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Les pathologies cardiovasculaires restent un des problèmes majeurs de santé publique qui justifie les recherches menées pour améliorer notre compréhension de la fonction cardiaque. Celles-ci nécessitent une bonne connaissance de la microarcInstitut de Technologie de Harbin - Chineecture myocardique afin de mieux comprendre les relations entre les fonctions mécanique, hémodynamique et les changements structuraux induits par les maladies cardiaques. Pour ce faire il est nécessaire d’accéder à une connaissance précise de l’arrangement spatial des composants du tissu. Cependant, notre compréhension de l’arcInstitut de Technologie de Harbin - Chineecture du coeur est limitée par le manque de description 3D de l’organisation des structures à l’échelle microscopique. Nous proposons d’explorer la structure 3D du tissu cardiaque en utilisant l’imagerie X synchrotron par contraste de phase disponible à l’ESRF. Pour la première fois, 9 échantillons de tissu de la paroi du ventricule gauche (VG) humain sont imagés à la résolution isotrope de 3,5 μm et analysés. Cette thèse est centrée sur la description 3D d’un des constituants principal du tissu: la matrice extracellulaire (MEC). La MEC inclue: l’endomysium qui entoure et sépare les myocytes et les capillaires de façon individuelle, le perimysium qui entoure et sépare des groupes de myocytes et l’épimysium qui enveloppe le muscle cardiaque dans son ensemble. Chaque échantillon reconstruit fait environ 30 Gb, ce qui représente une quantité importante de données à traiter et à visualiser. Pour ce faire, nous avons développé un algorithme automatique de traitement d’image pour binariser chaque échantillon et isoler la MEC. Ensuite, nous avons extrait des parametres statistiques relatifs à la microarcInstitut de Technologie de Harbin - Chineecture de l’ECM, principalement l’épaisseur des plans de clivage (PC) et les distances inter-PC. Les résultats montrent que l’arrangement local des PC diffère selon l’emplacement au sein du VG (postérieur, antérieur, septal) et de leur distance à l’apex (plus complexe). L’épaisseur des PC extraite de tous les échantillons va approximativement de 24 μm à 59 μm et la distance inter-PC de 70 μm à 280 μm avec une variation locale significative de la déviation standard. Ce sont de nouveaux marqueurs quantitatifs de la MEC du tissu cardiaque humain qui sont d’un intérêt majeur pour une meilleure compréhension de la fonction cardiaque. / Cardiovascular diseases remain one of the most serious health problems, motivating research to deepen our understanding of the myocardial function. To succeed, there is a need to get detailed information about the spatial arrangement of the cardiac tissue components. Currently, our understanding of the cardiac microarcInstitut de Technologie de Harbin - Chineecture is limited by the lack of 3D descriptions of the cardiac tissue at the microscopic scale. This thesis investigates the 3D cardiac tissue microstructure using X-Ray µ-CT phase contrast imaging available at the ESRF. For the first time, 9 human cardiac left ventricle (LV) wall samples are imaged at an isotropic resolution (3.5 µm) and analysed. We focus on the description of the cardiac extracellular matrix (CEM) that is one of the main components of the tissue. The CEM includes: the endomysium that surrounds and separates individual myocytes and capillaries, the perimysium that surrounds groups of myocytes and the epimysium that surrounds the entire heart muscle. Each reconstructed sample is about 30 Gb which represents a large amount of data to process and display. To succeed, we developed an automatic image processing algorithm to binarise each sample by selecting the CEM. We extract statistical features of the ECM, mainly the thickness of the cleavage planes (CP) and the inter-CP distances. The results show that the local 3D arrangement of the CP differs according to their location in the LV (posterior, anterior, septal) and their distance from the apex (more complex). The thickness of the CP extracted from all the samples roughly ranges from 24 µm to 59 µm and the inter-CP distances from 70 µm to 280 µm with significant local variations of the standard deviation. Those new quantitative markers of the ECM of the human cardiac are of main interest for a better understanding of the heart function.

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Coordinated electrical activation of the heart is essential for the maintenance of a regular cardiac rhythm and effective contractions. Action potentials spread from one cell to the next via gap junction channels. Because of the elongated shape of cardiomyocytes, longitudinal resistivity is lower than transverse resistivity causing electrical anisotropy. Moreover, non-uniformity is created by clustering of gap junction channels at cell poles and by non-excitable structures such as collagenous strands, vessels or fibroblasts. Structural changes in cardiac disease often affect passive electrical properties by increasing non-uniformity and altering anisotropy. This disturbs normal electrical impulse propagation and is, consequently, a substrate for arrhythmia. However, to investigate how these structural changes lead to arrhythmias remains a challenge. One important mechanism, which may both cause and prevent arrhythmia, is the mismatch between current sources and sinks. Propagation of the electrical impulse requires a sufficient source of depolarizing current. In the case of a mismatch, the activated tissue (source) is not able to deliver enough depolarizing current to trigger an action potential in the non-activated tissue (sink). This eventually leads to conduction block. It has been suggested that in this situation a balanced geometrical distribution of gap junctions and reduced gap junction conductance may allow successful propagation. In contrast, source-sink mismatch can prevent spontaneous arrhythmogenic activity in a small number of cells from spreading over the ventricle, especially if gap junction conductance is enhanced. Beside gap junctions, cell geometry and non-cellular structures strongly modulate arrhythmogenic mechanisms. The present review elucidates these and other implications of passive electrical properties for cardiac rhythm and arrhythmogenesis.

Cardiac tissue engineering promises to create therapeutic tissue replacements for repair of diseased native myocardium. The main goals of this thesis were four-fold: 1) to evaluate cardiac tissues engineered using multiple cell types including endothelial cells (EC), fibroblasts (FB), and cardiomyocytes (CM); 2) to spatiotemporally track cells in organoids and optimize their seeding percentages for improved function; 3) to enhance vascular cord formation through sequential versus simultaneous seeding of ECs and FBs; and 4) to perform mechanistic studies to elucidate the role of soluble factors in cell-cell communication. Microscale templates fabricated from photocrosslinkable poly(ethylene glycol) diacrylate (PEG-DA) were used for all studies for rapid screening. When ECs and FBs were precultured for two days prior to seeding enriched CMs, cells self-assembled into three-dimensional, beating organoids, compared to simultaneously tricultured EC/ FB / CM which formed non-contractile clusters. Fluorescent dyes were used to label and track each cell type for up to 4 days, demonstrating an even distribution of cells within precultured organoids versus EC clustering in simultaneous triculture. When ECs were seeded first, followed by FBs 24 hours later and CMs 48 hours later, vascular-like cords formed that persisted with time in a seeding density-dependent manner. Vascular endothelial growth factor (VEGF) signaling was quantified, showing higher endogenous VEGF secretion rates in sequential preculture (16.6 ng/mL/hr) compared to undetectable VEGF secretion in simultaneous triculture. Blocking of endogenous VEGF signaling through addition of VEGF antibody / VEGFR2 inhibitor resulted in a significant decrease in mRNA and protein expression of the key cardiac gap junctional marker connexin-43. These findings provide a foundation for future work into the mechanisms governing functional cardiac tissue engineering performance and may aid in the development of novel therapies for heart failure based on growth factor signaling and engineering of vascularized, clinically relevant cardiac tissue patches.

Cardiac tissue engineering promises to create therapeutic tissue replacements for repair of diseased native myocardium. The main goals of this thesis were four-fold: 1) to evaluate cardiac tissues engineered using multiple cell types including endothelial cells (EC), fibroblasts (FB), and cardiomyocytes (CM); 2) to spatiotemporally track cells in organoids and optimize their seeding percentages for improved function; 3) to enhance vascular cord formation through sequential versus simultaneous seeding of ECs and FBs; and 4) to perform mechanistic studies to elucidate the role of soluble factors in cell-cell communication. Microscale templates fabricated from photocrosslinkable poly(ethylene glycol) diacrylate (PEG-DA) were used for all studies for rapid screening. When ECs and FBs were precultured for two days prior to seeding enriched CMs, cells self-assembled into three-dimensional, beating organoids, compared to simultaneously tricultured EC/ FB / CM which formed non-contractile clusters. Fluorescent dyes were used to label and track each cell type for up to 4 days, demonstrating an even distribution of cells within precultured organoids versus EC clustering in simultaneous triculture. When ECs were seeded first, followed by FBs 24 hours later and CMs 48 hours later, vascular-like cords formed that persisted with time in a seeding density-dependent manner. Vascular endothelial growth factor (VEGF) signaling was quantified, showing higher endogenous VEGF secretion rates in sequential preculture (16.6 ng/mL/hr) compared to undetectable VEGF secretion in simultaneous triculture. Blocking of endogenous VEGF signaling through addition of VEGF antibody / VEGFR2 inhibitor resulted in a significant decrease in mRNA and protein expression of the key cardiac gap junctional marker connexin-43. These findings provide a foundation for future work into the mechanisms governing functional cardiac tissue engineering performance and may aid in the development of novel therapies for heart failure based on growth factor signaling and engineering of vascularized, clinically relevant cardiac tissue patches.

There is a growing consensus that life-threatening cardiac arrhythmias like ventricular tachycardia (VT) or ventricular fibrillation (VF) arise because of the formation of spiral waves of electrical activation in cardiac tissue; unbroken spiral waves are associated with VT and broken ones with VF. Several experimental studies have shown that inhomogeneities in cardiac tissue can have dramatic effects on such spiral waves. In this thesis we try to understand these experimental results by carrying out detailed and systematic studies of the interaction of spiral waves with different types of inhomogeneities in mathematical models for cardiac tissue. In Chapter 1 we begin with a general introduction to cardiac arrhythmias, the cardiac conduction system, and the connection between electrical activation waves in cardiac tissue and cardiac arrhythmias. As we have noted above, VT and VF are believed to be associated with spiral waves of electrical activation on cardiac tissue; such spiral waves form because cardiac tissue is an excitable medium. Thus we give an overview of excitable media, in which sub-threshold perturbations decay but super-threshold perturbations lead to an action potential that consists of a rapid stage of depolarization of cardiac cells followed by a slow phase of repolarization. During this repolarization phase the cells are refractory. We then give an overview of earlier studies of the effects of inhomogeneities in cardiac tissue; and we end with a brief description of the principal problems we study here. Chapter 2 describes the models we use in our work. We start with a general introduction to the cable equation and then discuss the Hodgkin-Huxley-formalism for the transport of ions across a cell membrane through voltage-gated ion channels. We then describe in detail the three models that we use for cardiac tissue, which are, in order of increasing complexity, the Panfilov model, the Luo Rudy Phase I (LRI) model, and the reduced Priebe Beuckelmann (RPB)model. We then give the numerical schemes we use for solving these model equations and the initial conditions that lead to the formation of spiral waves. For all these models we give representative results from our simulations and compare the states with spiral turbulence. In Chapter 3 we investigate the effects of conduction inhomogeneities (obstacles) in the three models introduced in Chapter 2. We outline first the experimental results that have provided the motivation for our study. We then discuss how we introduce obstacles in our simulations of the Panffilov, LRI, and RPB models for cardiac tissue. Next we present the results of our numerical studies of the effects, on spiral-wave dynamics, of the sizes, shapes, and positions of the obstacles. Our Principal result is that spiral-wave dynamics in these models depends sensitively on the position of the obstacle. We find, in particular, that, merely by changing the position of a conduction inhomogeneity, we may convert spiral turbulence (the analogue in our models of VF) to a single rotating spiral (the analogue of VT) anchored to the obstacle or vice versa; even more exciting is the possibility that, at the boundary between these two types of behaviour, we find a quiescent state Q with no spiral waves. Thus our study obtains all the possible qualitative behaviours found in experiments, namely, (1) VF might persist even in the presence of an obstacle, (2) it might be suppressed partially and become VT, or (3) it might be eliminated completely. In Chapter 4 we extend our work on conduction inhomogeneities (Chapter 3) to ionic inhomogeneities. Unlike conduction inhomogeneities, ionic inhomogeneities allow the conduction of activation waves. We find, nevertheless, that they too can lead to the anchoring of spiral waves or even the complete elimination of spiral-wave turbulence. Since spiral waves can enter the region in which there is an ionic inhomogeneity, their behaviours in the presence of such an inhomogeneity are richer than those with conduction inhomogeneities. We find, in particular, that a single spiral wave anchored at an ionic inhomogeneity can show temporal evolution that may be periodic, quasiperiodic, or even chaotic. In the last case the spiral wave shows a chaotic pattern inside the ionic inhomogeneity and a regular one outside it. Defibrillation is the control of arrhythmias such as VF. Most often defibrillation is effected electrically by administering a shock, either externally or via an internally implanted defibrillator. The development of low-amplitude defibrillation schemes, which minimise the deleterious effects of the applied shock, is a major challenge in the treatment of cardiac arrhythmias. Numerical studies of models for cardiac tissue provide us with convenient means of studying the elimination of spiral-wave turbulence by the application of external electrical stimuli; this is the numerical analogue of defibrillation. Over the years some low-amplitude defibrillation schemes have been suggested on the basis of such numerical studies. In Chapter 5 we discuss two such schemes that have been shown to suppress spiral-wave turbulence in two-dimensional models for cardiac tissue and also scroll-wave turbulence in three-dimensional models. One of these schemes uses local electrical pacing, typically in the centre of the simulation domain; the other applies the external electrical stimuli over a mesh. We study the efficacy of these schemes in the presence of conduction inhomogeneities. We find, in particular, that the local-pacing scheme, though effective in a homogeneous simulation domain, fails to control spiral turbulence in the presence of an obstacle and, indeed, might even facilitate spiral-wave break up. By contrast, the second scheme, which uses a mesh, succeeds in eliminating spiral-wave turbulence even in the presence of an obstacle. We end with some concluding remarks about the possible experimental implications of our study in Chapter 6.

Les maladies cardiovasculaires sont une des causes de mortalités les plus élevées mondialement. Parmi celles-ci, on retrouve l’infarctus du myocarde, qui n’a pour traitement que la transplantation cardiaque. Or, dû à la faible quantité de donneur, une solution alternative est recherchée. De ce fait, l’ingénierie tissulaire permet le développement de tissus et d’implants thérapeutiques tels les patchs cardiaques, qui peuvent être bioimprimés. Or, une des limitations actuelles de l’utilisation d’une telle stratégie est la vascularisation de tissu bioimprimés. Dans cette étude, la bioimpression 3D a été utilisée afin de bioimprimer des patchs perfusables de gélatine méthacrylate (GelMA) à utiliser potentiellement pour le tissu cardiaque. Il a été possible de développer une bioencre pouvant être utilisée pour une application dans le tissu cardiaque, d’évaluer l’imprimabilité de l’encre et de bioimprimer de patchs standards et perfusables. Pour ce faire, GelMA a été synthétisé et les propriétés mécaniques ont été évaluées pour finalement sélectionner une encre de 10 % GelMA, ayant un module de Young approprié pour le tissu cardiaque, de 23,7±5,1 kPa. Par la suite, les processus d’impression, standard et coaxial, de patchs standards et perfusables ont pu être optimisés. Finalement, des patchs perfusables de GelMA 10% et gélatine 2% ont pu être imprimés avec une viabilité cellulaire élevée, jusqu’à 79,7±8,7 % et 83,5±5,7 % obtenue aux jours 1 et 7 de culture respectivement, avec des fibroblastes 3T3. La présence de canaux vides et la perfusabilité des patchs démontrent le potentiel de cette méthode pour éventuellement bioimprimer des patchs cardiaques vascularisés épais. / Cardiovascular diseases are a leading cause of death worldwide. Myocardial infarction captures a significant segment of this population, and the end-stage myocardial infarction can only be treated by heart transplantation. However, due to the scarcity donors, tissue engineering has been considered as an alternative solution. Tissue engineering allows the development of tissues and therapeutic implants such as cardiac patches. However, one of the main hurdles in the use of such a strategy is the vascularization of bioprinted tissue. In this study, 3D bioprinting was used to bioprint perfusable gelatin methacrylate (GelMA) patches for a potential use in cardiac tissue. This work consists in the development of a bioink that can be used for the cardiac tissue, the evaluation of the printability of the ink, and the final bioprinting of standard and perfusable patches. For this purpose, GelMA was synthesized and a final concentration of 10 % was selected as it showed an appropriate Young's modulus for cardiac tissue, of 23.7±5.1 kPa, while maintaining high biocompatibility. Subsequently, the printing process of standard and perfusable patches could be optimized with the use of GelMA and gelatin inks. Finally, 10% GelMA and 2% gelatin vascularized patches could be printed with high cell viability, of up to 79,7±8,7 % and 83,5±5,7 % on days 1 and 7 of culture respectively for 3T3 fibroblasts. Additionally, the presence of hollow channels of the perfusable patches demonstrates the potential of this method to be eventually applied to the bioprinting of thick vascularized cardiac patches.