Cardiac multi-scale investigation of the right and left ventricle ex vivo: a review

The heart is a complex multi-scale system composed of components integrated at the subcellular, cellular, tissue and organ levels. The myocytes, the contractile elements of the heart, form a complex three-dimensional (3D) network which enables propagation of the electrical signal that triggers the contraction to efficiently pump blood towards the whole body. Cardiovascular diseases (CVDs), a major cause of mortality in developed countries, often lead to cardiovascular remodeling affecting cardiac structure and function at all scales, from myocytes and their surrounding collagen matrix to the 3D organization of the whole heart. As yet, there is no consensus as to how the myocytes are arranged and packed within their connective tissue matrix, nor how best to image them at multiple scales. Cardiovascular imaging is routinely used to investigate cardiac structure and function as well as for the evaluation of cardiac remodeling in CVDs. For a complete understanding of the relationship between structural remodeling and cardiac dysfunction in CVDs, multi-scale imaging approaches are necessary to achieve a detailed description of ventricular architecture along with cardiac function. In this context, ventricular architecture has been extensively studied using a wide variety of imaging techniques: ultrasound (US), optical coherence tomography (OCT), microscopy (confocal, episcopic, light sheet, polarized light), magnetic resonance imaging (MRI), micro-computed tomography (micro-CT) and, more recently, synchrotron X-ray phase contrast imaging (SR X-PCI). Each of these techniques have their own set of strengths and weaknesses, relating to sample size, preparation, resolution, 2D/3D capabilities, use of contrast agents and possibility of performing together with in vivo studies. Therefore, the combination of different imaging techniques to investigate the same sample, thus taking advantage of the strengths of each method, could help us to extract the maximum information about ventricular architecture and function. In this review, we provide an overview of available and emerging cardiovascular imaging techniques for assessing myocardial architecture ex vivo and discuss their utility in being able to quantify cardiac remodeling, in CVDs, from myocyte to whole organ

Detailed quantification of cardiac ventricular myocardial architecture in the embryonic and fetal mouse heart by application of structure tensor analysis to high resolution episcopic microscopic data

The mammalian heart, which is one of the first organs to form and function during embryogenesis, develops from a simple tube into a complex organ able to efficiently pump blood towards the rest of the body. The progressive growth of the compact myocardium during embryonic development is accompanied by changes in its structural complexity and organisation. However, how myocardial myoarchitecture develops during embryogenesis remain poorly understood. To date, analysis of heart development has focused mainly on qualitative descriptions using selected 2D histological sections. High resolution episcopic microscopy (HREM) is a novel microscopic imaging technique that enables to obtain high-resolution three-dimensional images of the heart and perform detailed quantitative analyses of heart development. In this work, we performed a detailed characterization of the development of myocardial architecture in wildtype mice, from E14.5 to E18.5, by means of structure tensor analysis applied to HREM images of the heart. Our results shows that even at E14.5, myocytes are already aligned, showing a gradual change in their helical angle from positive angulation in the endocardium towards negative angulation in the epicardium. Moreover, there is gradual increase in the degree of myocardial organisation concomitant with myocardial growth. However, the development of the myoarchitecture is heterogeneous showing regional differences between ventricles, ventricular walls as well as between myocardial layers, with different growth patterning between the endocardium and epicardium. We also found that the percentage of circumferentially arranged myocytes within the LV significantly increases with gestational age. Finally, we found that fractional anisotropy (FA) within the LV gradually increases with gestational age, while the FA within RV remains unchanged

Machine Learning in Fetal Cardiology: What to Expect

In fetal cardiology, imaging (especially echocardiography) has demonstrated to help in the diagnosis and monitoring of fetuses with a compromised cardiovascular system potentially associated with several fetal conditions. Different ultrasound approaches are currently used to evaluate fetal cardiac structure and function, including conventional 2-D imaging and M-mode and tissue Doppler imaging among others. However, assessment of the fetal heart is still challenging mainly due to involuntary movements of the fetus, the small size of the heart, and the lack of expertise in fetal echocardiography of some sonographers. Therefore, the use of new technologies to improve the primary acquired images, to help extract measurements, or to aid in the diagnosis of cardiac abnormalities is of great importance for optimal assessment of the fetal heart. Machine leaning (ML) is a computer science discipline focused on teaching a computer to perform tasks with specific goals without explicitly programming the rules on how to perform this task. In this review we provide a brief overview on the potential of ML techniques to improve the evaluation of fetal cardiac function by optimizing image acquisition and quantification/segmentation, as well as aid in improving the prenatal diagnoses of fetal cardiac remodeling and abnormalities

Structural coronary artery remodelling in the rabbit fetus as a result of intrauterine growth restriction

Intrauterine growth restriction (IUGR) is a fetal condition that affects up to 10% of all pregnancies and is associated with cardiovascular structural and functional remodelling that persists postnatally. Some studies have reported an increase in myocardial coronary blood flow in severe IUGR fetuses which has been directly associated to the dilatation of the coronary arteries. However, a direct measurement of the coronaries’ lumen diameter in IUGR has not been reported before. The aim of this paper is to perform, for the first time, a quantitative analysis of the effects of IUGR in cardiac geometry and coronary vessel size in a wellknown rabbit model of IUGR using synchrotron-based X-ray Phase Contrast Tomography Imaging (X-PCI). Eight rabbit fetal hearts were imaged non-destructively with X-PCI. 3D reconstructions of the coronary arterial tree were obtained after semi-automatic image segmentation. Different morphometric features including vessel lumen diameter of the three main coronaries were automatically quantified. IUGR fetuses had more globular hearts and dilated coronary arteries as compared to controls. We have quantitatively shown that IUGR leads to structural coronary vascular tree remodelling and enlargement as an adaptation mechanism in response to an adverse environment of restricted oxygen and nutrients and increased perfusion pressure

Assessment of Haemodynamic Remodeling in Fetal Aortic Coarctation Using a Lumped Model of the Circulation

Introduction: Aortic coarctation is one of the most difficult cardiac defects to diagnose before birth, and it accounts for 8% of congenital heart diseases. Antenatal diagnosis is crucial for early treatment of the neonate and to decrease the risk of morbidity and mortality; however the fetal hemodynamic changes are not fully understood and current imaging methods are limited to accurately diagnosis this congenital defect. Objective: We propose to use a lumped model of the fetal circulation to provide insights into the hemodynamic changes in fetuses with aortic coarctation, and thus helping to improve its diagnosis. Methods: To achieve this goal a patient-specific lumped model of the fetal circulation was implemented in OpenCOR, including the modeling of different types and degrees of aortic coarctation. A parametric study of degree and type of coarctation was performed, where blood flow distribution, cerebroplacental ratio, pressure drop over the coarctation and left ventricular pressure were quantified. Results: Obvious changes in the fetal hemodynamics were observed only from 80% of coarctation, corresponding to the clinically used cut-off for pressure drop of 20 mmHg. Furthermore, the observed hemodynamic changes were different depending on the location and degree of the coarctation

Microstructural Analysis of Cardiac Endomyocardial Biopsies with Synchrotron Radiation-Based X-Ray Phase Contrast Imaging

Nowadays, unexplained cardiovascular diseases (CVD) and heart transplant response are assessed by qualitative histological analysis of extracted endomyocardial biopsies (EMB), which is a time consuming procedure involving structural damage of the tissue and the analysis in only a few slices of a 3D structure. In this paper we propose synchrotron radiation-based X-ray phase contrast imaging (X-PCI) as a suitable technique for the analysis of different cardiac microstructures, such as collagen matrix, cardiomyocytes and microvasculature, and how they are affected in abnormal conditions. Following an established procedure in clinics, biopsies from Wistar Kyoto rats are extracted, imaged with X-PCI, and processed in order to show that the quantification of the endomysial collagen matrix, cardiomyocytes and microvasculature is possible, thus demonstrating that the intrinsic properties of X-PCI make it a powerful technique for cardiac microstructure imaging and a promising methodology for a faster and more accurate EMB analysis for CVD diagnosis and evaluation

Microstructural Analysis of Cardiac Endomyocardial Biopsies with Synchrotron Radiation-Based X-Ray Phase Contrast Imaging

Nowadays, unexplained cardiovascular diseases (CVD) and heart transplant response are assessed by qualitative histological analysis of extracted endomyocardial biopsies (EMB), which is a time consuming procedure involving structural damage of the tissue and the analysis in only a few slices of a 3D structure. In this paper we propose synchrotron radiation-based X-ray phase contrast imaging (X-PCI) as a suitable technique for the analysis of different cardiac microstructures, such as collagen matrix, cardiomyocytes and microvasculature, and how they are affected in abnormal conditions. Following an established procedure in clinics, biopsies from Wistar Kyoto rats are extracted, imaged with X-PCI, and processed in order to show that the quantification of the endomysial collagen matrix, cardiomyocytes and microvasculature is possible, thus demonstrating that the intrinsic properties of X-PCI make it a powerful technique for cardiac microstructure imaging and a promising methodology for a faster and more accurate EMB analysis for CVD diagnosis and evaluation

Shape Analysis and Computational Fluid Simulations to Assess Feline Left Atrial Function and Thrombogenesis

In humans, there is a well-established relationship between atrial fibrillation (AF), blood flow abnormalities and thrombus formation, even if there is no clear consensus on the role of left atrial appendage (LAA) morphologies. Cats can also suffer heart diseases, often leading to an enlargement of the left atrium that promotes stagnant blood flow, activating the clotting process and promoting feline aortic thromboembolism. The majority of pathological feline hearts have echocardiographic evidence of abnormal left ventricular filling, usually assessed with 2D and Doppler echocardiography and standard imaging tools. Actually, veterinary professionals have limited access to advanced computational techniques that would enable a better understanding of feline heart pathologies with improved morphological and haemodynamic descriptors. In this work, we applied state-of-the-art image processing and computational fluid simulations based on micro-computed tomography images acquired in 24 cases, including normal cats and cats with varying severity of cardiomyopathy. The main goal of the study was to identify differences in the LA/LAA morphologies and blood flow patterns in the analysed cohorts with respect to thrombus formation and cardiac pathology. The obtained results show significant differences between normal and pathological feline hearts, as well as in thrombus vs non-thrombus cases and asymptomatic vs symptomatic cases, while it was not possible to discern in congestive heart failure with thrombus and from non-thrombus cases. Additionally, in-silico fluid simulations demonstrated lower LAA blood flow velocities and higher thrombotic risk in the thrombus cases

Revealing Cardiac Microstructure in a Human Fetal Heart of 8 weeks gestation with Synchrotron-based X-Ray Phase Contrast Tomographic Imaging

Introduction: Understanding the complexity of heart morphogenesis and the associated functional consequences of congenital heart disease is essential for providing appropriate treatment strategies. Since our knowledge on the microstructure of the whole fetal & paediatric heart is limited, novel imaging approaches offered by synchrotron facilities can provide structural detail currently not available otherwise. Our aim is to visualise and quantify cardiac microstructure in fetal hearts at different stages of development using synchrotron-based X-ray Phase-Contrast tomography Imaging (X-PCI). Methods: A normal fetal heart of 8 weeks and 6 days of gestation was selected from the from the Ospedale Maggiore Policlinico (Milan, Italy). While the specimen was fixed in formalin, it was placed in water as supporting medium for acquisition. X-PCI was performed at 1.625μm resolution at TOMCAT Beamline (Swiss Light Source, Paul Scherer Institute, Villigen, Switzerland) using an energy of 20 keV. Several acquisitions were necessary to cover the whole heart along its long axis. The image series were reconstructed using Gridrec algorithm. Orientation of myocytes aggregates was computed using an in-house structure tensor algorithm. Results: Fig.1(a) Two images of the gross specimen with scale - base to apex 2mm. Fig.1(b)-(c) show longitudinal (4-chamber) and short axis maximum intensity projection slices, respectively, from the X-PCI image dataset showing detail of myocardial structure. The ventricular myocardium is composed mainly of trabeculations while the compact myocardium is thin and under-developed. Taking both trabecular and compact myocardium together there is organisation even at this early gestation (see Fig.1(d)) with a clear change in helical angle (from 60º to -60º) from endo to epicardium, especially the septal wall. Conclusions: We managed for the first time to image a normal fetal heart with high-resolution and in 3D at an early stage of development, resolving detail of myocyte aggregates and providing information on cardiac microstructure without the need for sample processing or sectioning

Myoarchitectural disarray of hypertrophic cardiomyopathy begins pre‐birth

Myoarchitectural disarray – the multiscalar disorganisation of myocytes, is a recognised histopathological hallmark of adult human hypertrophic cardiomyopathy (HCM). It occurs before the establishment of left ventricular hypertrophy (LVH) but its early origins and evolution around the time of birth are unknown. Our aim is to investigate whether myoarchitectural abnormalities in HCM are present in the fetal heart. We used wild-type, heterozygous and homozygous hearts (n = 56) from a Mybpc3-targeted knock-out HCM mouse model and imaged the 3D micro-structure by high-resolution episcopic microscopy. We developed a novel structure tensor approach to extract, display and quantify myocyte orientation and its local angular uniformity by helical angle, angle of intrusion and myoarchitectural disarray index, respectively, immediately before and after birth. In wild-type, we demonstrate uniformity of orientation of cardiomyocytes with smooth transitions of helical angle transmurally both before and after birth but with traces of disarray at the septal insertion points of the right ventricle. In comparison, heterozygous mice free of LVH, and homozygous mice showed not only loss of the normal linear helical angulation transmural profiles observed in wild-type but also fewer circumferentially arranged myocytes at birth. Heterozygous and homozygous showed more disarray with a wider distribution than in wild-type before birth. In heterozygous mice, disarray was seen in the anterior, septal and inferior walls irrespective of stage, whereas in homozygous mice it extended to the whole LV circumference including the lateral wall. In conclusion, myoarchitectural disarray is detectable in the fetal heart of an HCM mouse model before the development of LVH