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Cardiac progenitor cells (CPCs) are abundant in the embryonic heart and have hallmark features which include a rapid rate of cell division and the ability to differentiate into mature heart muscle cells (cardiomyocytes). Based on these features, CPCs are considered an attractive candidate cell type for transplantation therapies which aim to replenish the diseased heart muscle tissue (myocardium) with new muscle forming cells. A better understanding of how pharmacological drugs and endogenous hormones/signalling molecules modulate the balance between proliferation and differentiation of CPCs could be used to develop more effective cell based therapies for myocardial repair. Furthermore, this information could provide valuable new insight into molecular mechanisms regulating normal cardiogenesis during the embryonic period. The specific aims of the present study were to characterize the effects of the Ca2+ channel blocking drug nifedipine and the endogenous hormone/paracrine factor atrial natriuretic peptide (ANP) on CPC proliferation and differentiation. Results showed that primary cultured CPCs, isolated from the ventricles of embryonic day (E) 11.5 mouse embryos, underwent a reduction in cell cycle activity following exposure to nifedipine. Furthermore, systemic administration of nifedipine to adult mice receiving transplanted E11.5 ventricular cells (containing CPCs) was associated with smaller graft sizes compared to control animals that did not receive the drug. Results from the present study also demonstrated that ANP receptor mediated signalling systems are biologically active in E11.5 ventricular cells and have an antiproliferative effect on cultured E11.5 CPCs. Moreover, preliminary data provided evidence that genetic ablation of the ANP high affinity receptor (NPRA) may be associated with impaired development of the ventricular cardiac conduction system. Collectively, work from this thesis provides evidence that interactions between transplanted cells and pharmacological drugs could have a significant impact on the effectiveness of cell based therapies and that ANP signalling systems may play a critical role in cardiac ontogeny by regulating the balance between CPC proliferation and differentiation.

Cardiovascular disease is the leading cause of death in the United States. Millions of patients suffer each year from endothelial dysfunction and/or debilitating myocardial damage resulting in decreased quality of life and increased risk of death or disablement. Current pharmacological approaches are only partly effective at treating cardiovascular disease, and hence, better strategies are needed to provide significant improvements in treatment options. Cardiac stem/progenitor cells have the potential to regenerate myocardial tissue and repair damaged heart muscle. There are many different types of cardiac progenitor cells, and each may have certain unique properties and characteristics that would likely be useful for particular clinical applications. A current challenge in the field is to identify, isolate, and test specific cardiac stem/progenitor cell populations for their ability to repair/regenerate myocardial tissue. Our laboratory has discovered a new type of cardiac progenitor cell that expresses the enzyme, Phenylethanolamine-n-methyltransferase (Pnmt). My initial studies focused on identification of Pnmt+ cells based on knock-in of a nuclear-localized Enhanced Green Fluorescent Protein (nEGFP) reporter gene into exon 1 of the Pnmt gene in a stable recombinant Pnmt-nEGFP mouse embryonic stem cell (mESC) line. These cells were differentiated into cardiomyocytes, and I identified nEGFP+ cells using fluorescence, immunofluorescence, and phase-contrast microscopy techniques. Our results showed that only about 0.025% ( 1 per 4000) of the cardiac-differentiating stem cells expressed the nEGFP+ marker. Because of the relative rarity of these cells, optimization of isolation methods proved initially challenging. To overcome this technical barrier, I used a surrogate cell culture system to establish the methods of isolation based on expression of either a fluorescent cell marker (EGFP), or a unique cell surface receptor represented by an inactivated (truncated) version of the human low-affinity nerve growth factor receptor (LNGFR). Plasmid DNA containing these reporter genes was transiently transfected into a permissive cell line (RS1), and reporter gene expression was used to identify and isolate transfected from non-transfected cells using either Fluorescence-Activated Cell Sorting (FACS) or Magnetic-Activated Cell Sorting (MACS) methods. The main objective of the study was to establish the isolation techniques based on the expression of reporter genes (EGFP and LNGFR) in RS1 cells. Following transfection, EGFP+ cells were successfully isolated via FACS as verified by flow cytometric and microscopic analyses, which showed that approximately 96% of the isolated cells were indeed EGFP+. Despite the relative purity of the isolated cell population, however, their viability in culture following FACS was substantially compromised ( 50% attrition). In contrast, MACS enabled efficient isolation of LNGFR+ cells, and the vast majority of these ( 90%) retained viability in culture following MACS. The LNGFR expression was verified using RT-PCR. Further, MACS methods enabled isolation of marked cells in about 5-7 mins, whereas it took 2-4 hours to using FACS to perform similar isolations from the same amount of starting material (10^6 cells). In addition, MACS is a more economical method in that it does not require the use of an expensive laser-based instrument to perform the sorting. These results suggest that MACS was a more efficient, gentle, and feasible technique than FACS for isolation of reporter-tagged mammalian cells. Consequently, future studies aimed at isolation of Pnmt+ cardiac progenitor cells will thus primarily focus on MACS methods.

The myocardial infarction (MI) is one of the leading causes of death in the world today. Accumulated atherosclerotic plaque occluding cardiac blood vessels results in a lack of oxygen supply to parts of the heart, and consequentially the death cardiomyocytes. The damaged area is replaced by scar tissue because of the heart’s insufficient regenerative capability, and the contraction property of the post-MI heart is therefore compromised. The recent findings of an endogenous cardiac progenitor cell (CPC) population gives hope for the establishment of new methods for medical treatments of the post-MI heart. Compared to other stem/progenitor cell sources, the CPCs are committed to a cardiac fate which places them in the forefront of interesting cell sources for regenerative treatments. In this thesis, the distribution of stem cell antigen 1 (Sca-1) positive CPCs in the healthy mouse myocardium, as well as the healthy and post-MI rat left ventricle was determined and compared to the total amount of nuclei. An immunohistochemistry protocol for the detection of Sca-1+ cells was established, and the number of Sca-1+ cells and the total number of nuclei in the different mouse and rat tissue samples were counted using laser scanning cytometry (LSC). The results could conclude a significantly higher distribution of Sca-1+ cells in the mouse atrium compared to the mouse ventricle, and a significantly higher distribution of Sca-1+ cells in the 8 days post-MI rat left ventricle compared to the healthy rat left ventricle. Furthermore, a heterogeneous distribution within the 8 days post-MI rat left ventricle was observed.

Heart failure, predominately caused by myocardial infarction (MI), is the leading cause of death in the United States. Currently the only treatment for heart failure is cardiac transplantation, but studies show that progenitor cell, biomaterial, or combined therapies have improved cardiac function post-MI. The endogenous environment of CPCs is drastically different from commonly used culture conditions. Further the endogenous environment changes with age and disease state. We evaluated the behavior of CPCs cultured on a naturally-derived, cardiac extracellular matrix (cECM) as compared to the standard culture coating collagen I, that also mimics fibrotic tissue. In this study, CPCs cultured on cECM had improved cell numbers and cardiomyogenic maturation. However, the microenvironmental cues responsible for stimulating CPC activation are largely unknown. During development, aging and disease the myocardium changes in matrix composition and stiffness exposing endogenous cells to a wide variety of stimuli. In a combinatorial study, we evaluated the effect of cyclic strain and extracellular matrix composition on CPC behavior. The response of CPCs to signals from the microenvironment is complex, with more matrix-dependency observed at lower strains. Alignment, cell division and paracrine signaling are extracellular matrix and strain dependent. Extracellular matrix conditions affect CPC maturation and calcium signaling. Mechanotransduction pathways, including focal adhesion kinase and extracellular signal-regulated kinase, are activated through adhesion and maintained under cyclic strain. Insights from this work will advance pragmatic cell therapy attempts to regenerate healthy myocardium post-MI.

Nos études ont démontrées que la formation de la cicatrice et la guérison sont associées avec l’apparition de cellules de type myocytes cardiaques nestine(+) dans la région péri-infarcie. Présentement, l’étude examine le mécanisme, tel que l’hypoxie ou les hormones neuronales, possiblement impliqué dans leur recrutement et de dévoiler leur origine cellulaire. La présence de ces cellules a été détectée dans les coeurs infarcies d’une semaine et maintenue après neuf mois suite à une sujétion coronaire complète. Aussi, ces cellules de type myocytes cardiaques nestine(+) ont été observées dans le coeur infarci humain. L’hypoxie représente un événement prédominant suite à un infarctus de myocarde, mais l’exposition des rats normaux à un environnement hypoxique n’a pas pu promouvoir l’apparition de ces cellules. Autrement, l’infusion de l’agoniste -adrénergique non-sélectif isoprotérénol (ISO) dans les rats adultes Sprague-Dawley a augmenté la protéine nestine dans le ventricule gauche et a été associé avec la réapparition de cellules de type myocytes cardiaques nestine(+). Cela représente possiblement un effet secondaire suite à la nécrose des myocytes cardiaques par l’administration d’isoprotérénol. Dernièrement, on a identifié une sous-population de cellules nestine(+) dans le coeur normal du rat qui co-exprime les marqueurs de cellules cardiaques progénitrices Nkx-2.5 et GATA-4. Cette sous-population de cellules nestine/Nkx-2.5/GATA-4 pourrait représenter des substrats cellulaires qui puissent se différentier en cellules de type myocytes cardiaques nestine(+) suite à une ischémie. Mots clés: nestine, isoprotérénol, nécrose, cellule souche, cellule progénitrice, myocyte cardiaque / Studies from our lab demonstrated that scar formation and healing was associated with the appearance of nestin(+) cardiac myocyte-like cells predominantly at the peri-infarct region. The focus of the present study was to identify the underlying mechanism(s) (e.g. hypoxia, neurohormones) implicated in their recruitment and their cellular origin. The presence of these cells was detected as early as 1-week post-myocardial infarction (MI) and persisted 9 months after complete coronary artery ligation. Furthermore, nestin(+) cardiac myocyte-like cells were also detected in the infarcted human heart. Hypoxia represents a predominant stimulus following MI, however the exposure of normal rats to a hypoxic environment failed to promote the re-appearance of nestin(+) cardiac myocyte-like cells. By contrast, the infusion of the non-selective -adrenergic agonist isoproterenol (ISO) in the normal adult Sprague-Dawley rat increased nestin expression in the left ventricle and was associated with the reappearance of nestin(+) cardiac myocyte-like cells. However, the reappearance of nestin(+) cardiac myocyte-like cells may not represent a direct effect but was apparently secondary to cardiac myocyte necrosis mediated by isoproterenol. Lastly, we identified a subpopulation of nestin-immunoreactive cells in the normal rat heart that coexpressed cardiac progenitor cell markers Nkx-2.5 and GATA-4. This subpopulation of nestin/Nkx-2.5/GATA-4 cells may represent the progenitor pool that differentiates to a nestin(+) cardiac myocyte-like cell following an ischemic insult. Key words: nestin, isoproterenol, cardiac myocyte, cardiac progenitor, necrosis

Cardiovascular diseases are the leading cause of death throughout the world and various estimates predict that heart disease will remain the number one killer in the world. Pharmacotherapies have not shown significant long term survival benefits to the patients, therefore alternate therapeutic strategies such as bioactive agent delivery and cell therapy based approaches are being investigated. One of the major causes of heart failure is the disease progression after an ischemic event and any successful therapy will be needed over the course of several days/weeks. Oxidative stress is greatly increased in the myocardium following infarction. This plays a significant role in cardiac disease progression and it has also been implicated in the failure of implanted cell therapy. Therefore, reducing oxidative stress in damaged tissue using antioxidants may have broad clinical implications for both the treatment of cardiac dysfunction and for cardiac regeneration applications. This dissertation work examines the effect of sustained delivery of endogenous antioxidant superoxide dismutase (SOD) to the rat myocardium following ischemia/reperfusion (IR) using polyketal polymers as drug carriers. The second major objective of this dissertation is to examine the effects of oxidative stress on cardiac progenitor cells - a promising endogenous adult stem cell in cardiac cell therapy applications

Nos études ont démontrées que la formation de la cicatrice et la guérison sont associées avec l’apparition de cellules de type myocytes cardiaques nestine(+) dans la région péri-infarcie. Présentement, l’étude examine le mécanisme, tel que l’hypoxie ou les hormones neuronales, possiblement impliqué dans leur recrutement et de dévoiler leur origine cellulaire. La présence de ces cellules a été détectée dans les coeurs infarcies d’une semaine et maintenue après neuf mois suite à une sujétion coronaire complète. Aussi, ces cellules de type myocytes cardiaques nestine(+) ont été observées dans le coeur infarci humain. L’hypoxie représente un événement prédominant suite à un infarctus de myocarde, mais l’exposition des rats normaux à un environnement hypoxique n’a pas pu promouvoir l’apparition de ces cellules. Autrement, l’infusion de l’agoniste -adrénergique non-sélectif isoprotérénol (ISO) dans les rats adultes Sprague-Dawley a augmenté la protéine nestine dans le ventricule gauche et a été associé avec la réapparition de cellules de type myocytes cardiaques nestine(+). Cela représente possiblement un effet secondaire suite à la nécrose des myocytes cardiaques par l’administration d’isoprotérénol. Dernièrement, on a identifié une sous-population de cellules nestine(+) dans le coeur normal du rat qui co-exprime les marqueurs de cellules cardiaques progénitrices Nkx-2.5 et GATA-4. Cette sous-population de cellules nestine/Nkx-2.5/GATA-4 pourrait représenter des substrats cellulaires qui puissent se différentier en cellules de type myocytes cardiaques nestine(+) suite à une ischémie. Mots clés: nestine, isoprotérénol, nécrose, cellule souche, cellule progénitrice, myocyte cardiaque / Studies from our lab demonstrated that scar formation and healing was associated with the appearance of nestin(+) cardiac myocyte-like cells predominantly at the peri-infarct region. The focus of the present study was to identify the underlying mechanism(s) (e.g. hypoxia, neurohormones) implicated in their recruitment and their cellular origin. The presence of these cells was detected as early as 1-week post-myocardial infarction (MI) and persisted 9 months after complete coronary artery ligation. Furthermore, nestin(+) cardiac myocyte-like cells were also detected in the infarcted human heart. Hypoxia represents a predominant stimulus following MI, however the exposure of normal rats to a hypoxic environment failed to promote the re-appearance of nestin(+) cardiac myocyte-like cells. By contrast, the infusion of the non-selective -adrenergic agonist isoproterenol (ISO) in the normal adult Sprague-Dawley rat increased nestin expression in the left ventricle and was associated with the reappearance of nestin(+) cardiac myocyte-like cells. However, the reappearance of nestin(+) cardiac myocyte-like cells may not represent a direct effect but was apparently secondary to cardiac myocyte necrosis mediated by isoproterenol. Lastly, we identified a subpopulation of nestin-immunoreactive cells in the normal rat heart that coexpressed cardiac progenitor cell markers Nkx-2.5 and GATA-4. This subpopulation of nestin/Nkx-2.5/GATA-4 cells may represent the progenitor pool that differentiates to a nestin(+) cardiac myocyte-like cell following an ischemic insult. Key words: nestin, isoproterenol, cardiac myocyte, cardiac progenitor, necrosis

Une partie du cœur est formée à partir des cellules progénitrices du second champ cardiaque, qui permettent une élongation rapide du tube cardiaque. Des défauts dans le développement de ces cellules entrainent des malformations cardiaques congénitales. Ces cellules sont localisées dans le péricarde dorsal au sein du mésoderme pharyngé. Mon travail de thèse a permis de démontrer pour la première fois que ces cellules sont épithéliales et polarisées, et qu’elles forment des filopodes dynamiques du côté basal. La délétion du facteur de transcription Tbx1 perturbe la polarité des cellules et la formation des filopodes, et augmente le niveau de la protéine apicale aPKCζ. Le traitement avec un activateur de aPKCζ montre le lien entre l’intégrité épithéliale, la polarité et la formation des filopodes, et l’état progéniteur des cellules. J’ai également analysé la polarité planaire dans l’épithélium, et montrais que les cellules sont anisotropiques, étirées et allongées en direction du pole artériel. Cet étirement crée une tension orientée, révélée par une accumulation polarisée d’actomyosine, jouant le rôle de rétrocontrôle négatif. En absence d‘élongation du tube cardiaque cette tension orientée est absente. Nous avons identifié une région postérieure de l’épithélium où se trouvent une tension et une prolifération élevées, ainsi qu’une forte activité YAP/TAZ qui jouerait le rôle de relai entre tension et prolifération. La tension orientée oriente les divisions cellulaires et oriente ainsi la croissance du tissu, promouvant l’addition des cellules au pole artériel. La biomécanique des cellules du second champ cardiaque semble ainsi un moteur important pour l’élongation du cœur. / A major part of the heart is formed by progenitor cells called the second heart field, that contribute to rapid elongation of the heart tube. Defects in second heart field development leads to congenital heart malformations. Second heart field cells are localised in pharyngeal mesoderm in the dorsal pericardial wall. This study focuses on the epithelial properties of second heart field cells and first shows that these progenitors in the dorsal pericardial wall are epithelial and polarised, and form dynamic basal filopodia. Deletion of the transcription factor Tbx1 perturbs epithelial polarity and filopodia formation and upregulates the apical determinant aPKCζ. Treatment with an activator of aPKCζ reveals that epithelial integrity, polarity and basal filopodia are coupled to the progenitor status of second heart field cells. Next we evaluated planar polarity of second heart field cells in the dorsal pericardial wall. Cells are anisotropic, being stretched and elongated on an axis directed towards the arterial pole. This stretch results in oriented epithelial tension revealed by polarised actomyosin accumulation through a negative feedback loop. In the absence of cell addition to the cardiac poles oriented tension is absent. We identified a posterior region in the epithelium with high tension, elevated proliferation and a high level of active YAP/TAZ that may act as relay between tension and proliferation. Oriented tension orients the axis of cell division and the growth of the tissue on an axis toward the arterial pole, further promoting addition of the tissue to the pole. Biomechanical feedback may thus be an important driver of heart tube elongation.

Les récepteurs couplés aux protéines G représentent la plus grande famille de récepteurs membranaires. Parmi eux, nous avons choisi d’étudier deux récepteurs apparentés : les récepteurs de la prokinéticine 1 et 2. Ces deux récepteurs ont pour ligands des hormones de nature peptidique, divisées en deux sous-groupes : les prokinéticines 1 et 2. Ces deux prokinéticines sont impliquées dans plusieurs processus physiologiques en se liant à leurs récepteurs PKR1 et PKR2. Il a été récemment montré que la prokinéticine 2 pouvait stimuler la prolifération et la différenciation des cellules souches progénitrices cardiaques, via les récepteurs PKR1 et PKR2. Il a également été reporté que l’activation de PKR1 protège les cardiomyocytes et les cellules progénitrices cardiaques de l’apoptose. Afin d’étudier ces effets nous avons synthétisé des agonistes non-peptidiques du récepteur PKR1. Nous avons donc poursuivi les études de pharmacomodulation d’une première famille de composés et développé une seconde famille d’agonistes potentiels originaux, déterminée par des études de modélisation moléculaire. Une sonde fluorescente a été synthétisée afin d’évaluer la liaison de nouveaux composés. Au cours de ces travaux nous avons découvert une nouvelle réaction multi-composante permettant la synthèse d’un composé dihydropyrrole polyfonctionnel. Nous nous sommes alors intéressés à son mécanisme et à sa limitation chimique dans le but de former de nouveaux hétérocycles fonctionnalisés. / The G protein-coupled receptors represent the largest familly of membrane receptors. Among them,we choose to study two related receptors: prokineticin receptors 1 and 2. These two receptors have peptidic hormone ligands, divided in two sub-groups: prokineticins 1 and 2. Both prokineticins are involved in many physiological processes by binding to their receptors PKR1 and PKR2. It has recently been shown that prokineticin 2 could stimulate proliferation and differentiation of cardiac progenitor cells. It was also reported that activation of PKR1 protects cardiomyocytes and cardiac progenitor cells from apoptosis. To investigate these effects we synthesized non-peptidic receptor PKR1. We continued pharmacodulation studies of a first familly of compounds and developped a second familly of original potential agonists, determined by molecular modeling studies. A fluorescent probe was synthesized to access the binding of novel compounds. During this work we discovered a new multi-component reaction for the synthesis of a polyfunctional dihydrpyrrol compound. We then interested in the mechanism and its chemical limitation in order to form new functionalized heterocycles.