Lysosomal calcium loading promotes spontaneous calcium release by potentiating ryanodine receptors.

Spontaneous calcium release by ryanodine receptors (RyRs) due to intracellular calcium overload results in delayed afterdepolarizations, closely associated with life-threatening arrhythmias. In this regard, inhibiting lysosomal calcium release by two-pore channel 2 (TPC2) knockout has been shown to reduce the incidence of ventricular arrhythmias under ß-adrenergic stimulation. However, mechanistic investigations into the role of lysosomal function on RyR spontaneous release remain missing. We investigate the calcium handling mechanisms by which lysosome function modulates RyR spontaneous release, and determine how lysosomes are able to mediate arrhythmias by its influence on calcium loading. Mechanistic studies were conducted using a population of biophysically detailed mouse ventricular models including for the first time modeling of lysosomal function, and calibrated by experimental calcium transients modulated by TPC2. We demonstrate that lysosomal calcium uptake and release can synergistically provide a pathway for fast calcium transport, by which lysosomal calcium release primarily modulates sarcoplasmic reticulum calcium reuptake and RyR release. Enhancement of this lysosomal transport pathway promoted RyR spontaneous release by elevating RyR open probability. In contrast, blocking either lysosomal calcium uptake or release revealed an antiarrhythmic impact. Under conditions of calcium overload, our results indicate that these responses are strongly modulated by intercellular variability in L-type calcium current, RyR release, and sarcoplasmic reticulum calcium-ATPase reuptake. Altogether, our investigations identify that lysosomal calcium handling directly influences RyR spontaneous release by regulating RyR open probability, suggesting antiarrhythmic strategies and identifying key modulators of lysosomal proarrhythmic action.

Inhibition of adenylyl cyclase 1 by ST034307 inhibits IP3-evoked changes in sino-atrial node beat rate.

Atrial arrhythmias, such as atrial fibrillation (AF), are a major mortality risk and a leading cause of stroke. The IP3 signalling pathway has been proposed as an atrial-specific target for AF therapy, and atrial IP3 signalling has been linked to the activation of calcium sensitive adenylyl cyclases AC1 and AC8. We investigated the involvement of AC1 in the response of intact mouse atrial tissue and isolated guinea pig atrial and sino-atrial node (SAN) cells to the α-adrenoceptor agonist phenylephrine (PE) using the selective AC1 inhibitor ST034307. The maximum rate change of spontaneously beating mouse right atrial tissue exposed to PE was reduced from 14.5% to 8.2% (p = 0.005) in the presence of 1 µM ST034307, whereas the increase in tension generated in paced left atrial tissue in the presence of PE was not inhibited by ST034307 (Control = 14.2%, ST034307 = 16.3%; p > 0.05). Experiments were performed using isolated guinea pig atrial and SAN cells loaded with Fluo-5F-AM to record changes in calcium transients (CaT) generated by 10 µM PE in the presence and absence of 1 µM ST034307. ST034307 significantly reduced the beating rate of SAN cells (0.34-fold decrease; p = 0.003) but did not inhibit changes in CaT amplitude in response to PE in atrial cells. The results presented here demonstrate pharmacologically the involvement of AC1 in the downstream response of atrial pacemaker activity to α-adrenoreceptor stimulation and IP3R calcium release.

A modified density gradient proteomic-based method to analyze endolysosomal proteins in cardiac tissue.

The importance of lysosomes in cardiac physiology and pathology is well established, and evidence for roles in calcium signaling is emerging. We describe a label-free proteomics method suitable for small cardiac tissue biopsies based on density-separated fractionation, which allows study of endolysosomal (EL) proteins. Density gradient fractions corresponding to tissue lysate; sarcoplasmic reticulum (SR), mitochondria (Mito) (1.3 g/mL); and EL with negligible contamination from SR or Mito (1.04 g/mL) were analyzed using Western blot, enzyme activity assay, and liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis (adapted discontinuous Percoll and sucrose differential density gradient). Kyoto Encyclopedia of Genes and Genomes, Reactome, Panther, and Gene Ontology pathway analysis showed good coverage of RAB proteins and lysosomal cathepsins (including cardiac-specific cathepsin D) in the purified EL fraction. Significant EL proteins recovered included catalytic activity proteins. We thus present a comprehensive protocol and data set of guinea pig atrial EL organelle proteomics using techniques also applicable for non-cardiac tissue.

Combining tissue engineering and optical imaging approaches to explore interactions along the neuro-cardiac axis.

Interactions along the neuro-cardiac axis are being explored with regard to their involvement in cardiac diseases, including catecholaminergic polymorphic ventricular tachycardia, hypertension, atrial fibrillation, long QT syndrome and sudden death in epilepsy. Interrogation of the pathophysiology and pathogenesis of neuro-cardiac diseases in animal models present challenges resulting from species differences, phenotypic variation, developmental effects and limited availability of data relevant at both the tissue and cellular level. By contrast, tissue-engineered models containing cardiomyocytes and peripheral sympathetic and parasympathetic neurons afford characterization of cellular- and tissue-level behaviours while maintaining precise control over developmental conditions, cellular genotype and phenotype. Such approaches are uniquely suited to long-term, high-throughput characterization using optical recording techniques with the potential for increased translational benefit compared to more established techniques. Furthermore, tissue-engineered constructs provide an intermediary between whole animal/tissue experiments and in silico models. This paper reviews the advantages of tissue engineering methods of multiple cell types and optical imaging techniques for the characterization of neuro-cardiac diseases.

Synaptic Plasticity in Cardiac Innervation and Its Potential Role in Atrial Fibrillation.

Synaptic plasticity is defined as the ability of synapses to change their strength of transmission. Plasticity of synaptic connections in the brain is a major focus of neuroscience research, as it is the primary mechanism underpinning learning and memory. Beyond the brain however, plasticity in peripheral neurons is less well understood, particularly in the neurons innervating the heart. The atria receive rich innervation from the autonomic branch of the peripheral nervous system. Sympathetic neurons are clustered in stellate and cervical ganglia alongside the spinal cord and extend fibers to the heart directly innervating the myocardium. These neurons are major drivers of hyperactive sympathetic activity observed in heart disease, ventricular arrhythmias, and sudden cardiac death. Both pre- and postsynaptic changes have been observed to occur at synapses formed by sympathetic ganglion neurons, suggesting that plasticity at sympathetic neuro-cardiac synapses is a major contributor to arrhythmias. Less is known about the plasticity in parasympathetic neurons located in clusters on the heart surface. These neuronal clusters, termed ganglionated plexi, or "little brains," can independently modulate neural control of the heart and stimulation that enhances their excitability can induce arrhythmia such as atrial fibrillation. The ability of these neurons to alter parasympathetic activity suggests that plasticity may indeed occur at the synapses formed on and by ganglionated plexi neurons. Such changes may not only fine-tune autonomic innervation of the heart, but could also be a source of maladaptive plasticity during atrial fibrillation.

Caveolae in Rabbit Ventricular Myocytes: Distribution and Dynamic Diminution after Cell Isolation.

Caveolae are signal transduction centers, yet their subcellular distribution and preservation in cardiac myocytes after cell isolation are not well documented. Here, we quantify caveolae located within 100 nm of the outer cell surface membrane in rabbit single-ventricular cardiomyocytes over 8 h post-isolation and relate this to the presence of caveolae in intact tissue. Hearts from New Zealand white rabbits were either chemically fixed by coronary perfusion or enzymatically digested to isolate ventricular myocytes, which were subsequently fixed at 0, 3, and 8 h post-isolation. In live cells, the patch-clamp technique was used to measure whole-cell plasma membrane capacitance, and in fixed cells, caveolae were quantified by transmission electron microscopy. Changes in cell-surface topology were assessed using scanning electron microscopy. In fixed ventricular myocardium, dual-axis electron tomography was used for three-dimensional reconstruction and analysis of caveolae in situ. The presence and distribution of surface-sarcolemmal caveolae in freshly isolated cells matches that of intact myocardium. With time, the number of surface-sarcolemmal caveolae decreases in isolated cardiomyocytes. This is associated with a gradual increase in whole-cell membrane capacitance. Concurrently, there is a significant increase in area, diameter, and circularity of sub-sarcolemmal mitochondria, indicative of swelling. In addition, electron tomography data from intact heart illustrate the regular presence of caveolae not only at the surface sarcolemma, but also on transverse-tubular membranes in ventricular myocardium. Thus, caveolae are dynamic structures, present both at surface-sarcolemmal and transverse-tubular membranes. After cell isolation, the number of surface-sarcolemmal caveolae decreases significantly within a time frame relevant for single-cell research. The concurrent increase in cell capacitance suggests that membrane incorporation of surface-sarcolemmal caveolae underlies this, but internalization and/or micro-vesicle loss to the extracellular space may also contribute. Given that much of the research into cardiac caveolae-dependent signaling utilizes isolated cells, and since caveolae-dependent pathways matter for a wide range of other study targets, analysis of isolated cell data should take the time post-isolation into account.

High resolution structural evidence suggests the Sarcoplasmic Reticulum forms microdomains with Acidic Stores (lysosomes) in the heart.

Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) stimulates calcium release from acidic stores such as lysosomes and is a highly potent calcium-mobilising second messenger. NAADP plays an important role in calcium signalling in the heart under basal conditions and following ß-adrenergic stress. Nevertheless, the spatial interaction of acidic stores with other parts of the calcium signalling apparatus in cardiac myocytes is unknown. We present evidence that lysosomes are intimately associated with the sarcoplasmic reticulum (SR) in ventricular myocytes; a median separation of 20 nm in 2D electron microscopy and 3.3 nm in 3D electron tomography indicates a genuine signalling microdomain between these organelles. Fourier analysis of immunolabelled lysosomes suggests a sarcomeric pattern (dominant wavelength 1.80 µm). Furthermore, we show that lysosomes form close associations with mitochondria (median separation 6.2 nm in 3D studies) which may provide a basis for the recently-discovered role of NAADP in reperfusion-induced cell death. The trigger hypothesis for NAADP action proposes that calcium release from acidic stores subsequently acts to enhance calcium release from the SR. This work provides structural evidence in cardiac myocytes to indicate the formation of microdomains between acidic and SR calcium stores, supporting emerging interpretations of NAADP physiology and pharmacology in heart.

Ccoffinn: Automated Wave Tracking in Cultured Cardiac Monolayers.

Cardiac arrhythmias are one of the most frequent causes of death worldwide. A popular biological model used to study arrhythmogenesis is the cultured cardiac cell monolayer, which provides a good trade-off between physiological relevance and experimental access. Excitation wave patterns are imaged using high-bandwidth detectors, producing large data sets that are typically analyzed manually. To make such analysis less time consuming and less subjective, we have designed and implemented a toolkit for segmentation and tracking of cardiac waves in optical mapping recordings. The toolkit is optimized for high-resolution detectors to accommodate the growing availability of inexpensive high-resolution detectors for life science imaging applications (e.g., scientific CMOS cameras). The software extracts key features of propagating waves, such as wavefront speed and entropy. The methods have been validated using synthetic data, and real-world examples are provided, showing a difference in conduction velocity between two different types of cardiac cell cultures.

Resolving Fine Cardiac Structures in Rats with High-Resolution Diffusion Tensor Imaging.

Cardiac architecture is fundamental to cardiac function and can be assessed non-invasively with diffusion tensor imaging (DTI). Here, we aimed to overcome technical challenges in ex vivo DTI in order to extract fine anatomical details and to provide novel insights in the 3D structure of the heart. An integrated set of methods was implemented in ex vivo rat hearts, including dynamic receiver gain adjustment, gradient system scaling calibration, prospective adjustment of diffusion gradients, and interleaving of diffusion-weighted and non-diffusion-weighted scans. Together, these methods enhanced SNR and spatial resolution, minimised orientation bias in diffusion-weighting, and reduced temperature variation, enabling detection of tissue structures such as cell alignment in atria, valves and vessels at an unprecedented level of detail. Improved confidence in eigenvector reproducibility enabled tracking of myolaminar structures as a basis for segmentation of functional groups of cardiomyocytes. Ex vivo DTI facilitates acquisition of high quality structural data that complements readily available in vivo cardiac functional and anatomical MRI. The improvements presented here will facilitate next generation virtual models integrating micro-structural and electro-mechanical properties of the heart.

Mapping cardiac microstructure of rabbit heart in different mechanical states by high resolution diffusion tensor imaging: A proof-of-principle study.

Myocardial microstructure and its macroscopic materialisation are fundamental to the function of the heart. Despite this importance, characterisation of cellular features at the organ level remains challenging, and a unifying description of the structure of the heart is still outstanding. Here, we optimised diffusion tensor imaging data to acquire high quality data in ex vivo rabbit hearts in slack and contractured states, approximating diastolic and systolic conditions. The data were analysed with a suite of methods that focused on different aspects of the myocardium. In the slack heart, we observed a similar transmural gradient in helix angle of the primary eigenvector of up to 23.6°/mm in the left ventricle and 24.2°/mm in the right ventricle. In the contractured heart, the same transmural gradient remained largely linear, but was offset by up to +49.9° in the left ventricle. In the right ventricle, there was an increase in the transmural gradient to 31.2°/mm and an offset of up to +39.0°. The application of tractography based on each eigenvector enabled visualisation of streamlines that depict cardiomyocyte and sheetlet organisation over large distances. We observed multiple V- and N-shaped sheetlet arrangements throughout the myocardium, and insertion of sheetlets at the intersection of the left and right ventricle. This study integrates several complementary techniques to visualise and quantify the heart's microstructure, projecting parameter representations across different length scales. This represents a step towards a more comprehensive characterisation of myocardial microstructure at the whole organ level.

Human-based approaches to pharmacology and cardiology: an interdisciplinary and intersectorial workshop.

Both biomedical research and clinical practice rely on complex datasets for the physiological and genetic characterization of human hearts in health and disease. Given the complexity and variety of approaches and recordings, there is now growing recognition of the need to embed computational methods in cardiovascular medicine and science for analysis, integration and prediction. This paper describes a Workshop on Computational Cardiovascular Science that created an international, interdisciplinary and inter-sectorial forum to define the next steps for a human-based approach to disease supported by computational methodologies. The main ideas highlighted were (i) a shift towards human-based methodologies, spurred by advances in new in silico, in vivo, in vitro, and ex vivo techniques and the increasing acknowledgement of the limitations of animal models. (ii) Computational approaches complement, expand, bridge, and integrate in vitro, in vivo, and ex vivo experimental and clinical data and methods, and as such they are an integral part of human-based methodologies in pharmacology and medicine. (iii) The effective implementation of multi- and interdisciplinary approaches, teams, and training combining and integrating computational methods with experimental and clinical approaches across academia, industry, and healthcare settings is a priority. (iv) The human-based cross-disciplinary approach requires experts in specific methodologies and domains, who also have the capacity to communicate and collaborate across disciplines and cross-sector environments. (v) This new translational domain for human-based cardiology and pharmacology requires new partnerships supported financially and institutionally across sectors. Institutional, organizational, and social barriers must be identified, understood and overcome in each specific setting.

Optical control of excitation waves in cardiac tissue.

In nature, macroscopic excitation waves1,2 are found in a diverse range of settings including chemical reactions, metal rust, yeast, amoeba and the heart and brain. In the case of living biological tissue, the spatiotemporal patterns formed by these excitation waves are different in healthy and diseased states2,3. Current electrical and pharmacological methods for wave modulation lack the spatiotemporal precision needed to control these patterns. Optical methods have the potential to overcome these limitations, but to date have only been demonstrated in simple systems, such as the Belousov-Zhabotinsky chemical reaction4. Here, we combine dye-free optical imaging with optogenetic actuation to achieve dynamic control of cardiac excitation waves. Illumination with patterned light is demonstrated to optically control the direction, speed and spiral chirality of such waves in cardiac tissue. This all-optical approach offers a new experimental platform for the study and control of pattern formation in complex biological excitable systems.

Three-dimensional histology: tools and application to quantitative assessment of cell-type distribution in rabbit heart.

AIMS: Cardiac histo-anatomical organization is a major determinant of function. Changes in tissue structure are a relevant factor in normal and disease development, and form targets of therapeutic interventions. The purpose of this study was to test tools aimed to allow quantitative assessment of cell-type distribution from large histology and magnetic resonance imaging- (MRI) based datasets. METHODS AND RESULTS: Rabbit heart fixation during cardioplegic arrest and MRI were followed by serial sectioning of the whole heart and light-microscopic imaging of trichrome-stained tissue. Segmentation techniques developed specifically for this project were applied to segment myocardial tissue in the MRI and histology datasets. In addition, histology slices were segmented into myocytes, connective tissue, and undefined. A bounding surface, containing the whole heart, was established for both MRI and histology. Volumes contained in the bounding surface (called 'anatomical volume'), as well as that identified as containing any of the above tissue categories (called 'morphological volume'), were calculated. The anatomical volume was 7.8 cm(3) in MRI, and this reduced to 4.9 cm(3) after histological processing, representing an 'anatomical' shrinkage by 37.2%. The morphological volume decreased by 48% between MRI and histology, highlighting the presence of additional tissue-level shrinkage (e.g. an increase in interstitial cleft space). The ratio of pixels classified as containing myocytes to pixels identified as non-myocytes was roughly 6:1 (61.6 vs. 9.8%; the remaining fraction of 28.6% was 'undefined'). CONCLUSION: Qualitative and quantitative differentiation between myocytes and connective tissue, using state-of-the-art high-resolution serial histology techniques, allows identification of cell-type distribution in whole-heart datasets. Comparison with MRI illustrates a pronounced reduction in anatomical and morphological volumes during histology processing.

Quantifying distortions in two-photon remote focussing microscope images using a volumetric calibration specimen.

Remote focussing microscopy allows sharp, in-focus images to be acquired at high speed from outside of the focal plane of an objective lens without any agitation of the specimen. However, without careful optical alignment, the advantages of remote focussing microscopy could be compromised by the introduction of depth-dependent scaling artifacts. To achieve an ideal alignment in a point-scanning remote focussing microscope, the lateral (XY) scan mirror pair must be imaged onto the back focal plane of both the reference and imaging objectives, in a telecentric arrangement. However, for many commercial objective lenses, it can be difficult to accurately locate the position of the back focal plane. This paper investigates the impact of this limitation on the fidelity of three-dimensional data sets of living cardiac tissue, specifically the introduction of distortions. These distortions limit the accuracy of sarcomere measurements taken directly from raw volumetric data. The origin of the distortion is first identified through simulation of a remote focussing microscope. Using a novel three-dimensional calibration specimen it was then possible to quantify experimentally the size of the distortion as a function of objective misalignment. Finally, by first approximating and then compensating the distortion in imaging data from whole heart rodent studies, the variance of sarcomere length (SL) measurements was reduced by almost 50%.

Mechanism of reentry induction by a 9-V battery in rabbit ventricles.

Although the application of a 9-V battery to the epicardial surface is a simple method of ventricular fibrillation induction, the fundamental mechanisms underlying this process remain unstudied. We used a combined experimental and modelling approach to understand how the interaction of direct current (DC) from a battery may induce reentrant activity within rabbit ventricles and its dependence on battery application timing and duration. A rabbit ventricular computational model was used to simulate 9-V battery stimulation for different durations at varying onset times during sinus rhythm. Corresponding high-resolution optical mapping measurements were conducted on rabbit hearts with DC stimuli applied via a relay system. DC application to diastolic tissue induced anodal and cathodal make excitations in both simulations and experiments. Subsequently, similar static epicardial virtual electrode patterns were formed that interacted with sinus beats but did not induce reentry. Upon battery release during diastole, break excitations caused single ectopics, similar to application, before sinus rhythm resumed. Reentry induction was possible for short battery applications when break excitations were slowed and forced to take convoluted pathways upon interaction with refractory tissue from prior make excitations or sinus beats. Short-lived reentrant activity could be induced for battery release shortly after a sinus beat for longer battery applications. In conclusion, the application of a 9-V battery to the epicardial surface induces reentry through a complex interaction of break excitations after battery release with prior induced make excitations or sinus beats.

Fast measurement of sarcomere length and cell orientation in Langendorff-perfused hearts using remote focusing microscopy.

RATIONALE: Sarcomere length (SL) is a key indicator of cardiac mechanical function, but current imaging technologies are limited in their ability to unambiguously measure and characterize SL at the cell level in intact, living tissue. OBJECTIVE: We developed a method for measuring SL and regional cell orientation using remote focusing microscopy, an emerging imaging modality that can capture light from arbitrary oblique planes within a sample. METHODS AND RESULTS: We present a protocol that unambiguously and quickly determines cell orientation from user-selected areas in a field of view by imaging 2 oblique planes that share a common major axis with the cell. We demonstrate the effectiveness of the technique in establishing single-cell SL in Langendorff-perfused hearts loaded with the membrane dye di-4-ANEPPS. CONCLUSIONS: Remote focusing microscopy can measure cell orientation in complex 2-photon data sets without capturing full z stacks. The technique allows rapid assessment of SL in healthy and diseased heart experimental preparations.

Rearrangement of atrial bundle architecture and consequent changes in anisotropy of conduction constitute the 3-dimensional substrate for atrial fibrillation.

BACKGROUND: Anisotropy of conduction facilitates re-entry and is, therefore, a key determinant of the stability of atrial fibrillation (AF). Little is known about the effect of AF on atrial bundle architecture and consequent changes in anisotropy of conduction and maintenance of AF. METHODS AND RESULTS: Direct contact mapping was performed in left atria of goats with acute AF (n=6) or persistent AF (n=5). The degree and direction of anisotropic conduction were analyzed. Mapped tissue regions were imaged by high-resolution MRI for identification of endocardial and epicardial bundle directions. Correlation between endocardial and epicardial bundle directions and between bundle directions and anisotropic conduction was quantified. In persistent AF, epicardial bundles were oriented more perpendicularly to endocardial bundles than in acute AF (% angles<20° between epicardial and endocardial bundle directions were 7.63% and 21.25%, respectively; P<0.01). In acute AF, the direction of epicardially mapped anisotropic conduction correlated with endocardial but not with epicardial bundles. In persistent AF, the direction of anisotropic conduction correlated better with epicardial than with endocardial bundles (% angles<20° between direction of anisotropic conduction and bundle direction were 28.77% and 18.45%, respectively; P<0.01). CONCLUSIONS: During AF, atrial bundle rearrangement manifests itself in more perpendicular orientation of epicardial to endocardial bundles. Propagation of fibrillation waves is dominated by endocardial bundles in acute AF and by epicardial bundles in persistent AF. Together with the loss of endo-epicardial electrical connections, rearrangement of atrial bundles underlies endo-epicardial dissociation of electrical activity and the development of a 3-dimensional AF substrate.

Histo-anatomical structure of the living isolated rat heart in two contraction states assessed by diffusion tensor MRI.

Deformation and wall-thickening of ventricular myocardium are essential for cardiac pump function. However, insight into the histo-anatomical basis for cardiac tissue re-arrangement during contraction is limited. In this report, we describe dynamic changes in regionally prevailing cardiomyocyte (fibre) and myolaminar (sheet) orientations, using Diffusion Tensor Imaging (DTI) of ventricles in the same living heart in two different mechanical states. Hearts, isolated from Sprague-Dawley rats, were Langendorff-perfused and imaged, initially in their slack state during cardioplegic arrest, then during lithium-induced contracture. Regional fibre- and sheet-orientations were derived from DTI-data on a voxel-wise basis. Contraction was accompanied with a decrease in left-handed helical fibres (handedness relative to the baso-apical direction) in basal, equatorial, and apical sub-epicardium (by 14.0%, 17.3%, 15.8% respectively; p < 0.001), and an increase in right-handed helical fibres of the sub-endocardium (by 11.0%, 12.1% and 16.1%, respectively; p < 0.001). Two predominant sheet-populations were observed, with sheet-angles of either positive (ß+) or negative (ß-) polarity relative to a 'chamber-horizontal plane' (defined as normal to the left ventricular long-axis). In contracture, mean 'intersection'-angle (geometrically quantifiable intersection of sheet-angle projections) between ß+ and ß- sheet-populations increased from 86.2 ± 5.5° (slack) to 108.3 ± 5.4° (p < 0.001). Subsequent high-resolution DTI of fixed myocardium, and histological sectioning, reconfirmed the existence of alternating sheet-plane populations. Our results suggest that myocardial tissue layers in alternating sheet-populations align into a more chamber-horizontal orientation during contraction. This re-arrangement occurs via an accordion-like mechanism that, combined with inter-sheet slippage, can significantly contribute to ventricular deformation, including wall-thickening in a predominantly centripetal direction and baso-apical shortening.

Microscopic magnetic resonance imaging reveals high prevalence of third coronary artery in human and rabbit heart.

AIM: The human coronary tree is commonly assumed to have two roots: the left and right coronary arteries (LCA and RCA, respectively). However, a third coronary artery (TCA) has been observed in humans and animals, usually arising from the right anterior aortic sinus near the RCA. Using high-resolution magnetic resonance imaging, we identified TCA prevalence and characteristics in rabbit and human hearts. METHODS AND RESULTS: Third coronary artery presence was analysed in hearts from 11 New Zealand white rabbits and 7 human cadavers, using excised tissue that was fixed, gadolinium-treated, and agar-embedded for imaging-based reconstruction. A TCA was identified in all rabbit hearts and six of seven human hearts, originating either from an independent ostium (7 of 11 rabbits, 2 of 7 humans) or an ostium shared with the RCA (4 of 11 rabbits, 4 of 7 humans). Proximal TCA cross-sectional area in rabbits was 15.3 ± 6.0% of RCA area (mean ± SD, based on n = 9 rabbit hearts in which reliable measurements could be taken for both vessels), and 26.7 ± 10.1% in humans (n = 4). In all-but-one case where a TCA was observed, it originated ventral to the RCA, progressing towards the right ventricular outflow tract. In one rabbit, the TCA originated dorsal to the RCA and progressed towards the Crista terminalis in the right atrium. A fourth vessel, forming a separate aortic Vas vasorum was occasionally seen, originating from the right anterior aortic sinus either from an ostium common with (1 of 11 rabbits, 0 of 7 humans) or independent of (1 of 11 rabbits, 1 of 7 humans) the TCA. Pilot optical mapping experiments showed that TCA occlusion had variable acute effects on rabbit cardiac electrophysiology. CONCLUSION: Third coronary artery presence is common in rabbit and human hearts. Functional effects of disrupted TCA blood supply are ill-investigated, and the rabbit may be a suitable species for such research.

Rediscovering the third coronary artery.

A second right coronary artery is not at all unusual, as described here from Oxford, England.