Acute cold exposure increases the risk of sudden cardiac events, similarly exposure to constant light negatively affects the cardiovascular system. However, the individual effects of these factors and the effect of their combination on cardiomyocytes are not yet known. The thesis deals with the influence of a 3 day cold exposure and constant light on the expression of β-adrenergic receptors and associated G-proteins in association with apoptotic signals in the left ventricle of the Wistar rat heart. In this work apoptotic proteins BAX, BCL2, caspase 8 and important components of β-adrenergic signalization - β1, β2, G-proteins, Gas, Gi1/2 and Gi3 were determined. The relative expression of the proteins was analyzed by Western blotting. The results confirm the detrimental effect of cold and light exposure. However, the synergistic effect of these two stressors shows surprising results.

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No description available.

After myocardial infarction the innate immune response is pivotal in clearing of tissue debris as well as scar formation, but exaggerated cytokine and chemokine secretion with subsequent leukocyte infiltration also leads to further tissue damage. Here, we address the value of targeting a previously unknown a disintegrin and metalloprotease 10 (ADAM10)/CX3CL1 axis in the regulation of neutrophil recruitment early after MI. We show that myocardial ADAM10 is distinctly upregulated in myocardial biopsies from patients with ischemia-driven cardiomyopathy. Intriguingly, upon MI in mice, pharmacological ADAM10 inhibition as well as genetic cardiomycyte-specific ADAM10 deletion improves survival with markedly enhanced heart function and reduced scar size. Mechanistically, abolished ADAM10-mediated CX3CL1 ectodomain shedding leads to diminished IL-1β-dependent inflammation, reduced neutrophil bone marrow egress as well as myocardial tissue infiltration. Thus, our data shows a conceptual insight into how acute MI induces chemotactic signaling via ectodomain shedding in cardiomyocytes.

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<p dir="ltr">Embryonic development relies on the complex interplay of epigenetic regulation, timely expression of genes, signal transduction pathways, and diverse morphological changes. The heart is the first organ to form during mammalian embryonic development. The proper development of the heart is critical to supply nutrients and oxygen to other cell types of the organism. Most cells that comprise the heart originate from the mesoderm post-gastrulation. Cardiomyocytes are the predominant cell type and confer function to the heart via contractile activity. The development and proliferation of cardiomyocytes ceases shortly after birth, where cardiomyocytes only nucleate and increase in size. Consequently, cardiomyocyte insufficiency underlies most cardiovascular diseases, a leading cause of death globally.</p><p dir="ltr">Vascular endothelial zinc finger 1 (VEZF1) is a transcription factor expressed predominantly in mesoderm during development. Previous studies from our lab show that the loss of VEZF1 impairs the differentiation of embryonic stem cells into endothelial cells, a cell type derived from mesoderm. Other published studies also show that Vezf1 loss impairs cardiomyocyte growth in Zebrafish and hematopoietic cell differentiation. Our work here describes a detailed investigation of the role of Vezf1 in the differentiation of mesoderm and cardiomyocytes using mouse embryonic stem cell (ESC) differentiation as a mammalian model system. We initially developed an efficient method, known as the Wnt Switch method, to differentiate ESCs into cardiomyocytes. Our technique relies on the treatment of differentiating ESCs with small molecule inhibitors: i) CHIR99021, which induces mesoderm development via the activation of Wnt signaling in the first 48 hours of differentiation, followed by ii) XAV939, which inhibits Wnt signaling and drives mesoderm cells toward cardiomyocyte differentiation pathway. The Wnt Switch method significantly increases the efficiency of cardiomyocyte derivation (86%) from ESC compared to published methods (56%).</p><p dir="ltr">Interestingly, the Wnt Switch method showed that despite the external stimulation of Wnt signaling, Vezf1 KO cells are unable to differentiate into cardiomyocytes and show reduced expression of mesodermal genes 48 hrs post-differentiation. To better understand the stage-specific role of Vezf1 in cardiomyocyte development, we generated doxycycline-inducible Vezf1 knockdown clones that significantly reduce Vezf1 protein levels upon treatment with doxycycline. We found that the knockdown of Vezf1 prior to mesoderm induction significantly impaired ESC differentiation but had no significant effect on cardiomyocyte development after mesoderm induction. These data indicate that Vezf1 expression is crucial for proper mesoderm and, thus, mesodermal lineage development. Further, FACS analysis showed reduced mesoderm cell populations derived from Vezf1 null post-differentiation. We used high throughput sequencing methods to determine genome-wide Vezf1 binding by ChIP-SEQ and compared gene expression in WT and Vezf1 null cells using RNA-SEQ. The data indicated that VEZF1 binds near the promoters of numerous Wnt signaling genes after differentiation and that the expression of Wnt pathway genes decreases when Vezf1 is lost. Interestingly, supplementing WNT3A protein in culture media of Vezf1 null cells rescues the expression of Wnt target genes necessary for mesoderm formation.</p><p dir="ltr">Differentiating Vezf1 KO cells to endothelial or cardiomyocyte lineages also resulted in massive cell death. The surviving cells interestingly stained positive for alkaline phosphatase (AP) staining, indicating retention of the pluripotency in Vezf1 KO cells. Whereas, re-culturing of WT ESC in LIF media, after differentiating them for five days in the absence of LIF, results in cell death, Vezf1 KO cells proliferate and form AP-positive and SSEA-positive colonies. We further show the retention of pluripotency gene expression post-differentiation using RNA sequencing and RT-qPCR. Moreover, we show that the continued expression of pluripotency genes post-differentiation was not a consequence of reduced global DNA methylation in Vezf1 KO cells.</p><p dir="ltr">Interestingly, our data show that Vezf1 is a transcriptional activator and binds to key pro-differentiation pathways like the MAPK signaling and WNT signaling pathways. The loss of Vezf1 correlates with reduced expression of genes in the pro-differentiation pathways. We show that CTCF, an insulator-binding protein, opportunistically binds to VEZF1 sites on genes in the pro-differentiation signaling pathways in VEZF1 KO cells. Therefore, we hypothesized that this opportunistic CTCF binding is the mechanism that drives the repression of pro-differentiation signaling genes or compensates for the loss of Vezf1 binding to support basal gene expression in the absence of VEZF1. Given the dire consequences of pluripotency in cancer stem cells, we investigated the expression of Vezf1 in cancers. We found that Vezf1 expression is reduced in many cancers and is correlated with poor prognosis. We also show that MAPK3, a prominent member of the MAPK signaling pathway, is reduced in these cancers, highlighting a strong correlation between Vezf1 expression and Mapk3 gene expression in cancers. The data extend our observation of pluripotency in ESCs to cancers. To gain further insights into the role of Vezf1 in cancer, we utilized F9 embryonic carcinoma cells. F9 cells have been reported to retain pluripotency expression post-differentiation. Interestingly, the ectopic and transient expression of Vezf1 in F9 cells significantly reduced the expression of pluripotency genes, suggesting that Vezf1 is sufficient to repress pluripotency gene expression in F9 carcinoma cells. These data highlight the significant role of Vezf1 in pluripotency gene repression and provide an excellent avenue for treating cancer relapse caused by the occurrence of cancer stem cells.</p><p dir="ltr">In conclusion, our research elucidates the critical role of Vezf1 in cardiomyocyte formation and pluripotency regulation during embryonic development. Understanding the molecular mechanisms underlying Vezf1-mediated pathways provides insights into developmental processes and holds promise for therapeutic interventions for cardiomyocyte regeneration and against cancers.</p>

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Dissertation is focused on the application of optical measurement methods using techniques of optical microscopy and fluorescence microscopy in measurements of electromechanical characteristics of isolated cardiac cells and clusters of differentiated cardiomyocytes. The first proposed method uses a practical combination of fluorescence microscopy equipped with fluorescent fast and high-resolution camera and atomic force microscopy for simultaneous measurement of calcium transients and contraction of cardiomyocyte clusters. The signals obtained undergoes filtration, processing and analysis. Result function parameters obtained by analyzing signals after application of caffeine are evaluated by comparison with functional parameters obtained during the control measurement. The second proposed method is applied to the cardiomyocyte clusters for the purpose of cardiomyocyte contraction signals measurement. The signals obtained by optical methods are analyzed and compared with the reference signal obtained using atomic force microscopy. Optical measurement method of cell contractins based on detection of cell ends using adjusting of microscopy images by re-sharpening and fluorescence method for cardiomyocyte contractions measurements were designed to increase realiability in simultaneous measurement of cell contractions simultaneously with calcium transients in isolated cardiomyocytes experiments.

La moxonidine, un médicament antihypertenseur sympatholytique de type imidazolinique, agit au niveau de la médulla du tronc cérébral pour diminuer la pression artérielle, suite à l’activation sélective du récepteur aux imidazolines I1 (récepteur I1, aussi nommé nischarine). Traitement avec de la moxonidine prévient le développement de l’hypertrophie du ventricule gauche chez des rats hypertendus (SHR), associé à une diminution de la synthèse et une élévation transitoire de la fragmentation d’ADN, des effets antiprolifératifs et apoptotiques. Ces effets se présentent probablement chez les fibroblastes, car l’apoptose des cardiomyocytes pourrait détériorer la fonction cardiaque. Ces effets apparaissent aussi avec des doses non hypotensives de moxonidine, suggérant l’existence d’effets cardiaques directes. Le récepteur I1 se trouvé aussi dans les tissus cardiaques; son activation ex vivo par la moxonidine stimule la libération de l’ANP, ce qui montre que les récepteurs I1 cardiaques sont fonctionnels malgré l’absence de stimulation centrale. Sur la base de ces informations, en plus du i) rôle des peptides natriurétiques comme inhibiteurs de l’apoptose cardiaque et ii) des études qui lient le récepteur I1 avec la maintenance de la matrix extracellulaire, on propose que, à part les effets sympatholytiques centrales, les récepteurs I1 cardiaques peuvent contrôler la croissance-mort cellulaire. L’activation du récepteur I1 peut retarder la progression des cardiopathies vers la défaillance cardiaque, en inhibant des signaux mal adaptatifs de prolifération et apoptose. Des études ont été effectuées pour : 1. Explorer les effets in vivo sur la structure et la fonction cardiaque suite au traitement avec moxonidine chez le SHR et le hamster cardiomyopathique. 2. Définir les voies de signalisation impliquées dans les changements secondaires au traitement avec moxonidine, spécifiquement sur les marqueurs inflammatoires et les voies de signalisation régulant la croissance et la survie cellulaire (MAPK et Akt). 3. Explorer les effets in vitro de la surexpression et l’activation du récepteur I1 sur la survie cellulaire dans des cellules HEK293. 4. Rechercher la localisation, régulation et implication dans la croissance-mort cellulaire du récepteur I1 in vitro (cardiomyocytes et fibroblastes), en réponse aux stimuli associés au remodelage cardiaque : norépinephrine, cytokines (IL-1β, TNF-α) et oxydants (H2O2). Nos études démontrent que la moxonidine, en doses hypotensives et non-hypotensives, améliore la structure et la performance cardiaque chez le SHR par des mécanismes impliquant l’inhibition des cytokines et des voies de signalisation p38 MAPK et Akt. Chez le hamster cardiomyopathique, la moxonidine améliore la fonction cardiaque, module la réponse inflammatoire/anti-inflammatoire et atténue la mort cellulaire et la fibrose cardiaque. Les cellules HEK293 surexprimant la nischarine survivent et prolifèrent plus en réponse à la moxonidine; cet effet est associé à l’inhibition des voies ERK, JNK et p38 MAPK. La surexpression de la nischarine protège aussi de la mort cellulaire induite par le TNF-α, l’IL-1β et le H2O2. En outre, le récepteur I1 s’exprime dans les cardiomyocytes et fibroblastes, son activation inhibe la mort des cardiomyocytes et la prolifération des fibroblastes induite par la norépinephrine, par des effets différentiels sur les MAPK et l’Akt. Dans des conditions inflammatoires, la moxonidine/récepteur aux imidazolines I1 protège les cardiomyocytes et facilite l’élimination des myofibroblastes par des effets contraires sur JNK, p38 MAPK et iNOS. Ces études démontrent le potentiel du récepteur I1/nischarine comme cible anti-hypertrophique et anti-fibrose à niveau cardiaque. L’identification des mécanismes cardioprotecteurs de la nischarine peut amener au développement des traitements basés sur la surexpression de la nischarine chez des patients avec hypertrophie ventriculaire. Finalement, même si l’effet antihypertenseur des agonistes du récepteur I1 centraux est salutaire, le développement de nouveaux agonistes cardiosélectifs du récepteur I1 pourrait donner des bénéfices additionnels chez des patients non hypertendus. / Moxonidine, an antihypertensive sympatholytic imidazoline compound, reduces blood pressure by selective activation of non-adrenergic imidazoline I1-receptors (also known as nischarin) in brainstem medulla. Moxonidine prevents left ventricular hypertrophy development in hypertensive rats, associated with reduced cardiac DNA synthesis and early transient increase in DNA fragmentation. It is likely that the anti-proliferative and apoptotic effects occur in fibroblasts, as cardiomyocyte apoptosis may deteriorate cardiac function. The effects also occurred to sub-hypotensive doses, suggesting a blood-pressure-independent mechanism and pointing to a local cardiac action. Imidazoline I1-receptors have been identified in cardiac tissues, and their ex vivo activation by moxonidine stimulates ANP release, demonstrating that cardiac imidazoline I1-receptors are functional without the contribution of the central nervous system. Based on the above studies and on i) the role of natriuretic peptides in inhibition of myocardial cell apoptosis and ii) studies linking imidazoline I1-receptors to the maintenance of the extracellular matrix and PC12 cell survival, we propose that apart from centrally-mediated sympatholytic function, imidazoline I1-receptors in the heart may control cell growth and death. Activation of imidazoline receptors may delay the progression of cardiac pathologies into heart failure by inhibition of maladaptive proliferative signalling and downstream apoptotic pathways. In order to test this hypothesis studies were performed to: 1. Explore the in vivo effects of moxonidine on cardiac structure and function in SHR and cardiomyopathic hamsters. 2. Define the pathways involved in the observed changes following moxonidine treatment, specifically, on inflammatory markers and pathways involved in LVH and cardiac cell survival/death (MAPK and Akt). 3. Explore in vitro the effect of imidazoline I1-receptor activation by moxonidine, on cell survival by over-expressing nischarin in HEK293 cells, to circumvent the lack of specific imidazoline I1-receptor agonists and antagonists. 4. Investigate in vitro, imidazoline I1-receptor localization (cardiomyocytes and fibroblasts), regulation and implication in cell growth/death in response to cardiac remodelling-associated stimuli: norepinephrine, cytokines (IL-1β, TNF-α), and oxidants (H2O2). The studies reveal that hypotensive and sub-hypotensive concentrations of moxonidine improve cardiac structure and performance in SHR by mechanisms that involve inhibition of cytokines, p38MAPK, and Akt signalling pathways. In cardiomyopathic hamsters moxonidine improves cardiac performance, in association with differential inflammatory/anti-inflammatory responses that culminate in attenuated cardiomyocyte death and fibrosis and altered collagen type expression. HEK293 cells, transfected with nischarin cDNA, show increased viability/proliferation in response to moxonidine. The overall survival response is associated with moxonidine’s inhibition of ERK, JNK, and p38MAPK. Nischarin also opposes the reduced cell viability in response to oxidative stimuli (TNF-α, IL-1β and H2O2), with differential responses to moxonidine. Furthermore, the imidazoline I1-receptor is expressed in cardiac fibroblasts and myocytes and its activation inhibits norepinephrine-induced cardiomyocyte death and fibroblast proliferation, through differential effects on MAPKs and Akt. Moxonidine/imidazoline I1-receptor protects cardiomyocytes and facilitates elimination of myofibroblasts in inflammatory conditions, through opposite effects on JNK, p38MAPK and iNOS activity. These studies emphasize the potential importance of imidazoline I1-receptor/nischarin as an anti-hypertrophic and anti-fibrotic target. Identification of the cardio-protective mechanisms of cardiac nischarin could result in specifically-tailored cell/gene-driven nischarin treatments, which could be important for patients with heart disease. Also, while the antihypertensive action of centrally acting compounds is appreciated, new cardiac-selective I1-receptor agonists may confer additional benefit.