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of the dissertation Impact of minimally invasive approach on pulmonary function in patients undergoing aortic valve replacement MUDr. Ján Gofus The most common minimally invasive approach to aortic valve replacement is upper hemisternotomy, which has been implemented at our department, as well. Preserving the lower half of thoracic cage could lead to lower postoperative drop of pulmonary function, apart from other benefits. Nevertheless, publications on this topic are insufficient and controversial. Our aim was to perform a prospective randomized trial comparing upper hemisternotomy with standard (median) sternotomy in terms of pulmonary function changes perioperatively. We also added a novel exercise tolerance test, one-minute sit-to-stand test, and a quality of life evaluation to the study. We included patients indicated for elective isolated aortic valve replacement with bioprosthesis who were older than 65 years, signed informed consent, and in which both surgical approaches were technically feasible. Exclusion criteria were re-do surgery and concomitant cardiac surgery. Patients were randomized to minimally invasive and standard group in 1:1 ratio. On the day of admission, on the 7th postoperative day and 3 months postoperatively, the patients underwent pulmonary function testing and one-minute...

Les maladies valvulaires constituent une cause importante de morbidité et de mortalité. Dans les pays industrialisés, l’insuffisance mitrale et la sténose aortique sont les pathologies valvulaires les plus fréquentes et leur prévalence augmentent avec l’âge. Étant donné l’augmentation de l’espérance de vie dans ces pays, la prévalence des valvulopathies dégénératives deviendra plus importante et aura un impact non négligeable sur la santé publique. Les avancées en chirurgie cardiaque ainsi que les nouvelles percées en cardiologie interventionnelle ont modifié considérablement la prise en charge des patients avec des valvulopathies en offrant des approches minimalement invasives, surtout pour les patients à haut risque chirurgical. Dans le cadre de ce mémoire, deux études rétrospectives de cohorte ont été réalisées. La première consiste à comparer les résultats postopératoires et sur trois ans de la chirurgie conventionnelle par rapport à la procédure transcathéter MitraClip chez 259 patients avec une insuffisance mitrale ischémique sévère. La deuxième étude compare les résultats postopératoires de trois approches de remplacement de la valve aortique, soit la sternotomie, la ministernotomie et la minithoracotomie. La première étude permet de conclure que la procédure MitraClip a un taux de mortalité postopératoire et sur 3 ans inférieur à celui de la chirurgie mais qu’elle est associée à un plus haut taux de récurrence de l’insuffisance mitrale après 3 ans. La deuxième étude démontre que les deux approches minimalement invasives, la ministernotomie et la mini-thoracotomie, ont un taux équivalent de mortalité intra-hospitalier à la sternotomie. La mini-thoracotomie est associée à moins de saignement périopératoire et moins de douleur au repos que la sternotomie. En conclusion, les approches minimalement invasives offrent une excellente alternative à la chirurgie conventionnelle dans le traitement de la maladie valvulaire. Les bénéfices cliniques sont d’autant plus évidents lorsque les patients sont adéquatement sélectionnés; d’où l’importance d’une ‘Heart Team’ qui collabore pour une meilleure prise en charge des patients. / Valvular heart disease is an important cause of morbidity and mortality. In western countries, mitral regurgitation and aortic stenosis are the most frequent valvular pathologies and their prevalence increases with age. With the increase in life expectancy in these countries, the prevalence of degenerative valve disease will increase with a significant burden on healthcare systems. Advances in cardiac surgery as well as new breakthroughs in interventional cardiology have considerably modified the management of patients with valvular disease, by offering minimally invasive approaches, especially for patients at high surgical risk. In this thesis, two retrospective cohort studies were carried out. The first compares the postoperative and 3 years outcomes of mitral valve surgery vs MitraClip, a transcatheter procedure, in 259 patients with severe ischemic mitral regurgitation. The second study compares the postoperative results of two minimally invasive techniques (ministernotomy and minithoracotomy) for aortic valve replacement to conventional sternotomy. In the first study, MitraClip procedure had lower postoperative and 3-year mortality rate than surgery, but it was associated with higher recurrence rate of mitral regurgitation after 3 years. The second study showed that the two minimally invasive approaches had similar intrahospital mortality rate to sternotomy. Minithoracotomy was associated with less perioperative bleeding and less pain at rest than sternotomy. In conclusion, minimally invasive approaches offer an excellent alternative to conventional surgery in the treatment of valvular disease. The clinical benefits are more highlighted when patients are properly selected; hence the importance of a "Heart Team" that collaborates for better patient care.

À l’heure actuelle, plusieurs centres de chirurgie cardiaque ont noté une augmentation du nombre de patients âgés orientés pour une chirurgie. Les baisses majeures de la natalité et de la mortalité dans tous les groupes d’âge ont contribué au vieillissement progressif des populations des pays industrialisés. Il est bien connu que la prévalence des pathologies cardiovasculaires augmente avec l’âge et fait en sorte que ce groupe de patients âgés soit à haut risque opératoire. Trois études ont été réalisées dans le cadre de ce travail dédié aux deux types de chirurgie les plus fréquemment pratiquées pour ce groupe de population : la chirurgie de la valve aortique et la chirurgie de revascularisation coronarienne. Le substitut valvulaire aortique idéal pour une personne âgée considérée à risque opératoire élevé suscite toujours un débat. Depuis quelques années, avec les progrès technologiques en chirurgie cardiaque, les valves sans suture (V-SS) en général et la Perceval en particulier, constituent une excellente alternative à la prothèse standard chez ce sous-groupe de patients. Tout en diminuant le temps opératoire, la Perceval ne compromet ni la qualité ni la sécurité de l’acte chirurgical et elle présente des bénéfices cliniques et hémodynamiques aussi bien à court et à long terme. Pour le démontrer nous vous présentons les résultats d’une étude de cohorte regroupant 215 patients consécutifs qui a permis d’évaluer l’efficacité et la durabilité de la Perceval. Notre étude se veut l’appui qui soutiendra la place et l’apport de la Perceval dans l’arsenal thérapeutique du remplacement valvulaire aortique (RVA). Comme toutes les bioprothèses, la Perceval est soumise à un risque faible, mais constant à long terme de dégénérescence structurale et qui pourrait éventuellement nécessiter une réintervention. Pour clarifier cette problématique, nous vous présentons une étude descriptive rapportant notre expérience dans la prise en charge de la détérioration structurale de la prothèse Perceval. De plus, cette étude a permis de mettre en lumière la complémentarité parfois inattendue (dans des conditions urgentes) de ces approches thérapeutiques ( TAVI et Perceval) chez les patients à risque élevé. De nos jours, un certain pourcentage des malades orientés vers une revascularisation chirurgicale présente une pathologie coronarienne assez diffuse et complexe qui peut compromettre la revascularisation coronarienne souhaitable. A cet effet, l’endartériectomie coronarienne (EC), une ancienne technique largement utilisée auparavant, puis délaissée par la suite, mérite d’être reconsidérée chez ces patients. La troisième étude présentée dans ce mémoire rapporte les résultats d’une cohorte de 147 patients atteints de maladie coronarienne diffuse. En plus d’être sécuritaire, notre étude documente la perméabilité à court et à moyen terme d’un sous groupe de patient étudié avec un angioscanner coronaire. / Currently, several cardiac surgery centers have noted an increase in the number of elderly patients referred for surgery. The major decline in birth rates and deaths in all age groups has contributed to the gradual aging of populations in industrialized countries. It is well known that the prevalence of cardiovascular pathologies increases with age and puts this group of elderly patients at high risk for surgery. Three studies were carried out as part of this work dedicated to the two types of surgery most frequently performed for this population group: aortic valve surgery and coronary revascularization surgery. The ideal aortic valve substitute for an elderly person considered to be at high operative risk is still a subject of debate. In recent years, with technological advances in cardiac surgery, sutureless valves (V-SS) in general and the Perceval in particular have been an excellent alternative to the standard prosthesis in this subgroup of patients. While reducing operating time, Perceval does not compromise the quality or safety of the surgical procedure and it offers clinical and hemodynamic benefits both in the short and long term. To demonstrate this, we present to you the results of a cohort study involving 215 consecutive patients which made it possible to evaluate the effectiveness and durability of Perceval. Our study is intended to support the place and contribution of Perceval in the therapeutic arsenal of aortic valve replacement (AVR). Like all bioprostheses, Perceval is subject to a low, but constant long-term risk of structural degeneration which may eventually require reoperation. To clarify this problem, we present to you a descriptive study reporting our experience in the management of structural deterioration of the Perceval prosthesis. In addition, this study shed light on the sometimes unexpected complementarity (in urgent conditions) of these therapeutic approaches (TAVI and Perceval) in high-risk patients. Nowadays, a certain percentage of patients referred for surgical revascularization present a fairly diffuse and complex coronary pathology which can compromise the desirable coronary revascularization. To this end, coronary endarterectomy (CE), an old technique widely used before, then abandoned thereafter, deserves to be reconsidered in these patients. The third study presented in this thesis reports the results of a cohort of 147 patients with diffuse coronary artery disease. In addition to being safe, our study documents the short- and medium-term patency of a subgroup of patients studied with a coronary CT angiography.

réalisé en cotutelle avec le Dr. Marie Kmita et Dr. Marco Horb / Chez l’humain, les maladies congénitales cardiaques (MCC) sont présentes chez 3-4% des nouveaux nés et sont une cause importante de mortalité infantile et de morbidité dans le monde. La majorité des MCCs implique les valves et les septums, qui proviennent des cellules endocardiques. Les valves aortiques bicuspides (VAB) sont la MCC la plus fréquente chez l’humain, avec un taux estimé de 1-2% dans la population. Cependant, les gènes et les mécanismes moléculaires qui causent cette malformation demeurent obscures. Le facteur de transcription GATA5 est exprimé dans les cellules et les coussins endocardiques de façon transitoire durant la septation et la formation des compartiments cardiaques. Chez le poisson zèbre, des mutations dans le gène Gata5 causent des malformations cardiaques sévères incluant l’absence de cellules endocardiques. In vitro, l’inhibition de Gata5 bloque la différentiation endocardique. Ces études suggéraient donc un rôle important de GATA5 dans la formation du cœur. Dans le cadre de ce projet de doctorat, nous avons analysé le rôle de GATA5 dans le développement du cœur en produisant des lignées de souris chez lesquelles le gène Gata5 était inactif soit dans toutes les cellules ou uniquement dans les cellules endocardiques. Les souris possédant 2 allèles mutées du gène Gata5 étaient viables mais plus de 26% des souris Gata5-/- ont développé des VABs. Par ailleurs, une incidence similaire de VABs a été obtenue chez les souris ayant une délétion spécifique de Gata5 des cellules endocardiques, obtenue en croisant les souris Gata5WT/Flox avec les souris transgéniques Tie2-Cre. Sur le plan mécanistique, une réduction significative de JAG1, un corécepteur pour Notch1, ainsi qu’une augmentation marquée de Rbj un répresseur de cette voie, ont été détectés chez les souris Gata5-/- et Tie2- cre+;Gata5Flox/Flox, suggérant qu’une dérégulation de la voie Notch dans les cellules endocardiques puisse être la cause des VABs. Ces résultats démontrent l’importance de GATA5 pour le développement endocardique et la formation de la valve aortique. De plus, ils identifient GATA5 comme gène candidat de MCCs chez l’humain. Environ 12-14% des MCCs sont causés par le développement anormal de la voie de chasse, menant aux malformations telles que la transposition des grandes artères, la tétralogie de Fallot ou le syndrome du ventricule droit à double issue. Des mutations dans Gata4 et Gata6 sont associés à des défauts de la voie de chasse, dans plusieurs espèces incluant l’humain. Nous avons examiné si GATA5 interagit avec GATA4 ou GATA6 dans le développement de la voie de chasse. Alors que les souris hétérozygotes pour Gata5, Gata4 ou Gata6 ont des défauts cardiaques subtiles et sont viables, les embryons Gata4+/-Gata5+/- et Gata5+/-Gata6+/- démontrent une létalité embryonnaire et périnatale due à des défauts cardiaques, tel qu’un ventricule droit à double issue et des défauts de septation ventriculaire. Ces résultats indiquent l'importance des interactions génétiques entre GATA5 et les autres facteurs GATA pour la rotation et l’alignement de la voie de chasse au cours du développement cardiaque et soulèvent la possibilité que des changements subtiles de l'activité de 2 facteurs GATA puissent mener à des MCCs chez l'humain. / Congenital heart defect (CHD) in humans occur in 3-4% of live birth and is a major cause of infant mortality and morbidity in the world. The majority of CHD involves the valves and septa, which originate from endocardial cells. Bicuspid aortic valve (BAV) is the most common CHD in humans with an estimated rate of 1-2% in the population. However, very few genes have been linked to this defect and the mechanisms underlying BAV formation remain undefined. GATA5, a member of the GATA family of transcription factors, is expressed in a spatial and temporal manner in the developing heart where it is predominantly found in endocardial cells and endocardial cushions (ECs) of the outflow tract (OFT) and atrioventricular canal between E9.5-E12.5 in the mouse. Mutations in the Gata5 gene in zebrafish (faust mutants) cause cardia bifida and lead to endocardial cell depletion. In vitro studies using antisense mRNA against Gata5 revealed a critical role for this gene in differentiation of endocardial cells. In the context of the present doctoral research project, we investigated the role of GATA5 in mammalian heart development by generating a mouse line with a null Gata5 allele. Gata5 null mice are viable but over 26% of them developed BAVs. Endocardial specific deletion of Gata5 obtained by crossing mice with floxed (Flox) Gata5 alleles with Tie2-cre transgenic mice resulted in a similar incidence of BAVs. RNA profiling revealed that Jag-1, a co-receptor for Notch1, is significantly downregulated in both Gata5 null and Tie2-cre+;Gata5Flox/Flox mice, suggesting that disruption of Notch signaling in endocardial cells may be the underlying mechanism of disease. These findings reveal an important function for GATA5 in endocardial cell development and aortic valve formation and identify GATA5 as an important candidate CHD causing gene. Abnormal development of the OFT accounts for about 12-14% of all CHDs, leading to malformations such as persistent truncus arteriosus (PTA), tetralogy of Fallot (TOF), double outlet right ventricle (DORV) and transposition of the great arteries (TGA). Both GATA4 and GATA6 play important role in OFT development. We tested whether GATA5 might interact genetically with GATA4 and GATA6 for proper heart morphogenesis. We found that, whereas mice lacking a single copy of Gata5, Gata4 or Gata6 have subtle cardiac defects, the Gata4+/-Gata5+/- and Gata5+/-Gata6+/- mutant embryos show embryonic and perinatal lethality due to severe heart defects, including double outlet right ventricle and ventricular septal defects. These findings reveal the importance of genetic interactions between GATA5 and the other cardiac GATA factors in the normal rotation and patterning of the OFT during heart development in vivo. The results raise the possibility that subtle alterations in the level or activity of 2 cardiac GATA factors might lead to congenital heart disease in human.

As doenças cardíacas são a principal causa de morte em todo o mundo. Os hormônios tireoidianos desempenham um papel chave no metabolismo miocárdico e na fisiologia do sistema cardiovascular. A doença cardíaca aguda ou crônica promove uma queda sistêmica da concentração dos hormônios tireoidianos que se associa a um prognóstico pior da doença e aumento da sua mortalidade. Essa redução dos hormônios tireoidianos pode ocorrer na presença de função normal da tireóide, entidade clínica conhecida por síndrome da doença não-tireoidiana ou síndrome do enfermo eutireoideo (SEE). A participação do músculo cardíaco na patogênese da SEE é desconhecida. O entendimento do papel do músculo cardíaco na SEE é essencial para o tratamento das doenças cardíacas. Este estudo se propõe a avaliar a variação dos hormônios tireoidianos promovida pelo metabolismo cardíaco nos pacientes submetidos a cirurgias cardíacas com diferentes graus de isquemia miocárdica aguda, bem como estudar os principais mecanismos envolvidos nessa variação. Para avaliar a variação sistêmica de hormônios tireoideanos induzida pela cirurgia cardíaca com e sem circulação extracorpórea (CEC), 35 pacientes com estenose aórtica grave e doença coronariana submetidos à cirurgia com CEC e 12 pacientes submetidos à cirurgia de revascularização miocárdica sem CEC tiveram as concentrações sistêmicas dos hormônios tireoidianos dosadas no início do procedimento cirúrgico, imediatamente antes do clampeamento da aorta, 3 minutos após o desclampeamento da aorta, 6 e 24h após o procedimento. Além disso, a avaliação da participação isolada do coração foi feita pela dosagem dos hormônios tireoidianos na raiz da aorta e no seio coronário antes e após a isquemia miocárdica aguda induzida pelo clampeamento da aorta. Foram ainda quantificadas, em amostras do tecido miocárdico colhidas após a CEC, a expressão do gene das desiodades, enzimas responsáveis pela conversão dos hormônios tireoidianos nos tecidos periféricos. Essas medidas sanguíneas foram comparadas, bem como a expressão das desiodases presentes no músculo cardíaco, relacionando a sua expressão à variação transcardíaca dos hormônios tireoidianos. O estudo demonstrou uma queda significativa de 37,6% da concentração periférica de T3 associada a uma elevação de 261,6% do rT3 e manutenção das concentrações séricas de T4 livre ao longo do acompanhamento perioperatório nos três grupos. Os resultados não mostraram diferença da variação periférica dos hormônios tireoidianos entre os grupos. Nas amostras centrais, observou-se uma redução transcardíaca de 4,6% de T3 com incremento de 6,9% do rT3, sem alterações do T4 total no grupo estenose aórtica antes do início da CEC. Esse comportamento, no entanto, não foi visto nos pacientes com doença arterial coronariana antes da CEC. Após cerca de 3 minutos de reperfusão miocárdica depois do término da CEC, as variações de concentração de T3 e de rT3 entre a aorta e o seio coronário se perderam. A análise do mRNA do tecido miocárdico indicou expressão significativa da desiodase tipo III com ausência de expressão da desiodase tipo II nos três grupos, sem diferença significativa entre elas. Dessa forma, pode-se concluir que as cirurgias cardíacas com CEC ou sem CEC estão associadas ao desenvolvimento da SEE e que a intensidade desse distúrbio metabólico é similar nos três tipos de procedimento, independente da CEC. Em relação à contribuição do coração para este fenômeno, a expressão das enzimas relacionadas à síndrome no tecido cardíaco foi observada em todos os grupos estudados, mas somente o grupo estenose aórtica demonstrou variação hormonal transcardíaca pré-CEC, com a isquemia miocárdica possivelmente neutralizando esse efeito após a CEC. É possível que a isquemia crônica provavelmente devido à hipertrofia miocárdica, e não a isquemia aguda causada pela CEC, tenha uma capacidade de modificar as concentrações dos hormônios tireoidianos / Heart diseases are the main cause of death over the world and thyroid hormones are key elements in myocardial metabolism and cardiovascular physiology. In heart disease patients, low thyroid hormone levels lead to a worse prognosis and increase in the mortality, even with regular thyroid function, in a condition known as Euthyroid Sick Syndrome (ESS). There is no evidence that myocardial tissue is involved in ESS pathophysiology. The better understanding of heart role might be important to optimal treatment of heart disease. The current study aims to evaluate thyroid hormones variation induced by myocardial metabolism in patients submitted to several acute myocardial ischemic intensities and study the main mechanisms associated to this condition. To reach this objective, 35 stable severe aortic stenosis coronary artery disease submitted to in-pump cardiac surgery and 12 patients submitted to off-pump myocardial revascularization surgery were analyzed at the procedure beginning, before aortic clamping, 3 minutes after aortic cross-clamp release, six and 24h after procedure by measuring thyroid hormones concentration in systemic circulation. Therefore, cardiac metabolism was evaluated alone by the thyroid hormones concentration measurement in aortic root and coronary sinus just before and after myocardial ischemia induced by aortic clamping, as well the gene expression of thyroid hormones metabolism related enzyme in myocardial tissue samples. There was a significant 37.6% reduction in T3 systemic concentration, a 261.6% elevation in rT3 and no variation in free T4 systemic values during the observation time in three groups. However, there were no statistically differences among the groups. Central analysis showed a 4.6% significant reduction in T3 and 6.9% increase in rT3 in coronary sinus, compared to aortic root, in aortic stenosis group before cardiopulmonary bypass. The same behavior was not observed in coronary artery disease before aortic cross clamping. After cardiopulmonary bypass, no differences were seen in any group. However, Deiodinase Type III, which is responsible for the T3 concentration decrease, gene RNA-m expression was detected in all myocardial tissue biopsies, and the Deiodinase Type II, which produces T3 from T4, was absent in myocardial tissue during the heart surgery. In conclusion, in- or off-pump heart surgeries are associated to similar systemic ESS intensities and to ESS-enzyme related gene expressions in myocardial tissue. However, myocardial metabolism in aortic stenosis patients is able to change thyroid hormones concentrations, probably due to myocardial hypertrophy and chronic ischemia assault, which were no observed in coronary disease patients

Calcific aortic valve disease (CAVD) is one of the most common causes of cardiovascular disease in North America. Mechanical factors have been closely linked to the pathogenesis of CAVD and may contribute to the disease by actively regulating the mechanobiology of valve interstitial cells (VICs). Mechanical forces affect VIC function through interactions between the VIC and the extracellular matrix (ECM). Studies have shown that the transfer of mechanical stimulus during cell-ECM interaction depends on the local material properties at hierarchical length scales encompassing tissue, cell and cytoskeleton. In this thesis, biomechanical tools were developed and applied to investigate hierarchical cell-ECM interactions, using VICs and valve tissue as a model system. Four topics of critical importance to understanding VIC-ECM interactions were studied: focal biomechanical material properties of aortic valve tissue; viscoelastic properties of VICs; transduction of mechanical deformation from the ECM to the cytoskeletal network; and the impact of altered cell-ECM interactions on VIC survival. To measure focal valve tissue properties, a micropipette aspiration (MA) method was implemented and validated. It was found that nonlinear elastic properties of the top layer of a multilayered biomaterial can be estimated by MA by using a pipette with a diameter smaller than the top layer thickness. Using this approach, it was shown that the effective stiffness of the fibrosa layer is greater than that of the ventricularis layer in intact aortic valve leaflets (p<0.01). To characterize the viscoelastic properties of VICs, an inverse FE method of single cell MA was developed and compared with the analytical half-space model. It was found that inherent differences in the half-space and FE models of single cell MA yield different cell viscoelastic material parameters. However, under particular experimental conditions, the parameters estimated by the half-space model are statistically indistinguishable from those predicted by the FE model. To study strain transduction from the ECM to cytoskeleton, an improved texture correlation algorithm and a uniaxial tension release device were developed. It was found that substrate strain fully transfers to the cytoskeletal network via focal adhesions in live VICs under large strain tension release. To study the effects of cell-ECM interactions on VIC survival, two mechanical stimulus systems that can simulate the separate effects of cell contraction and cell monolayer detachment were developed. It was found that cell sheet detachment and disrupted cell-ECM signaling is likely responsible for the apoptosis of VICs grown in culture on thin collagen matrices, leading to calcification. The studies presented in this thesis refine existing biomechanical tools and provide new experimental and analytical tools with which to study cell-ECM interactions. Their application resulted in an improved understanding of hierarchical valve biomechanics, mechanotransduction, and mechanobiology.

Calcific aortic valve disease (CAVD) is one of the most common causes of cardiovascular disease in North America. Mechanical factors have been closely linked to the pathogenesis of CAVD and may contribute to the disease by actively regulating the mechanobiology of valve interstitial cells (VICs). Mechanical forces affect VIC function through interactions between the VIC and the extracellular matrix (ECM). Studies have shown that the transfer of mechanical stimulus during cell-ECM interaction depends on the local material properties at hierarchical length scales encompassing tissue, cell and cytoskeleton. In this thesis, biomechanical tools were developed and applied to investigate hierarchical cell-ECM interactions, using VICs and valve tissue as a model system. Four topics of critical importance to understanding VIC-ECM interactions were studied: focal biomechanical material properties of aortic valve tissue; viscoelastic properties of VICs; transduction of mechanical deformation from the ECM to the cytoskeletal network; and the impact of altered cell-ECM interactions on VIC survival. To measure focal valve tissue properties, a micropipette aspiration (MA) method was implemented and validated. It was found that nonlinear elastic properties of the top layer of a multilayered biomaterial can be estimated by MA by using a pipette with a diameter smaller than the top layer thickness. Using this approach, it was shown that the effective stiffness of the fibrosa layer is greater than that of the ventricularis layer in intact aortic valve leaflets (p<0.01). To characterize the viscoelastic properties of VICs, an inverse FE method of single cell MA was developed and compared with the analytical half-space model. It was found that inherent differences in the half-space and FE models of single cell MA yield different cell viscoelastic material parameters. However, under particular experimental conditions, the parameters estimated by the half-space model are statistically indistinguishable from those predicted by the FE model. To study strain transduction from the ECM to cytoskeleton, an improved texture correlation algorithm and a uniaxial tension release device were developed. It was found that substrate strain fully transfers to the cytoskeletal network via focal adhesions in live VICs under large strain tension release. To study the effects of cell-ECM interactions on VIC survival, two mechanical stimulus systems that can simulate the separate effects of cell contraction and cell monolayer detachment were developed. It was found that cell sheet detachment and disrupted cell-ECM signaling is likely responsible for the apoptosis of VICs grown in culture on thin collagen matrices, leading to calcification. The studies presented in this thesis refine existing biomechanical tools and provide new experimental and analytical tools with which to study cell-ECM interactions. Their application resulted in an improved understanding of hierarchical valve biomechanics, mechanotransduction, and mechanobiology.

Chez l’humain, les maladies congénitales cardiaques (MCC) sont présentes chez 3-4% des nouveaux nés et sont une cause importante de mortalité infantile et de morbidité dans le monde. La majorité des MCCs implique les valves et les septums, qui proviennent des cellules endocardiques. Les valves aortiques bicuspides (VAB) sont la MCC la plus fréquente chez l’humain, avec un taux estimé de 1-2% dans la population. Cependant, les gènes et les mécanismes moléculaires qui causent cette malformation demeurent obscures. Le facteur de transcription GATA5 est exprimé dans les cellules et les coussins endocardiques de façon transitoire durant la septation et la formation des compartiments cardiaques. Chez le poisson zèbre, des mutations dans le gène Gata5 causent des malformations cardiaques sévères incluant l’absence de cellules endocardiques. In vitro, l’inhibition de Gata5 bloque la différentiation endocardique. Ces études suggéraient donc un rôle important de GATA5 dans la formation du cœur. Dans le cadre de ce projet de doctorat, nous avons analysé le rôle de GATA5 dans le développement du cœur en produisant des lignées de souris chez lesquelles le gène Gata5 était inactif soit dans toutes les cellules ou uniquement dans les cellules endocardiques. Les souris possédant 2 allèles mutées du gène Gata5 étaient viables mais plus de 26% des souris Gata5-/- ont développé des VABs. Par ailleurs, une incidence similaire de VABs a été obtenue chez les souris ayant une délétion spécifique de Gata5 des cellules endocardiques, obtenue en croisant les souris Gata5WT/Flox avec les souris transgéniques Tie2-Cre. Sur le plan mécanistique, une réduction significative de JAG1, un corécepteur pour Notch1, ainsi qu’une augmentation marquée de Rbj un répresseur de cette voie, ont été détectés chez les souris Gata5-/- et Tie2- cre+;Gata5Flox/Flox, suggérant qu’une dérégulation de la voie Notch dans les cellules endocardiques puisse être la cause des VABs. Ces résultats démontrent l’importance de GATA5 pour le développement endocardique et la formation de la valve aortique. De plus, ils identifient GATA5 comme gène candidat de MCCs chez l’humain. Environ 12-14% des MCCs sont causés par le développement anormal de la voie de chasse, menant aux malformations telles que la transposition des grandes artères, la tétralogie de Fallot ou le syndrome du ventricule droit à double issue. Des mutations dans Gata4 et Gata6 sont associés à des défauts de la voie de chasse, dans plusieurs espèces incluant l’humain. Nous avons examiné si GATA5 interagit avec GATA4 ou GATA6 dans le développement de la voie de chasse. Alors que les souris hétérozygotes pour Gata5, Gata4 ou Gata6 ont des défauts cardiaques subtiles et sont viables, les embryons Gata4+/-Gata5+/- et Gata5+/-Gata6+/- démontrent une létalité embryonnaire et périnatale due à des défauts cardiaques, tel qu’un ventricule droit à double issue et des défauts de septation ventriculaire. Ces résultats indiquent l'importance des interactions génétiques entre GATA5 et les autres facteurs GATA pour la rotation et l’alignement de la voie de chasse au cours du développement cardiaque et soulèvent la possibilité que des changements subtiles de l'activité de 2 facteurs GATA puissent mener à des MCCs chez l'humain. / Congenital heart defect (CHD) in humans occur in 3-4% of live birth and is a major cause of infant mortality and morbidity in the world. The majority of CHD involves the valves and septa, which originate from endocardial cells. Bicuspid aortic valve (BAV) is the most common CHD in humans with an estimated rate of 1-2% in the population. However, very few genes have been linked to this defect and the mechanisms underlying BAV formation remain undefined. GATA5, a member of the GATA family of transcription factors, is expressed in a spatial and temporal manner in the developing heart where it is predominantly found in endocardial cells and endocardial cushions (ECs) of the outflow tract (OFT) and atrioventricular canal between E9.5-E12.5 in the mouse. Mutations in the Gata5 gene in zebrafish (faust mutants) cause cardia bifida and lead to endocardial cell depletion. In vitro studies using antisense mRNA against Gata5 revealed a critical role for this gene in differentiation of endocardial cells. In the context of the present doctoral research project, we investigated the role of GATA5 in mammalian heart development by generating a mouse line with a null Gata5 allele. Gata5 null mice are viable but over 26% of them developed BAVs. Endocardial specific deletion of Gata5 obtained by crossing mice with floxed (Flox) Gata5 alleles with Tie2-cre transgenic mice resulted in a similar incidence of BAVs. RNA profiling revealed that Jag-1, a co-receptor for Notch1, is significantly downregulated in both Gata5 null and Tie2-cre+;Gata5Flox/Flox mice, suggesting that disruption of Notch signaling in endocardial cells may be the underlying mechanism of disease. These findings reveal an important function for GATA5 in endocardial cell development and aortic valve formation and identify GATA5 as an important candidate CHD causing gene. Abnormal development of the OFT accounts for about 12-14% of all CHDs, leading to malformations such as persistent truncus arteriosus (PTA), tetralogy of Fallot (TOF), double outlet right ventricle (DORV) and transposition of the great arteries (TGA). Both GATA4 and GATA6 play important role in OFT development. We tested whether GATA5 might interact genetically with GATA4 and GATA6 for proper heart morphogenesis. We found that, whereas mice lacking a single copy of Gata5, Gata4 or Gata6 have subtle cardiac defects, the Gata4+/-Gata5+/- and Gata5+/-Gata6+/- mutant embryos show embryonic and perinatal lethality due to severe heart defects, including double outlet right ventricle and ventricular septal defects. These findings reveal the importance of genetic interactions between GATA5 and the other cardiac GATA factors in the normal rotation and patterning of the OFT during heart development in vivo. The results raise the possibility that subtle alterations in the level or activity of 2 cardiac GATA factors might lead to congenital heart disease in human. / réalisé en cotutelle avec le Dr. Marie Kmita et Dr. Marco Horb

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