunction with the collagen network in extended blocks of myocardium. Rat hearts were fixed with Bouin's solution and perfusion-stained with picrosirius red. Transmural blocks from the left ventricular free wall were embedded in Agar 100 resin and mounted securely in an ultramicrotome chuck. Confocal fluorescence laser scanning microscopy was used to obtain 3D images to a depth of 60 microns in a contiguous mosaic across the surface. Approximately 50 microns was then cut off the surface of the block with an ultramicrotome. This sequence was repeated 20 times. Images were assembled and registered in 3D to form an extended volume 3800 x 800 x 800 microns 3 spanning the heart wall from epicardium to endocardium. Examples are given of how digital reslicing and volume rendering methods can be applied to the resulting dataset to provide quantitative structural information about the 3D organization of myocytes, extracellular collagen matrix and blood vessel network of the heart.

Laminar structure of the heart: a mathematical model.

A mathematical description of cardiac anatomy is presented for use with finite element models of the electrical activation and mechanical function of the heart. The geometry of the heart is given in terms of prolate spheroidal coordinates defined at the nodes of a finite element mesh and interpolated within elements by a combination of linear Lagrange and cubic Hermite basis functions. Cardiac microstructure is assumed to have three axes of symmetry: one aligned with the muscle fiber orientation (the fiber axis); a second set orthogonal to the fiber direction and lying in the newly identified myocardial sheet plane (the sheet axis); and a third set orthogonal to the first two, in the sheet-normal direction. The geometry, fiber-axis direction, and sheet-axis direction of a dog heart are fitted with parameters defined at the nodes of the finite element mesh. The fiber and sheet orientation parameters are defined with respect to the ventricular geometry such that 1) they can be applied to any heart of known dimensions, and 2) they can be used for the same heart at various states of deformation, as is needed, for example, in continuum models of ventricular contraction.

Three-dimensional changes in left and right ventricular geometry in chronic mitral regurgitation.

Regional three-dimensional (3-D) right (RV) and left ventricular (LV) geometry was studied in eight dogs before and 5-6 mo after induction of mitral regurgitation (MR). Ventricular shape changes were quantified with a 3-D finite-element model fitted to chamber contours traced on cardiac magnetic resonance images. MR increased LV end-diastolic volume (LVEDV; 99 vs. 57 ml; P < 0.001) and LV stroke volume (LVSV; 55 vs. 26 ml; P < 0.001). In contrast, RVEDV decreased (45 vs. 55 ml; P < 0.01), whereas SV was maintained. LV mass (free wall plus septum) increased (115 vs. 94 g; P < 0.05), whereas RV free-wall mass was relatively unchanged. Shape changes due to MR were characterized by a marked (7.4-mm) rightward shift of the septum relative to the lateral LV free wall at end diastole. In contrast, the distance from the RV free wall to the lateral LV free wall was relatively unchanged (2.7 mm). The distance between the LV lateral free wall and septum increased more than the distance between the anterior and posterior LV walls (22 vs. 15%; P = 0.04). During systole, the displacement of the septum into the LV increased significantly (7.3 vs. 2.9 mm; P < 0.01). Consistent with the end-diastolic dimension changes, LV endocardial circumferential curvature was decreased at end diastole to a greater extent in the anterior and posterior walls than in the septal and lateral walls (P < 0.01). Thus chronic MR produced an asymmetric LV dilatation with regional variation in geometry. The septum increased its contribution to the LVSV at the expense of RVEDV. RVSV was maintained, possibly by ventricular interaction.

Laminar structure of the heart: ventricular myocyte arrangement and connective tissue architecture in the dog.

We have studied the three-dimensional arrangement of ventricular muscle cells and the associated extracellular connective tissue matrix in dog hearts. Four hearts were potassium-arrested, excised, and perfusion-fixed at zero transmural pressure. Full-thickness segments were cut from the right and left ventricular walls at a series of precisely located sites. Morphology was visualized macroscopically and with scanning electron microscopy in 1) transmural planes of section and 2) planes tangential to the epicardial surface. The appearance of all specimens was consistent with an ordered laminar arrangement of myocytes with extensive cleavage planes between muscle layers. These planes ran radially from endocardium toward epicardium in transmural section and coincided with the local muscle fiber orientation in tangential section. Stereological techniques were used to quantify aspects of this organization. There was no consistent variation in the cellular organization of muscle layers (48.4 +/- 20.4 microns thick and 4 +/- 2 myocytes across) transmurally or in different ventricular regions (23 sites in 6 segments), but there was significant transmural variation in the coupling between adjacent layers. The number of branches between layers decreased twofold from subepicardium to midwall, whereas the length distribution of perimysial collagen fibers connecting muscle layers was greatest in the midwall. We conclude that ventricular myocardium is not a uniformly branching continuum but a laminar hierarchy in which it is possible to identify three axes of material symmetry at any point.

Estimation of epicardial strain using the motions of coronary bifurcations in biplane cinéangiography.

A quantitative method is described for estimating epicardial deformation from the motion of the superficial arteries. A structural model of the time-varying surface is constructed using tensor product basis functions which are bicubic Hermite in the spatial domain and sinusoidal in the temporal domain. The loci of the superficial coronary arteries are reconstructed interactively at diastasis and the bifurcations are tracked semiautomatically throughout a cardiac cycle. An initial surface is fitted to the vessels at diastasis and is subsequently deformed under the influence of the bifurcations. The Lagrange-Green strain tensor is used to obtain a complete description of surface strain over the entire region spanned by the model. The calculated deformation field varies smoothly over space and time and is not constrained by assumptions of isotropy or piecewise homogeneity. Results are presented for a single cycle of a human heart.

Mechanical effects of coronary perfusion in the passive canine left ventricle.

Mechanical effects of coronary perfusion on passive left ventricular filling were studied in seven isolated potassium-arrested dog hearts subjected to static pressure loading. With the use of a biplane video method, midanterior epicardial deformations were measured before, during, and after perfusion of the coronary circulation with a cardioplegic solution. During perfusion, there was a highly significant reduction (P less than 0.001) in ventricular compliance; the mean cavity volume change decreased by 50% at a filling pressure of 12 mmHg. The loss of compliance was reversible and increased with coronary artery pressure. The magnitudes of the principal epicardial extensions, determined by homogeneous strain analysis, also decreased significantly (P less than 0.001) by an average of 30-40% at ventricular pressures of 4-12 mmHg. But there was no change in the pattern of epicardial deformations. These findings, and similar significant falls in epicardial in-plane rotation (P less than 0.05) and angular translation (P less than 0.001), suggest that the main mechanical effect of the coronary circulation is homogeneous and uniform and is, therefore, probably associated with the microcirculation. We propose that this effect may be modeled by treating the myocardium as a porous elastic medium swollen with an incompressible fluid rather than by an increase in ventricular wall thickness due to filling of the coronary vessels.

An anatomical heart model with applications to myocardial activation and ventricular mechanics.

A 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 16 by McCulloch and co-workers.

Biaxial testing of membrane biomaterials: testing equipment and procedures.

A testing facility for measuring the biaxial mechanical properties of highly deformable membranes is described. Forces are applied, via strain-gauge force transducers, to four points on each side of an initially square 12 to 25 mm membrane sample to produce biaxial extensions of up to 80 percent of undeformed length. Strain is estimated from the displacement of markers bounding a 1 to 2 mm central square. The accuracy of stress and strain field measurements has been assessed by finite element analysis of a biaxially-loaded isotropic elastic membrane. Major advantages of the present system over those previously described in the literature are that 1) sample mounting procedures are simplified, 2) there is provision for independent adjustment of stress field uniformity and measurement of the applied point forces and 3) faster strain rates can be imposed on the relatively small samples tested.

Mathematical model of geometry and fibrous structure of the heart.

We developed a mathematical representation of ventricular geometry and muscle fiber organization using three-dimensional finite elements referred to a prolate spheroid coordinate system. Within elements, fields are approximated using basis functions with associated parameters defined at the element nodes. Four parameters per node are used to describe ventricular geometry. The radial coordinate is interpolated using cubic Hermite basis functions that preserve slope continuity, while the angular coordinates are interpolated linearly. Two further nodal parameters describe the orientation of myocardial fibers. The orientation of fibers within coordinate planes bounded by epicardial and endocardial surfaces is interpolated linearly, with transmural variation given by cubic Hermite basis functions. Left and right ventricular geometry and myocardial fiber orientations were characterized for a canine heart arrested in diastole and fixed at zero transmural pressure. The geometry was represented by a 24-element ensemble with 41 nodes. Nodal parameters fitted using least squares provided a realistic description of ventricular epicardial [root mean square (RMS) error less than 0.9 mm] and endocardial (RMS error less than 2.6 mm) surfaces. Measured fiber fields were also fitted (RMS error less than 17 degrees) with a 60-element, 99-node mesh obtained by subdividing the 24-element mesh. These methods provide a compact and accurate anatomic description of the ventricles suitable for use in finite element stress analysis, simulation of cardiac electrical activation, and other cardiac field modeling problems.