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Maggiorani, Damien. "Caractérisation de la sénescence des cardiomyocytes et identification de marqueurs associés." Thesis, Toulouse 3, 2017. http://www.theses.fr/2017TOU30320/document.
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Le vieillissement de l'organisme prédispose à de nombreuses pathologies chroniques telles que l'insuffisance cardiaque (IC). Des études récentes ont montré que l'accumulation de cellules sénescentes dans les organes au cours du vieillissement est associée à l'apparition de ces pathologies. La sénescence cellulaire a initialement été décrite comme un arrêt stable du cycle cellulaire permettant de limiter la prolifération des cellules dont l'ADN est endommagé. Ce processus s'accompagne de profondes modifications de la fonction cellulaire, avec notamment l'acquisition d'un phénotype sécrétoire associé à la sénescence. La sénescence peut être induite par un raccourcissement des télomères ou par l'exposition à des signaux de stress, tels que le stress oxydant ou l'irradiation, qui entrainent l'activation de la réponse cellulaire aux dommages de l'ADN et l'expression des gènes suppresseurs de tumeurs (p16INK4a, p21CIP1, p53). Ces inhibiteurs du cycle cellulaire sont classiquement utilisés comme marqueur de sénescence car leur expression augmente de manière ubiquitaire au cours du vieillissement. Toutefois, ces marqueurs ne sont pas spécifiques du tissu concerné et un des objectifs de ma thèse a été d'identifier de nouveaux marqueurs de sénescence tissu-spécifiques qui pourraient caractériser un vieillissement cardiaque pathologique. Le vieillissement cardiaque se caractérise par une hypertrophie des cardiomyocytes, une sensibilité accrue au stress et une prédisposition à l'IC. Les cardiomyocytes étant des cellules post-mitotiques, les mécanismes de sénescence mis en jeu, les marqueurs associés et leur rôle potentiel dans l'IC demeurent à l'heure actuelle peu caractérisés. Au cours de ce travail de thèse nous avons donc entrepris : 1) d'étudier le rôle des télomères et des dysfonctions mitochondriales dans l'induction de la sénescence du cardiomyocyte et 2) d'identifier des marqueurs spécifiques. Nous avons tout d'abord montré que les cardiomyocytes de souris âgées expriment les marqueurs classiques de la sénescence comme p16INK4a, p53 et p21CIP1. Concernant les mécanismes inducteurs, nous avons étudié l'implication des dommages télomériques (telomere associated foci, TAF). Au cours du vieillissement, nous avons observé une augmentation du nombre de TAFs par cardiomyocytes en association avec l'hypertrophie. De plus, l'induction de TAFs in vitro est suffisante à l'activation de la voie de sénescence p53/p21CIP1 et l'hypertrophie dans une lignée de cardiomyoblastes H9c2. La formation des TAFs est augmentée chez des souris avec une dysfonction mitochondriale et est associée à l'activation des voies p53/p21CIP1. Par ailleurs, les cardiomyocytes âgés présentent une dérégulation des gènes impliqués dans la biologie mitochondriale pouvant rendre compte de l'augmentation des TAFs. Par l'analyse haut débit du transcriptome (RNAseq) nous avons identifié six nouveaux gènes qui sont surexprimés dans les cardiomyocytes sénescents (Prom2, Kcnk1, Pah, Edn3, Gdf15, Tgfb2). La comparaison d'expression de ces gènes dans le cœur avec d'autres tissus et avec le stroma cardiaque lors vieillissement a permis de confirmer la spécificité d'expression de ces marqueurs au niveau des cardiomyocytes. Nous avons validé cette signature dans deux modèles in vitro de sénescence induite par le stress et démontré que l'expression de certains de ces marqueurs est dépendante de la voie p53. De plus, l'expression de Prom2 est associée à l'hypertrophie des cardiomyocytes. En conclusion, nous avons démontré, qu'avec le vieillissement, les cardiomyocytes présentent un programme de sénescence associé à une dysfonction mitochondriale et une augmentation des TAFs. Cette sénescence se caractérise par l'activation des voies classiques de sénescence (p16INK4, p53/p21CIP1), une hypertrophie et l'acquisition d'une signature spécifique. Ces marqueurs offrent de nouvelles perspectives dans la compréhension de la sénescence cardiaque et dans son implication potentielle dans l'ICAgeing of the organism is associated with several chronic pathologies such as heart failure (HF). Recent studies have demonstrated the link between the accumulation of senescent cells during ageing and age-associated diseases. Cellular senescence, originally defined as a stable cell cycle arrest, acts as a tumorigenic repressor by limiting the proliferation of DNA damaged cells. Despite this protective effect, senescence is characterized by deep remodeling of cell biology which drives functional disorders, such as the acquisition of a senescence-associated secretory phenotype (SASP). Senescence can be induced by telomeric attrition and by exposition to cellular stress signals such as oxidative stress or irradiation, which induce telomeric damage, activation of the DNA Damage Response (DDR) and increased expression of antitumoral genes (p16INK4a, p21CIP1, p53). These genes are classically used as markers of senescence because their expression increases in several tissues during ageing but they are not tissue-specific. Therefore, At the cardiac level, ageing is characterized by cardiomyocytes hypertrophy, increased sensitivity to stress and highest risk of developing HF. Cardiomyocytes are post- mitotic cells and the senescence inductor mechanisms, specifics markers and their role in HF remains poorly understood. This thesis project is articulated around two aims, 1/ studying the role of telomeric damages and mitochondrial dysfunction in triggering cardiomyocyte senescence and 2/ identification of specifics markers. Fisrtly, we shown that aged cardiomyocytes overexpress classic markers of senescence such as p16INK4a, p53 et p21CIP1. Concerning the inductors mechanisms, we studied the implication of telomeric damages (telomere associated foci, TAF). During ageing, we found an increased number of TAFs per cardiomyocytes and their association with hypertrophy. Moreover, TAF- induction in cardiac H9c2 in vitro activated the p53/p21 pathway and induced senescence. These data confirmed the role of TAFs in cardiomyocyte senescence induction. Furthermore, aged cardiomyocytes exhibit a global alteration of genes involved in mitochondrial biology, oxidative stress and metabolism in aged cardiomyocytes that could play a prominent role in TAF accumulation with ageing. In a second part of the study, by using a next generation sequencing method (RNA-seq) we identified 6 new genes highly expressed in senescent cardiomyocytes (Prom2, Kcnk1, Pah, Edn3, Gdf15 and Tgfb2). Expression comparison with other senescent organs and cardiac stromal cells confirmed these new genes as cardiomyocyte specific. Thanks to an in vitro approach, we validate this signature by using different models of stress-induced senescence in cardiac H9c2 cells and demonstrated the implication of the p53 in the regulation of some of these genes. Moreover, Prom2 expression is associated with cardiomyocytes hypertrophy. In conclusion, we demonstrated that, with ageing, cardiomyocytes display a senescence phenotype associated with mitochondrial dysfunction and TAFs. This process is characterized by classic markers (p16INK4, p53/p21CIP1), hypertrophy and new identified signature. These new markers offer innovative perspectives in the understanding and the identification of the cardiac senescence and their potential deleterious role in heart failure
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Nelson, Brandon John. "MicroRNA analysis of human embryonic stem cell derived cardiomyocytes and neonatal rat ventricular cardiomyocytes." Diss., Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 2007. http://wwwlib.umi.com/cr/ucsd/fullcit?p1447322.
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Thesis (M.S.)--University of California, San Diego, 2007.Title from first page of PDF file (viewed January 15, 2008). Available via ProQuest Digital Dissertations. Includes bibliographical references (p. 45-48).
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Bond, Richard. "Cellular electrophysiology of rat pulmonary vein cardiomyocytes : a comparative study with left atrial cardiomyocytes." Thesis, University of Bristol, 2015. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.685358.
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Although atrial fibrillation (AF) is the most common sustained arrhythmia, its pathophysiology is complex and remains poorly understood. Most episodes of AF are initiated by ectopic beats originating from the pulmonary veins (PVs). It has been suggested that the distinct electrophysiological properties of the PVs and their rich innervation by noradrenergic sympathetic fibres contribute to the susceptibility of this region to ectopic activity. This study aimed to determine whether the cellular electrophysiological responses to noradrenaline (NA) of card iomyocytes isolated from the PVs differed to those isolated from the left atrium (LA).
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Fijnvandraat, Arnoldus Cornelis. "Embryonic stem cell-derived cardiomyocytes." [S.l. : Amsterdam : s.n.] ; Universiteit van Amsterdam [Host], 2003. http://dare.uva.nl/document/68354.
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Risto, Morten. "Modelling hypertrophy in dystrophic cardiomyocytes." Thesis, University of Newcastle upon Tyne, 2016. http://hdl.handle.net/10443/3402.
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Duchenne Muscular Dystrophy (DMD) is an X-linked disorder, caused by mutations in the DMD gene. This gene encodes dystrophin, a structural protein that links the sarcomere to the extracellular matrix via a trans-membrane protein complex. In the absence of dystrophin the associated glycoprotein complex fails to assemble, leading to sarcolemmal instability, impaired ion handling, skeletal muscle wasting and fibrosis. Patients become non-ambulant in their teens and seldom live past their third decade. Cardiac failure is one of the leading causes of death. The heart initially compensates for reduced functional capacity by becoming hypertrophic, but eventually becomes fibrotic and develops dilated cardiomyopathy. Several proposed therapies have now reached clinical trial phase, but there is still no cure available for all DMD patients. Some of these therapies target skeletal muscle better than the heart. Sample availability restricts research into cardiac mechanisms of disease and testing treatments. This thesis presents a model that can potentially be used as an in vitro outcome measure for testing DMD therapies. Cardiomyocytes isolated from hearts collected from the DMD mouse model (mdx) embryos became larger than control mouse embryo-derived cardiomyocytes in response to serum starvation in culture. Control and mdx cardiomyocytes were collected at five timepoints of serum starvation and RNA-Seq was performed on the samples to identify pathways responsible for this hypertrophic response observed in dystrophic cells. Several pharmacological compounds as well as a proposed gene therapy method were trialled for their ability to reduce the hypertrophic response. Serum starved cardiomyocytes from mdx mouse embryos were transduced with adeno-associated viruses containing a gene construct expressing a functional internally truncated version of dystrophin. The viral rescue therapy and some pharmacological compounds significantly reduced the dystrophic hypertrophy caused by serum starvation. This model of mdx cardiomyocyte hypertrophy could therefore be used for testing therapies in pre-clinical trials.
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De, Marco Margot. "BAG3 role in cardiomyocytes physiopathology." Doctoral thesis, Universita degli studi di Salerno, 2013. http://hdl.handle.net/10556/896.
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2010 - 2011The anti-apoptotic protein BAG3 is expressed at high levels in skeletal and cardiac muscle in vivo. Our group recently focused its interest on BAG3 role in myocardiocyte proliferation, survival and response to stressful stimuli. We found that BAG3 is upregulated during the differentiation of cardiomyoblasts. Our results prompted us to verify whether bag3 silencing could affect the differentiation state of cardiocytes and we found that bag3 silencing resulted in highly reducing the levels of myogenin. Furthermore, we analyzed BAG3 expression and localization following cell exposure to oxidative stress. In particular, we found that epinephrine in vitro increases BAG3 expression in adult human cardiomyocytes. We evaluated whether BAG3 could be involved in the Tako-tsubo cardiomyopathy (or stress cardiomyopathy) pathogenesis that is characterized by left ventricular dysfunction, with symptoms that can mimic an acute coronary syndrome. The absence of significant cardiovascular risk factors in patients affected by stress cardiomyopathy suggested that it might be associated with a possible genetic etiology. Therefore, we sequenced bag3 gene to check for polymorphisms in 29 patients and 1043 healthy donors. Three polymorphism were highly represented among patients (R71Q, C151R, P407L). We also showed for the first time that BAG3 protein is released from stressed cardiomyocytes and is found in chronic heart failure (HF) patients’ sera. Since anti-BAG3 antibodies are also present in patients’ sera, we developed an ELISA test for their specific detection. In serum samples from chronic HF patients, we found significantly higher values of anti-BAG3 antibodies respect to samples from healthy donors. The presence of anti-BAG3 antibodies in chronic HF patients’ sera and the availability of an ELISA test for their detection can contribute a novel tool for diagnostic and prognostic evaluations. [edited by author]
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Driesen, Ronald Bertie Mario Antonio. "Adaptive remodeling of cardiomyocytes under stress." [Maastricht] : Maastricht : Universitaire Pers Maastricht ; University Library, Universiteit Maastricht [host], 2008. http://arno.unimaas.nl/show.cgi?fid=11068.
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Kemeny, Naomi. "Alendronate affects calcium dynamics in cardiomyocytes." Thesis, McGill University, 2009. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=40684.
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Alendronate (ALN) is effective in the treatment of osteoporosis. However, ALN has been recently associated with an increased risk of serious atrial fibrillation. We investigated the effects of ALN on cytosolic free calcium concentration ([Ca2+]i) in cardiomyocytes. HL-1 atrial cardiomyocytes were loaded with fura-2 and examined using microspectrofluorimetry. ALN (10-8, 10-7, 10-6 M) induced transitory high frequency oscillations of [Ca2+]i with greater frequency for 10-8 M ALN (61 ± 10 mHz) compared to 10-6 ALN (42 ± 4 mHz). In cells treated with 10-6 M ALN responses to subsequent application of caffeine were delayed, and exhibited a decrease in the rate and amplitude of [Ca2+]i increase. Long term (48 h) exposure to 10-8 M Alendronate resulted in delay of caffeine-induced Ca2+ transients and decreased rate of [Ca2+]i increase, followed by oscillations in [Ca2+]i of 54 ± 8 mHz versus those observed at higher concentrations of Alendronate (35 ± 5 mHz). Changes in calcium dynamics were accompanied by significant changes in the expression of sarcoendoplasmic reticulum ATPase (SERCA2a), calsequestrin and calreticulin.Alendronate est efficace dans le traitement de l’ostéoporose. Alendronate a récemment été associe avec une risque élevé de fibrillation auriculaire sérieux. On a examine les effets d’Alendronate sur la de calcium cytosolique ([Ca2+]i) dans les cellules musculaires cardiaques. Les cellules musculaires cardiaques HL-1 étaient chargés avec Fura-2 et examinés par microspectrofluorimetrie. Alendronate (10-8, 10-7, 10-6 M) ont provoqués des oscillations de [Ca2+]i fugaces et haute fréquences à 10-8 M Alendronate (61 ± 10 mHz) comparé aux concentrations plus hautes (42 ± 4 mHz). Dans les cellules traits avec 10-6 M ALN, la réponse à l’application de caféine était avec délai, et a manifesté un diminution dans la rythme et amplitude d’augmentation de [Ca2+]i. L’exposition a l’ALN à long terme (48 h) ont provoqué un délai des élévations de calcium transitoires, et un diminution du rythme d’augmentation de [Ca2+]i suites par les oscillations de [Ca2+]i, caractérisés par un augmentation de fréquences avec 10-8 M Alendronate (54 ± 8 mHz) compare aux concentrations plus hautes (35 ± 5 mHz). Le changement des dynamiques de calcium étaient accompagnés par les changements considérables dans l’expression d’ATPase (SERCA2a), calsequestrin et calreticulin du réticulum sarcoendoplasmique.
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Bowers, Keith Cyril. "Pathophysiology of ATP in single cardiomyocytes." Thesis, University of Liverpool, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.316576.
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Bowman, Peter Ronald Thomas. "Regulation of glucose transport in cardiomyocytes." Thesis, University of Glasgow, 2019. http://theses.gla.ac.uk/41002/.
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Major common complications of diabetes such as myocardial infarction arise from the onset of vascular disease. However, there is also evidence of a direct impairment of cardiac contractile function in diabetic individuals in the absence of atherosclerosis and hypertension, termed diabetic cardiomyopathy (DCM). This is characterised by early diastolic dysfunction that progresses to systolic dysfunction and heart failure through a pathological remodelling process. The earliest identified mechanism underlying this disease is the onset of metabolic perturbations such as cardiac insulin resistance. However, currently there are no specific treatments available, partly due to the lack of an appropriate experimental model with which translational research could be performed. iPSC-CM are a recently developed technology, whereby human dermal fibroblasts can be reliably harvested, dedifferentiated into a pluripotent form, and then differentiated into cardiomyocytes. These cells have an established intracellular calcium handling system and contractile capacity, however are generally considered to be at a foetal stage of development. A key aim of this project was to characterise the metabolic phenotype of these cells, in order to assess their potential suitability as the basis of a novel cellular model of DCM. Specifically, it was investigated if these cells exhibited robust insulin stimulated glucose uptake through insulin sensitive intracellular trafficking of the glucose transporter GLUT4, as impairment of this response is a central feature of any diabetic model. After adaptation of a [3H]-2-deoxyglucose uptake assay to a 96-well plate format, and optimisation of experimental factors, it was determined that iPSC-CM could not display robust insulin (or IGF-1) stimulated glucose uptake. Inhibition of the spontaneous contractile capacity of these cells did not induce a response upon subsequent insulin stimulation. iPSC-CM were found to express and activate central insulin signalling molecules such as Akt and Erk1/2, and also possess elements of the GLUT4 trafficking machinery such as the SNARE proteins Syntaxin 4 and SNAP23. However, the critically limiting factor identified was an approximate 10-fold lower expression of GLUT4 in iPSC-CM compared to primary adult cardiomyocytes, accompanied by strong expression of GLUT1. This data was supported by the finding that inhibition of GLUT4 had no impact on glucose uptake in iPSC-CM, whereas inhibition of GLUT1 significantly reduced uptake by ~50%. This phenotype suggests that iPSC-CM are also at a foetal-like stage of development with regards to their metabolic capacity, and are currently not suitable for modelling DCM. Subsequently, initial interventions based upon the literature were implemented in order to try and increase iPSC-CM GLUT4 content. However, neither increasing metabolic reliance upon fatty acid (rather than glucose) nor exposure to triiodothyronine were successful. In contrast, Lipofectamine 2000 mediated transfection of a customised GLUT4 plasmid facilitated a reliable 3-5 fold increase in iPSC-CM GLUT4 content. This increased basal glucose uptake, however did not induce an insulin response. It was concluded that a further increase in expression levels may be required. Finally, it was demonstrated that iPSC-CM are highly amenable to lentiviral mediated infection, and initial steps were taken towards the generation of a virus targeting the overexpression of GLUT4. Additionally, SNARE proteins are essential in facilitating insulin stimulated GLUT4 expression at the plasma membrane. Therefore they represent a possible mechanism by which cardiac insulin resistance could occur in disease states such as DCM. On account of this, the expression of a wide range of SNARE protein isoforms was assessed in cardiac lysates generated from 2 diabetic mouse models (db/db and high fat diet induced). The expression of SNAP29 and VAMP5 were found to differ in lysates from the high fat diet model, although the role of these proteins in GLUT4 trafficking is unclear. In contrast, in the more severe diabetic db/db model GLUT4 protein content was found to be significantly reduced, but SNARE protein content was unaffected. Finally, there is also an established link between glycemic control and both the risk of developing and subsequent prognosis for myocardial infarction (MI). There is a line of evidence suggesting that cardiac insulin sensitivity may also be highly relevant in this disease context. Accordingly, it was demonstrated that cardiomyocytes isolated from a clinically relevant 8-12 weeks post-MI rabbit model exhibited impaired insulin stimulated glucose uptake. This strengthens the association between MI and cardiac metabolic parameters. However, insulin stimulated phosphorylation of Akt, GLUT4 levels, and SNARE protein expression were unaffected post-MI. Therefore future work must identify both the underlying mechanism and clinical relevance of this finding.
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McDougal, Anthony Drew. "Modeling oxygen requirements in ischemic cardiomyocytes." Thesis, Massachusetts Institute of Technology, 2015. http://hdl.handle.net/1721.1/100105.
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Thesis: S.M., Massachusetts Institute of Technology, Department of Mechanical Engineering, 2015.Cataloged from PDF version of thesis.
Includes bibliographical references (pages 36-40).
Ischemic heart disease remains a leading cause of death globally and in the US. The most common ischemic event is a heart attack, where one or more arteries are obstructed and the cardiac muscle is deprived of oxygen. Although removing the blockage and allowing reperfusion can prevent death, at the same time it can cause significant damage through "reperfusion injury." To date, there are limited methods to predict the viability of the myocardial muscle cell (myocyte) and its quantitative conditions during ischemia. Here, we explore the viability of heart cells using a model for cellular metabolism. We use this model to predict conditions that will sustain viable concentrations of adenosine triphosphate (ATP) and compare these conditions to baseline energy consumption rates. Glycolytic metabolism is modeled using a system of coupled ordinary differential equations that describe the individual metabolic reactions that occur within the cardiac myocyte and its surrounding tissue. Over 200 conditions were simulated to characterize a range of reduced oxygen levels and ATP consumption rates. These conditions were organized according to their steady-state level of [ATP], and reveal a distinct transition region between low levels of ATP that are sustainable and depleted ATP levels that lead to cell death. Our simulations and analysis illustrate how very low concentrations of oxygen in the extracellular tissue allow the cells to perform essential survival functions. The model contains 58 of the molecular species within the cell, so that the conditions of the cell at the time of reperfusion can be predicted. We find the oxygen level required for viability increases roughly linearly with the ATP consumption rate, and is smaller than one would have expected based on previous results. An external tissue level 02 concentration of around 0.007 mM is sufficient to sustain cardiomyocyte viability in the absence of beating. This level of oxygen could be achieved through collateral circulation. This model of ischemia will also provide future investigations of the reperfusion process to proceed from a known metabolic and molecular state of the cardiomyocytes preceding re-oxygenation.
by Anthony Drew McDougal.
S.M.
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Giraldo, Ramirez Diego Alejandro. "Regulation of ATF3 expression in cardiomyocytes." Thesis, Imperial College London, 2008. http://hdl.handle.net/10044/1/1300.
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The stress responses of cardiomyocytes are likely to constitute a significant aspect of the development of cardiac pathologies. Oxidative stress is a common theme in the pathophysiology of ischaemic and non-ischaemic cardiomyopathy. On the other hand, hypertrophic agonists (e.g. endothelin-1, ET-1) are important mediators of the hypertrophic response to hemodynamic overloading, and cardiac pathologies such as heart failure are usually preceded by cardiac hypertrophy. Microarray studies indicate that one of the genes most potently induced by H2O2 (as an oxidative stress) or ET-1 in cardiac myocytes is the transcription factor ATF3. This thesis examines the regulation and role of ATF3 in neonatal rat ventricular cardiomyocytes. The increase in expression of ATF3 mRNA was confirmed in neonatal rat cardiomyocytes exposed to H2O2 or ET-1 using RT-PCR and Q-PCR. Using immunoblotting, it was confirmed that H2O2 or ET-1 increased ATF3 protein expression. ATF3 is an immediate early gene since the upregulation of ATF3 mRNA by ET-1 was not inhibited by cycloheximide (20 μM). Upregulation of ATF3 was inhibited by U0126 (10 μM), suggesting that signalling through ERK1/2 (extracellular signal-regulated kinases 1/2) was required. Other studies suggest that ATF3 downregulates interleukin-6 (IL-6) in human cells. The rat IL-6 promoter possesses an ATF consensus sequence, and chromatin immunoprecipitation (ChIP) analysis indicated that ET-1 or H2O2 increased the association of ATF3 with the IL-6 promoter. IL-6 mRNA and protein expression was transiently upregulated in cardiomyocytes exposed to H2O2 or ET-1, and this was shown to be an immediate early gene response. Following IL-6 mRNA upregulation, its expression was more rapidly downregulated than that of ATF3. The peak of ATF3 protein expression coincided with the return of IL-6 mRNA to basal levels after stimulation with either ET-1 or H2O2. This suggests that ATF3 operates in a negative feedback loop to downregulate IL-6 mRNA expression in cardiomyocytes. This was confirmed by adenoviral-mediated overexpression of full-length ATF3 antisense RNA, which attenuated ATF3 mRNA and protein expression following stimulation with ET-1. Inhibition of ATF3 expression was associated with superinduction of IL-6 mRNA as shown by Q-PCR. Other potential downstream targets of ATF3 were identified from microarray studies using bioinformatics. Those with ATF/CRE consensus sequences in their promoters included epiregulin and leukaemia inhibitory factor (LIF) (also shown to be immediate early genes). Epiregulin and LIF mRNA expression was superinduced by ATF3 antisense RNA. Taken together, these experiments indicate that ATF3 operates in a negative feedback loop by downregulating a cluster of immediate early genes induced by ET-1. Thus, ATF3 may play an important role in the establishment and fine-tuning of an organised and compensatory hypertrophic response in the cardiomyocyte secondary to hypertrophic stimulation.
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Marshall, Andrew Keith. "Signalling through Rho GTPases in cardiomyocytes." Thesis, Imperial College London, 2011. http://hdl.handle.net/10044/1/6962.
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Endothelin-1 (ET-1) promotes changes in gene/protein expression in cardiomyocytes leading to hypertrophy. This results from activation of intracellular signalling pathways including small G proteins that activate protein kinases. Thus, ET-1 activates RhoA that stimulates ROCK and PKN, and Ras that promotes activation of extracellular signal-regulated kinases 1/2 (ERK1/2). Microarrays were used to dissect the roles of ERK1/2 vs RhoA in the cardiomyocyte transcriptomic response to ET-1 using PD184352 and C3 endotoxin from C. botulinum (C3T) for selective inhibition of the ERK1/2 cascade and RhoA, respectively. Microarray data were analysed using GeneSpring and data were validated by qPCR. ERK1/2 signalling positively regulated ~65% of the early gene expression response to ET-1 with a small (~2%) negative effect, whereas RhoA signalling positively regulated ~11% of the early gene expression response to ET-1 with a greater (~14%) negative contribution. Of RNAs non-responsive to ET-1, 66 or 448 were regulated by PD184352 or C3T, respectively, indicating that RhoA had a more significant effect on baseline RNA expression. mRNAs upregulated by ET-1 encoded several receptor ligands (e.g. Ereg, Areg) and transcription factors (e.g. Abra/STARS, Srf) that potentially propagate the response. Published studies suggest that PKN1 (activated by RhoA) is important in cardiomyocyte gene expression. Adenoviruses were generated to overexpress FLAG-tagged PKN1 in cardiomyocytes for protein kinase studies. Unexpectedly, PKN1 was not activated by ET-1, but was activated by oxidative stress, insulin, or hyperosmotic shock, stimuli that do not activate RhoA. Thus, PKN1 is not necessarily an effector of RhoA in cardiomyocytes. In conclusion, ERK1/2 dominates over RhoA in the early transcriptomic response to ET-1. RhoA plays a major role in maintaining baseline RNA expression but, as with upregulation of Abra/Srf by ET-1, RhoA may regulate changes in RNA expression over longer times. However, the effects of RhoA on cardiomyocyte gene expression are unlikely to be mediated through PKN1.
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Cavalli, Amy Lynn. "Characterization of sphingolipid receptors in cardiomyocytes /." Diss., Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 2002. http://wwwlib.umi.com/cr/ucsd/fullcit?p3071179.
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Ellis, Charlotte Elizabeth. "Subcellular effects of pavetamine on rat cardiomyocytes." Thesis, University of Pretoria, 2010. http://hdl.handle.net/2263/26785.
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The aim of this study was to investigate the mode of action of pavetamine on rat cardiomyocytes. Pavetamine is the causative agent of gousiekte (“quick-disease”), a disease of ruminants characterized by acute heart failure following ingestion of certain rubiaceous plants. Two in vitro rat cardiomyocyte models were utilized in this study, namely the rat embryonic cardiac cell line, H9c2, and primary neonatal rat cardiomyocytes. Cytotoxicity of pavetamine was evaluated in H9c2 cells using the MTT and LDH release assays. The eventual cell death of H9c2 cells was due to necrosis, with LDH release into the culture medium after exposure to pavetamine for 72 h. Pavetamine did not induce apoptosis, as the typical features of apoptosis were not observed. Electron microscopy was employed to study ultrastructural alterations caused by pavetamine in H9c2 cells. The mitochondria and sarcoplasmic reticula showed abnormalities after 48 h exposure of the cells to pavetamine. Abundant secondary lysosomes with electron dense material were present in treated cells. Numerous vacuoles were also present in treated cells, indicative of autophagy. During this exposure time, the nuclei appeared normal, with no chromatin condensation as would be expected for apoptosis. Abnormalities in the morphology of the nuclei were only evident after 72 h exposure. The nuclei became fragmented and plasma membrane blebbing occurred. The mitochondrial membrane potential was investigated with a fluorescent probe, which demonstrated that pavetamine caused significant hyperpolarization of the mitochondrial membrane, in contrast to the depolarization caused by apoptotic inducers. Pavetamine did not cause opening of the mitochondrial permeability transition pore, because cyclosporine A, which is an inhibitor of the mitochondrial permeability transition pore, did not reduce the cytotoxicity of pavetamine significantly. Fluorescent probes were used to investigate subcellular changes induced by pavetamine in H9c2 cells. The mitochondria and sarcoplasmic reticula showed abnormal features compared to the control cells, which is consistent with the electron microscopy studies. The lysosomes of treated cells were more abundant and enlarged. The activity of cytosolic hexosaminidase was nearly three times higher in the treated cells than in the control cells, which suggested increased lysosomal membrane permeability. The activity of acid phosphatase was also increased in comparison to the control cells. In addition, the organization of the cytoskeletal F-actin of treated cells was severely affected by pavetamine. Rat neonatal cardiomyocytes were labelled with antibodies to detect the three major contractile proteins (titin, actin and myosin) and cytoskeletal proteins (F-actin, desmin and β-tubulin). Cells treated with pavetamine had degraded myosin and titin, with altered morphology of sarcomeric actin. Vacuoles appeared in the β-tubulin network, but the appearance of desmin was normal. F-actin was severely disrupted in cardiomyocytes treated with pavetamine and was degraded or even absent in treated cells. Ultrastructurally, the sarcomeres of rat neonatal cardiomyocytes exposed to pavetamine were disorganized and disengaged from the Z-lines, which can also be observed in the hearts of ruminants that have died of gousiekte. It is concluded that the pathological alteration to the major contractile and cytoskeleton proteins caused by pavetamine could explain the cardiac dysfunction that characterizes gousiekte. F-actin is involved in protein synthesis and therefore can play a role in the inhibition of protein synthesis in the myocardium of ruminants suffering from gousiekte. Apart from inhibition of protein synthesis in the heart, there is also increased degradation of cardiac proteins in an animal with gousiekte. The mitochondrial damage will lead to an energy deficiency and possibly to generation of reactive oxygen species. The sarcoplasmic reticula are involved in protein synthesis and any damage to them will affect protein synthesis, folding and post-translational modifications. This will activate the unfolded protein response (UPR) and sarcoplasmic reticula-associated protein degradation (ERAD). If the oxidizing environment of the sarcoplasmic reticula is disturbed, it will activate the ubiquitin-proteasome pathway (UPP) to clear aggregated and misfolded proteins. Lastly, the mitochondria, sarcoplasmic reticula and F-actin are involved in calcium homeostasis. Any damage to these organelles will have a profound influence on calcium flux in the heart and will further contribute to the contractile dysfunction that characterizes gousiekte.Thesis (PhD)--University of Pretoria, 2010.
Paraclinical Sciences
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Kovacs, Yoann. "Rôle de la protéine découplante mitochondriale Ucp2 dans la régulation du métabolisme et de la maturation des cardiomyocytes néonataux." Electronic Thesis or Diss., Sorbonne université, 2022. https://accesdistant.sorbonne-universite.fr/login?url=https://theses-intra.sorbonne-universite.fr/2022SORUS391.pdf.
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Au cours du développement, le cœur utilise un métabolisme essentiellement glycolytique. A la naissance, l’exposition à l’O2 atmosphérique et le passage à l’alimentation lactée riche en acides gras entraînent un remodelage métabolique vers la phosphorylation oxydative mitochondriale. Les cardiomyocytes adoptent alors un métabolisme oxydatif, majoritairement dépendant de la β-oxydation. Ce remodelage s’accompagne de changements transcriptomiques permettant, parallèlement à cette induction de la β-oxydation, de promouvoir la maturation des cardiomyocytes. Celle-ci, qui permet d’optimiser la production d’énergie et la fonction de contraction, requiert une induction de la biogénèse mitochondriale, une augmentation et une modulation de l’expression des constituants de la machinerie contractile, responsable de l’hypertrophie des cardiomyocytes néonataux, et un arrêt de leur prolifération. De nombreuses hypothèses suggèrent que l’activité métabolique de la mitochondrie participe à la signalisation de cette maturation, régulant notamment l’arrêt de prolifération des cardiomyocytes néonataux et leur hypertrophie. Dans une perspective thérapeutique, la compréhension de cette signalisation est essentielle. Elle permettrait notamment de promouvoir l’utilisation des modèles de cardiomyocytes in vitro en améliorant leur différenciation terminale, ou au contraire de stimuler la prolifération de cardiomyocytes adulte in vivo pour permettre la régénération cardiaque. Mes travaux de thèse portent sur la protéine mitochondriale Ucp2, spécifiquement exprimée dans le cœur néonatal, et sur son rôle dans la maturation des cardiomyocytes. Ucp2 est un transporteur localisé dans la membrane interne de la mitochondrie. Il possède deux fonctions (1) de régulation de la production d’espèces réactives mitochondriales et (2) de régulation de l’orientation métabolique de la cellule via l’export d’intermédiaires du cycle de Krebs de la matrice vers le cytosol. Dans cette étude, nous avons montré que la délétion d’Ucp2 dans le cœur murin néonatal entraîne un retard de maturation des cardiomyocytes. Celui-ci est marqué par un défaut d’initiation du programme transcriptomique associé à la maturation, entrainant une altération de la biogénèse mitochondriale, une absence d’utilisation de la β-oxydation et des défauts de contractilité. L’analyse de l’activité métabolique a révélé qu’en absence d’Ucp2, le cœur néonatal utilise peu les acides gras et le pyruvate, mais devient dépendant de la glutamine via le GABA shunt. Cela entraîne un déséquilibre de la présence relative des intermédiaires du cycle de Krebs, notamment l’α-cétoglutarate et le succinate . Ces métabolites sont des régulateurs essentiels des TET, enzymes qui activent l’expression génique et sont impliquées dans la maturation métabolique et contractile des cardiomyocytes néonataux. Ces résultats démontrent le rôle essentiel d’Ucp2 dans la régulation métabolique des cardiomyocytes néonataux, et révèlent l’existence d’un nouveau type de signalisation mitochondriale initiant la maturation cardiaque post-nataleDuring development, the heart relies mainly on glycolysis for ATP production. At birth, exposure to atmospheric O2 levels and nutritional switch towards the fatty acid-rich maternal milk triggers a metabolic remodeling towards mitochondrial oxidative phosphorylation. Cardiomyocytes then rely on oxidative metabolism, particularly β-oxidation. The remodeling is concomitant with transcriptional changes allowing increasing mitochondrial biogenesis and maturation of the contractile machinery, thus optimizing energy production and heart contractile function. The augmentation of mitochondrial metabolic activity participates in the signaling ensuring the emergence of mature phenotypes, including cardiomyocyte cell-cycle arrest and transition to hypertrophy. Thus, in a long-term therapeutic view, a better understanding of mitochondrial signaling during maturation appears critical to improve cardiomyocyte in vitro models relevancy as well as to stimulate their proliferation in the adult in order to sustain heart regeneration. My PhD project is focused on the mitochondrial uncoupling protein 2 (Ucp2), specifically expressed in the neonatal heart, and its role during cardiomyocyte maturation. Ucp2 is a carrier located on the inner mitochondrial membrane, where it exerts 2 functions. (1) It regulates mitochondrial reactive oxygen species production and (2) it is involved in cell fuel preferences via Krebs metabolite export from the matrix to the cytosol. We have shown that Ucp2 deletion in neonatal mouse heart induces defective cardiomyocyte maturation. Indeed, Ucp2-/- cardiomyocytes display inability to induce the post-natal transcriptional program, including impaired mitochondrial biogenesis, absence of β-oxidation and defective contractility. Metabolic activity analysis revealed that Ucp2 mutant -heart poorly relies on pyruvate or fatty acids for ATP production. Instead, it shows metabolic dependency to glutamine through the GABA shunt. This is associated to unbalanced Krebs intermediates relative abundance, in particular α-ketoglutarate and succinate. These metabolites are important regulators of TET enzymes activity which activate gene expression and participate in neonatal cardiomyocyte metabolic and contractile maturation. These results demonstrate that Ucp2 plays a critical role in regulating metabolic activity of neonatal cardiomyocytes, and unravel a new type of mitochondrial signaling initiating postnatal heart maturation
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Ponzielli, Romina. "Etude du contrôle génétique de la morphogenèse du tube cardiaque chez la Drosophile." Aix-Marseille 2, 2002. http://www.theses.fr/2002AIX22073.
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Blandin, Camille. "Rôle de la protéine d'échafaudage CASK dans le remodelage adaptatif et pathologique, chez le rongeur et l'Homme." Electronic Thesis or Diss., Sorbonne université, 2023. http://www.theses.fr/2023SORUS503.
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Le disque intercalaire est l’entité moléculaire qui connecte deux cardiomyocytes adjacents au niveau de leurs extrémités terminales, à la fois électriquement et mécaniquement, via des complexes protéiques spécifiques. Cette organisation du disque, qui assure la cohésion des cellules myocardiques pendant les cycle de contraction et de relaxation, signe le degré de maturité et la fonctionnalité du cardiomyocyte. La désorganisation du disque est une caractéristique commune à de nombreuses pathologie cardiaques, d’origines héréditaires ou acquises. La compréhension des processus moléculaires impliqués dans l’organisation de ce domaine membranaire, et sa désorganisation dans le contexte pathologique, constituent un champ de recherche très actif. Dans ce contexte, nous avons identifié une protéine de la membrane latérale, CASK, qui contrôle l’organisation de protéines du disque intercalaire en régulant leur trafic antérograde. Dans des modèles in vitro (cardiomyocytes de rats néonataux ou cardiomyocytes dérivés de cellules souches pluripotentes humaines induites), l’invalidation de CASK promeut l’organisation et la stabilité des protéines du disque. L’invalidation de CASK chez le rongeur favorise également l’organisation des protéines du disque et conduit à une amélioration de la performance cardiaque. Chez l’Homme, dans le contexte la cardiomyopathie arythmogène du ventricule droit (CAVD), une pathologique associé à une perte de cohésion entre cardiomyocytes due à une altération des contacts mécaniques de type desmosome, l’expression de CASK est augmentée. In vitro, dans un modèle cellulaire humain de CAVD, l’invalidation de CASK restaure l’intégrité des contacts desmosomaux. L’ensemble de nos résultats suggère que CASK est un régulateur majeur du trafic intracellulaire, de l’adressage et de l’organisation des protéines du disque intercalaireThe intercalated disc is the molecular entity that connects two adjacent cardiomyocytes at their terminal ends, both electrically and mechanically, via specific protein complexes. The organization of the disc, which ensures the cohesion of myocardial cells during contraction and relaxation cycles, is a hallmark of the maturity and functionality of the cardiomyocyte. Disorganization of the disc is a common feature of many cardiac pathologies, both inherited and acquired. Understanding the molecular processes involved in the organization of this membrane domain, and its disorganization in the pathological context, is a very active field of research. In this context, we have identified a lateral membrane protein, CASK, which controls the organization of intercalated disc proteins by regulating their anterograde trafficking. In in vitro models (neonatal rat cardiomyocytes or cardiomyocytes derived from induced human pluripotent stem cells), invalidation of CASK promotes the organization and stability of disc proteins. Invalidation of CASK in rodents also promotes disc protein organization and leads to improved cardiac performance. In human arrhythmogenic right ventricular cardiomyopathy (ARVC), a pathological condition associated with loss of cohesion between cardiomyocytes due to altered desmosome-type mechanical contacts, CASK expression is increased. In vitro, in a human cell model of ARVC, CASK invalidation restores the integrity of desmosomal contacts. Taken together, our results suggest that CASK is a major regulator of intracellular trafficking, addressing and organization of intercalated disc proteins
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Lau, Siu-ling. "Characterization of adenosine transport in rat cardiomyocytes, H9c2 /." View the Table of Contents & Abstract, 2005. http://sunzi.lib.hku.hk/hkuto/record/B31494894.
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Kane, Émilie. "Protein-protein regulation of calsequestrin expression in cardiomyocytes." Thesis, McGill University, 2012. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=107782.
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Heart failure is the leading cause of death in both men and women of Western countries. The pathophysiology of heart failure is associated with abnormalities in intracellular calcium control. Calsequestrin (CSQ2), a calcium storage protein in cardiomyocytes, is negatively regulated by the transcription factor Egr-1 thus altering calcium availability for cardiac contraction/relaxation. Here, we tested the hypothesis that the proteins complexed to Egr-1 and/or their post-translational modifications would affect regulation of CSQ2 expression. Egr-1 and Sp1 compete for binding at the CSQ2 promoter, but also bind one another. In fact, together they form a complex with another ubiquitous transcription factor YBX-1. This complex was identified in vivo and in vitro by a series of co-immunoprecipitations. To test the idea that complex formation and CSQ2 expression could be affected by acetylation, histone acetyltransferase (HAT) inhibitors and histone deacetylase (HDAC) inhibitors were used to respectively decrease or increase acetylation within cells. We found that acetylation did not impact the formation of the Egr-1: Sp1: YBX-1 complex thought to regulate CSQ2 expression. However, changes in CSQ2 expression were observed when acetylation was modified by HAT and HDAC inhibitors. We conclude that acetylation modifies CSQ2 expression although not by means of the Egr-1: Sp1: YBX-1 complex even though Egr-1 is known to be acetylated.La maladie du coeur est la cause principale de mortalité chez les femmes et les hommes en Occident. L'arrêt cardiac est souvent associé à des anomalies en control de calcium intracellulaire. Le facteur de transcription Egr-1 est connu pour son contrôle négatif de l'expression de calsequestrine (CSQ2), une protéine réservoir de calcium. Un manque de CSQ2 impacte les quantités de calcium disponible pour un bon fonctionnement cardiaque. Ici nous avons examiné l'hypothèse que les protéines liées à Egr-1 et que ses modifications post-translationelles. Egr-1 et Sp1 sont en compétition pour le promoteur de CSQ2 mais sont aussi liés l'un à l'autre. En fait, ensemble ils forment un complexe avec le facteur de transcription universel YBX-1. Ce complexe a été identifié en vivo et en vitro par co-immunoprécipitations. Considérant que la formation de ce complexe ainsi que l'expression de CSQ2 peuvent être affectés par l'acétylation, des inhibiteurs d'acétyltransferase d'histone (ATH) et de déacétylase d'histone (DACH) ont été respectivement utilisés pour réduire et augmenter l'acétylation en cellules. Nous avons trouvés que la formation du complexe Egr-1: Sp1: YBX-1 qui est possiblement responsable de l'expression de CSQ2 n'est pas affecté par les changements en acétylation. Par contre, des changements ont été aperçus dans l'expression de CSQ2 quand l'acétylation a été modifiée par ATH ou par DACH. Nous croyons que l'acétylation impacte l'expression de CSQ2 mais pas par entremise du complexe Egr-1 : Sp1 : YBX-1 malgré l'acétylation de la protéine Egr-1 elle-même.
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Kilborn, Michael John. "Postnatal changes in electrophysiological properties of rat cardiomyocytes." Thesis, University of Oxford, 1990. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.292673.
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Lau, Siu-ling, and 劉少玲. "Characterization of adenosine transport in rat cardiomyocytes, H9c2." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2005. http://hub.hku.hk/bib/B45010237.
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Païta, Lucille. "Caractérisation du canal cationique TRPV1 dans les cardiomyocytes." Thesis, Lyon, 2016. http://www.theses.fr/2016LYSE1329/document.
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L'infarctus du myocarde, une des causes majeures de mortalité à travers le monde, engendre une mort irréversible du muscle cardiaque suite à une ischémie. Cette ischémie, c'est-à-dire une privation de dioxygène et de nutriments, déclencher un stress réticulaire qui perturbe l'équilibre calcique de la cellule cardiaque. Plusieurs pompes et canaux calciques situés à la membrane plasmique ou réticulaire sont des intervenants clés dans le maintien de l'homéostasie calcique. Parmi eux, il existe des canaux de fuites calciques passives, comme les TRPs, et peu d'informations sont actuellement connus à propos de leur rôle précis au cours de l'infarctus du myocarde.TRPV1 est un canal cationique non sélectif qui est activé par la capsaïcine, le pH et la chaleur nocive (>42°C). Dans le muscle squelettique, nous avions démontré que TRPV1 est situé dans la partie longitudinale du réticulum sarcoplasmique et qu'il répond à différentes stimulations physiologiques et pharmacologiques (Lotteau et al., 2013). Ici, nous nous interrogeons sur un éventuel rôle similaire de TRPV1 dans la physiologie cardiaque. Des analyses biochimiques et des mesures de calcium intracellulaire furent réalisées sur des cardiomyocytes issus de souris WT et KO TRPV1. Nos résultats in vitro montrent que: (a) TRPV1 est exprimé dans les cellules cardiaques; (b) une activation de TRPV1 engendre une réduction de la concentration calcique réticulaire et que (c) TRPV1 pourrait être une cible directe de l'isoflurane.Dans la mesure où TRPV1 peut être modulé par de nombreuses molécules pharmacologiques, il pourrait constituer une cible thérapeutique pour réduire la taille d'infarctus. De nombreuses études antérieures ont déjà mis en évidence un rôle cardioprotecteur de TRPV1 dans le système nerveux entourant le cœur. Le but de cette étude est de décrire le fonctionnement des canaux TRPV1 dans des cardiomyocytes adultesAcute myocardial infarction (MI), a leading cause of morbidity and mortality worldwide, is the irreversible death of heart muscle secondary to ischemia. This ischemia, i.e. oxygen and nutrients deprivation, triggers a reticular stress disrupting the Ca2+ balance of the cardiac cell. Several Ca2+ pumps and channels located at the sarcolemma or at the reticulum membrane are key players in this maintenance of Ca2+ homeostasis. Among them, we find passive leak channels, such as TRPs and little is known about their precise role in MI.TRPV1 represents a non-selective cation channel that is activated by capsaicin, pH and noxious heat. In skeletal muscle, we previously demonstrated that TRPV1 is located in the longitudinal part of the SR and respond to pharmacological and physiological activations (Lotteau et al., 2013). We questioned here whether TRPV1 might have a similar role in heart physiology. Biochemical analysis and intracellular Ca2+ measurements were performed on cardiomyocytes from wild-type and TRPV1-KO mice. Our in vitro results show that: (i) TRPV1 is expressed in cardiac cells; (ii) an increase in intracellular calcium concentration ([Ca2+]i) is elicited under TRPV1 activation; (iii) TRPV1 could be a direct target of isoflurane. In parallel, our in vivo results indicate that a pharmacological preconditioning by isoflurane decrease the infarct size, probably though activation of TRPV1. According to the fact that TRPV1 activity can be modulated by a lot of pharmacological molecules, TRPV1 may serve as therapeutic target to reduce the infarct size. Most of published data have already evidenced this TRPV1 cardioprotective role in the peripheral heart system. The aim of the present work is to describe how TRPV1 channels behave in adult cardiomyocytes
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Kranzhöfer, David K. [Verfasser], Lutz [Akademischer Betreuer] Hein, and Christian [Akademischer Betreuer] Flotho. "DNA demethylation in cardiomyocytes during development and maturation." Freiburg : Universität, 2021. http://d-nb.info/1226091261/34.
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Kanaan, Georges. "Mitochondrial Dysfunction: From Mouse Myotubes to Human Cardiomyocytes." Thesis, Université d'Ottawa / University of Ottawa, 2018. http://hdl.handle.net/10393/37582.
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Mitochondrial dysfunction is a common feature in a wide range of disorders and diseases from obesity, diabetes, cancer to cardiovascular diseases. The overall goal of my doctoral research has been to investigate mitochondrial metabolic dysfunction in skeletal and cardiac muscles in the context of chronic disease development.
Perinatal nutrition is well known to affect risk for insulin resistance, obesity, and cardiovascular disease during adulthood. The underlying mechanisms however, are poorly understood. Previous research from our lab showed that the in utero maternal undernutrition mouse model is one in which skeletal and cardiac muscle physiology and metabolism is impaired. Here we used this model to study the impact of in utero undernutrition on offspring skeletal primary muscle cells and to determine if there is a cell autonomous phenotype. Metabolic analyses using extracellular flux technologies revealed a shift from oxidative to glycolytic metabolism in primary myotubes. Gene expression profiling identified significant changes in mRNA expression, including an upregulation of cell stress and OXPHOS genes and a downregulation of cell division genes. However, there were no changes in levels of marker proteins for mitochondrial oxidative phosphorylation (OXPHOS). Findings are consistent with the conclusion that susceptibility to metabolic disease in adulthood can be caused at least in part by muscle defects that are programmed in utero and mediated by impaired mitochondrial function.
In my second project, the effects of the absence of glutaredoxin-2 (Grx2) on redox homeostasis and on mitochondrial dynamics and energetics in cardiac muscle from mice were investigated. Previous work in our lab established that Grx2-deficient mice exhibit fibrotic cardiac hypertrophy, and hypertension, and that complex I of OXPHOS is defective in isolated mitochondria. Here we studied the role of Grx2 in the control of mitochondrial structure and function in intact cells and tissue, as well as the role of GRX2 in human heart disease. We demonstrated that the absence of Grx2 impacts mitochondrial fusion, ultrastructure and energetics in mouse primary cardiomyocytes and cardiac tissue and that provision of the glutathione precursor, N-acetylcysteine (NAC) did not restore glutathione redox or prevent impairments. Furthermore we used data from the human Genotype-Tissue Expression consortium to show that low GRX2 expression is associated with increased fibrosis, hypertrophy, and infarct in the left ventricle. Altogether, our results indicate that GRX2 plays a major role in cardiac mitochondrial structure and function, and protects against left ventricle pathologies in humans.
In my third project, we collaborated with cardiac surgeon, Dr. Calum Redpath, of the Ottawa Heart Institute to study atrial mitochondrial metabolism in atrial fibrillation patients with and without type 2 diabetes (T2DM). T2DM is a major risk factor for atrial fibrillation, but the causes are poorly understood. Atrial appendages from coronary artery bypass graft surgery were collected and analyzed. We showed an impaired complex I respiration in diabetic patients with atrial fibrillation compared to diabetic patients without atrial fibrillation. In addition, and for the first time in atrial fibrillation patients, mitochondrial supercomplexes were studied; results showed no differences in the assembly of the “traditional” complexes but a decrease in the formation of “high oligomeric” complexes. A strong trend for increased protein oxidation was also observed. There were no changes in markers for OXPHOS protein levels. Overall findings reveal novel aspects of mitochondrial dysfunction in atrial fibrillation and diabetes in humans.
Overall, our results reveal that in utero undernutrition affects the programming of skeletal muscle primary cells, thereby increasing susceptibility to metabolic diseases. In addition, we show that GRX2 impacts cardiac mitochondrial dynamics and energetics in both mice and humans. Finally, we show impaired mitochondrial function and supercomplex assembly in humans with atrial fibrillation and T2DM. Ultimately, understanding the mechanisms causing mitochondrial dysfunction in muscle tissues during chronic disease development will increase our capacity to identify effective prevention and treatment strategies.
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Hurst, MaryKathryn. "VEGF-R1 AGONISTS PROTECT CARDIOMYOCYTES AGAINST OXIDATIVE STRESS." Master's thesis, Temple University Libraries, 2014. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/239774.
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Biomedical SciencesM.S.
VEGF-R1 Agonists protect Cardiomyocytes against Oxidative Stress Background: Selective agonists of the vascular endothelial growth factor receptor 1 (VEGFR-1) display cytoprotective and anti-apoptotic effects in the failing heart. Since a major determinant of myocardial damage in heart failure is oxidative stress, we tested the hypothesis that VEGFR-1 mediates anti-oxidant mechanisms. Methods: Freshly prepared cardiac tissue slices were obtained from dogs with pacing-induced heart failure that had been previously transduced with the VEGFR-1 selective ligand VEGF-B. Dihydroetidium (DHE) fluorescence was used to monitor the production of reactive oxygen species. In addition, cultured rat neonatal cardiomyocytes were tested with two major mediators causing oxidative stress in the failing heart, namely angiotensin II (10-8 M for 24 hours) and norepinephrine (50 µM for 24 hours). The experiments were performed in the absence or in the presence of the VEGFR-1 agonists VEGF-B and PlGF or of the mixed VEGFR-1 and VEGFR-2 agonist VEGF-A or of the selective VEGFR-2 agonist VEGF-E. Mitochondrial superoxide and cytosolic hydrogen peroxide were measured, respectively, as MitoSox and DCF fluorescence intensity. Results: In fresh cardiac tissue slices, DHE fluorescence indicated that superoxide production was significantly reduced in VEGF-B treated hearts compared to control failing hearts. In cultured cardiomyocytes, VEGF-B and PlGF, but not VEGF-A or VEGF-E, prevented mitochondrial superoxide and cytosolic hydrogen peroxide overproduction in response to angiotensin II or norepinephrine. These findings were consistent with the induction of mitochondrial superoxide dismutase and glutathione peroxidase-1 overexpression in VEGF-B-treated cells. Conclusions: VEGF-R1 activation can reduce oxidative stress both in vivo and in vitro. Our results provide insights in the cardioprotective mechanisms activated by VEGF-B gene therapy in the failing heart.
Temple University--Theses
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Jeziorowska, Dorota. "Analyse des voies de régulation de la cardiogenèse et de la différenciation cardiomyocytaire." Thesis, Paris 6, 2016. http://www.theses.fr/2016PA066631/document.
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L'objectif général de ce travail de doctorat a été centré sur l'utilisation des cellules pluripotentes induites humaines dans la modélisation et l'évaluation thérapeutique des pathologies cardiaques. Depuis leur découverte en 2006, les iPSC offrent une opportunité pour le développement de modèles cellulaires humains et spécifiques de patients pour l'étude des mécanismes physiopathologiques, l'évaluation de réponses pharmacologiques et le génération de cellules redifférenciées (ici en cardiomyocytes) pour des applications thérapeutiques cellulaires. Dans ce travail nous avons démontré que la quantité mais aussi la qualité finale des cardiomyocytes dérivés d'iPSC dépend des conditions spatiales et pharmacologiques utilisées durant les différentes étapes de différenciation. L'utilisation d'un protocole de différentiation en monocouche avec blocage simultané et transitoire de l'ensemble des voies Wnt (canoniques et non canoniques) permet d'obtenir une maturation plus importante du sarcomère, étape essentielle pour la modélisation des sarcomèropathies La différenciation des iPSC en cardiomyocytes peut aussi être obtenue par une approche moléculaire ciblée visant à activer spécifiquement un programme cardiogénique. Celle-ci est obtenue via l'utilisation d'une protéine Cas9 mutée et couplée à un système transactivateur et permettant le ciblage simultané de 3 facteurs de transcription clés de la cardiogénèse (Gata4, Mef2c et Tbx5). Cette approche moléculaire est renforcée par la combinaison avec une stimulation pharmacologique ciblant la voie WntThe general objective of this work was centered on the use of human induced pluripotent cells in modeling and therapeutic evaluation of cardiac pathologies. Since their discovery in 2006, the iPSC provide an opportunity for the development of human cellular models and specific patients for the study of pathophysiological mechanisms, evaluation of pharmacological responses and the generation redifférenciées cells (cardiomyocytes here) for applications cellular therapeutic. In this work we demonstrated that the quantity but also the final quality of cardiomyocytes derived from iPSC depends on the spatial and pharmacological conditions used during the various stages of differentiation. The use of a monolayer differentiation protocol with simultaneous and transient blocking of all Wnt pathways (canonical and noncanonical) allows to obtain a higher maturation of the sarcomere, an essential step for modeling sarcomeropathies IPSC differentiation into cardiomyocytes can also be obtained by targeted molecular approach to specifically activate cardiogenic program. This is achieved through the use of a mutated Cas9 protein and coupled with transactivator system. This allows simultaneous targeting of 3 key cardiogenesis transcription factors (Gata4, MEF2C and Tbx5). This molecular approach is enhanced by the combination with a pharmacological stimulation targeting the Wnt pathway. Beyond modeling of monogenic cardiac disease, cardiomyocytes derived from iPSC can reproduce more complex and multigenic diseases
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Anwar, Attia. "Functional role of NFAT in ventricular cardiomyocytes of rat." Giessen VVB Laufersweiler, 2006. http://geb.uni-giessen.de/geb/volltexte/2006/3817/index.html.
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Potter, Ryan Wayne. "Protamine alters sodium and potassium currents in isolated cardiomyocytes /." Title page and abstract only, 2002. http://web4.library.adelaide.edu.au/theses/09SB/09sbp868.pdf.
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Huang, Brandon Pei Han. "The regulation of protein synthesis in adult rat cardiomyocytes." Thesis, University of British Columbia, 2008. http://hdl.handle.net/2429/976.
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Protein synthesis (mRNA) is tightly regulated under numerous conditions in cardiomyocytes. It can be activated by hormones such as insulin and also by other agents such as phenylephrine (PE) that activates hypertrophy in the heart. Cardiac hypertrophy involves an increase in the muscle mass of the heart, principally in the left ventricular muscle, and the increase is due to enlarged cell size, not increased cell number. A pivotal element of cardiac hypertrophy is an elevation in the rates of protein synthesis, which drives the increase in cell size causing hypertrophy. Unfortunately, we currently lack the understanding of the basic mechanisms that drives hyperactivated protein synthesis. Cardiac hypertrophy is clinically important because it is a major risk factor for heart failure. It initially serves as an adaptive response to increase cardiac output in response to higher demand, but ultimately leads to deterioration of contractility of the heart if hypertrophy is sustained. The main goal of this research project is to understand how hypertrophic agents, such as phenylephrine (PE), activate protein synthesis using adult rat ventricular cardiomyocytes as a model. Specifically, this study focuses on how the translational initiation is controlled by upstream signalling pathways.
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Schulson, Meredith Nicole. "The structure of excitation-contraction coupling in atrial cardiomyocytes." Thesis, University of British Columbia, 2009. http://hdl.handle.net/2429/3981.
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Standard local control theory, which describes Ca²⁺ release during excitation-contraction coupling (ECC), assumes that all Ryanodine Receptor (RyR) complexes are equivalent. Recent data from our laboratory has called this assumption into question. Specifically, we have shown that RyR complexes in ventricular myocytes differ depending on their location within the cell. This, and other data, has led us to hypothesize that similar differences occur within the rat atrial cell.
To test this hypothesis, we have triple-labeled enzymatically-isolated, fixed myocytes to examine the distribution and colocalization of RyR, calsequestrin (CSQ), voltage-gated Ca²⁺ channels (Cav1.2), sodium-calcium exchangers (NCX), and caveolin-3 (cav-3). All images were acquired on a wide-field microscope, deconvolved, and subject to extensive analysis, including a novel method of measuring statistical significance of the recorded colocalization values.
Overall, eight surface RyR populations were identified, depending on its binding partners. One of these groups, in which RyR, Cav1.2, and NCX colocalize, may provide the structural basis for ‘eager’ sites of Ca²⁺ release in atria, while other groups were defined based on their association with cav-3, and are therefore highly likely to be under the influence of other signaling molecules located within caveolae. Importantly, although a small portion of the surface RyR in atria do colocalize with NCX alone, the majority are tightly linked to Cav1.2 alone or Cav1.2 and NCX together. Therefore, it appears likely that Cav1.2-mediated calcium-induced calcium release (CICR) is the primary method of initiating Ca²⁺ release from the SR during EC coupling.
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Chan, Sai Yen Victor, and 陳世欽. "Effect of homocysteine on nitric oxide production in cardiomyocytes." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2001. http://hub.hku.hk/bib/B31970321.
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Plamondon, Philippe. "La MAP kinase p38γ influence la structure des cardiomyocytes." Mémoire, Université de Sherbrooke, 2014. http://savoirs.usherbrooke.ca/handle/11143/5307.
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Le cœur est un organe central au fonctionnement du système cardiovasculaire. Il est physiologiquement compartimenté et est constitué de cellules spécialisées qui régulent les impulsions électriques ainsi que la contraction du myocarde. Le cœur adapte le flux sanguin en fonction des besoins du corps. En condition pathologique, le cœur recourt toutefois à des mécanismes compensatoires. Au niveau physiologique, la compensation s’observe par l’hypertrophie des cardiomyocytes qui, bien que bénéfique à court terme, exacerbe à long terme la fonction cardiaque. L’activation des « mitogen activated protein kinases » (MAPK) contribue autant au maintien de la fonction physiologique qu’à la détérioration pathologique du myocarde et serait également une cause de l’hypertrophie observée. Parmi les 5 groupes de MAPK connues, la MAPK p38 est formée de 4 isoformes dont les sérine/thréonine kinases p38α et p38γ sont exprimées de façon prédominante dans le cœur. Les p38 partagent les mêmes activateurs, mais leurs effecteurs diffèrent. Bien que le rôle de p38α semble impliqué dans l’aggravement des troubles cardiaques, celui de p38γ ne semble pas redondant à p38α et demeure incompris. Cette isoforme possède un motif de liaison aux domaines PDZ, unique chez les MAP kinases. Également, chez les cellules cardiaques, elle transloque au noyau en condition de stress. Le but de l’étude ici est de comprendre le rôle de p38γ et de ses motifs uniques sur la structure et la taille des cardiomyocytes. Afin de répondre au but de l’étude, plusieurs mutants adénoviraux de p38 ont été conçus. Un des mutants ne possède pas le motif de liaison aux domaines PDZ, deux autres contrôlent la localisation cellulaire soit au noyau, soit au cytoplasme, et un autre mutant est muté au site de phosphorylation. Des cardiomyocytes en culture ont été infectés par les différents mutants en présence de leur activateur en amont ou de la β-galactosidase. Les réseaux d’α-actinine, ainsi que la taille des cardiomyocytes, ont été observés par microscopie. Les observations effectuées montrent que p38γ entraîne une désorganisation des réseaux d’α-actinine lorsqu’il est phosphorylé. Également, il facilite l’hypertrophie des cardiomyocytes en présence de son activateur s’il est forcé hors du noyau ou en l’absence de son motif de liaison aux domaines PDZ. En conclusion, les résultats obtenus suggèrent que p38γ exerce bel et bien un rôle dans le maintien structural des cardiomyocytes par l’intermédiaire de l’α-actinine.
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Miller, Stewart L. W. "A study of excitation contraction coupling in rabbit cardiomyocytes." Thesis, University of Glasgow, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.404445.
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Bettencourt, Carvalho Dias Monica. "Plasticity of the differentiated state in adult newt cardiomyocytes." Thesis, University College London (University of London), 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.274931.
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Susaeta, Isidro Allue. "Rigor, cytosolic ATP and pH in metabolically poisoned cardiomyocytes." Thesis, University of Liverpool, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.262588.
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Motion, J. P. Michael (Joseph Patrick Michael Motion Champana). "Mechanically-induced intercellular remodeling of cardiomyocytes by magnetic micromanipulation." Thesis, Massachusetts Institute of Technology, 2006. http://hdl.handle.net/1721.1/37202.
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Thesis (M. Eng.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, 2006.Includes bibliographical references (leaves 68-72).
Gap junctions are responsible for providing and maintaining a pathway for intercellular communication. This is critical in the heart where gap junctions are responsible for maintaining electrical impulse propagation. Connexin43 (Cx43) is the most abundant gap junction in the heart, and studies have shown that spatial heterogeneity of Cx43 may promote electrical instability and anisotropic conduction pathways that may cause cardiac arrhythmias. Structural and electrical remodeling of gap junctions have been linked to increases in stresses in conditions such as hypertrophy. Understanding how local mechanical forces influence the remodeling of gap junctions can provide insight into arrhythmias and reentry circuits. In this work, I describe a system for exerting local mechanical forces on cardiomyocytes to study gap junction remodeling and I show that cell-to-cell movement and subsequent remodeling of Cx43 can occur. The system consisted of patterned linear strands on polyacrylamide gels and mechanical stimulation using magnetic micromanipulation. Cardiomyocytes were patterned on polyacrylamide gel using 25pm and 50pm microchannels. Mechanical stimulation was induced in sections with high densities of magnetic beads.
(cont.) With a maximal input current of 1.5A, the system generated 1.5nN at 100pm distance from the magnetic trap, and this was sufficient to induce cell-to-cell movement. Cell-to-cell movement was measured to be 0.032±0.03pm/min, three times faster than the average cell-to-cell movement under no applied force. Remodeling of Cx43 was also shown using Cx43-YFP transfected cells while a local force induced cell-to-cell movement. Changes in both the distribution and expression of the protein were seen throughout time as the linear strand was pulled by the magnetic force. We conclude that this system can induce remodeling of Cx43 by an applied local force. This work establishes a system to allow to quantification of applied mechanical loads and resultant Cx43 remodeling.
by J.P. Michael Motion.
M.Eng.
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Humphrey, Peter Saah. "Signal transduction mechanisms for stem cell differentation into cardiomyocytes." Thesis, University of Hertfordshire, 2009. http://hdl.handle.net/2299/3760.
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Cardiovascular diseases are among the leading causes of death worldwide and particularly in the developed World. The search for new therapeutic approaches for improving the functions of the damaged heart is therefore a critical endeavour. Myocardial infarction, which can lead to heart failure, is associated with irreversible loss of functional cardiomyocytes. The loss of cardiomyocytes poses a major difficulty for treating the damaged heart since terminally differentiated cardiomyocytes have very limited regeneration potential. Currently, the only effective treatment for severe heart failure is heart transplantation but this option is limited by the acute shortage of donor hearts. The high incidence of heart diseases and the scarcity donor hearts underline the urgent need to find alternative therapeutic approaches for treating cardiovascular diseases. Pluripotent embryonic stem (ES) cells can differentiate into functional cardiomyocytes. Therefore the engraftment of ES cell-derived functional cardiomyocytes or cardiac progenitor cells into the damaged heart to regenerate healthy myocardial tissues may be used to treat damaged hearts. Stem cell-based therapy therefore holds a great potential as a very attractive alternative to heart transplant for treating heart failure and other cardiovascular diseases. A major obstacle to the realisation of stem cell-based therapy is the lack of donor cells and this in turn is due to the fact that, currently, the molecular mechanisms or the regulatory signal transduction mechanisms that are responsible for mediating ES cell differentiation into cardiomyocytes are not well understood. Overcoming this huge scientific challenge is absolutely necessary before the use of stem cell-derived cardiomyocytes to treat the damaged heart can become a reality. Therefore the aim of this thesis was to investigate the signal transduction pathways that are involved in the differentiation of stem cells into cardiomyocytes. The first objective was the establishment and use of cardiomyocyte differentiation models using H9c2 cells and P19 stem cells to accomplish the specific objectives of the thesis. The specific objectives of the thesis were, the investigation of the roles of (i) nitric oxide (ii) protein kinase C (PKC), (iii) p38 mitogen-activated protein kinase (p38 MAPK) (vi) phosphoinositide 3-kinase (PI3K) and (vi) nuclear factor-kappa B (NF-kB) signalling pathways in the differentiation of stem cells to cardiomyocytes and, more importantly, to identify where possible any points of convergence and potential cross-talk between pathways that may be critical for differentiation to occur. P19 cells were routinely cultured in alpha minimal essential medium (α-MEM) supplemented with 100 units/ml penicillin /100 μg/ml streptomycin and 10% foetal bovine serum (FBS). P19 cell differentiation was initiated by culturing the cells in microbiological plates in medium containing 0.8 % DMSO to form embryoid bodies (EB). This was followed by transfer of EBs to cell culture grade dishes after four days. H9c2 cells were cultured in Dulbecco’s Modified Eagle’s medium (DMEM) supplemented with 10% FBS. Differentiation was initiated by incubating the cells in medium containing 1% FBS. In both models, when drugs were employed, they were added to cells for one hour prior to initiating differentiation. Cell monolayers were monitored daily over a period of 12 or 14 days. H9c2 cells were monitored for morphological changes and P19 cells were monitored for beating cardiomyocytes. Lysates were generated in parallel for western blot analysis of changes in cardiac myosin heavy chain (MHC), ventricular myosin chain light chain 1(MLC-1v) or troponin I (cTnI) using specific monoclonal antibodies. H9c2 cells cultured in 1% serum underwent differentiation as shown by the timedependent formation of myotubes, accompanied by a parallel increase in expression of both MHC and MLC-1v. These changes were however not apparent until 4 to 6 days after growth arrest and increased with time, reaching a peak at day 12 to 14. P19 stem cells cultured in DMSO containing medium differentiated as shown by the timedependent appearance of beating cardiomyocytes and this was accompanied by the expression of cTnI. The differentiation of both P19 stem cells and H9c2 into cardiomyocytes was blocked by the PI3K inhibitor LY294002, PKC inhibitor BIM-I and the p38 MAPK inhibitor SB2035800. However when LY294002, BIM-I or SB2035800 were added after the initiation of DMSO-induced P19 stem cell differentiation, each inhibitor failed to block the cell differentiation into beating cardiomyocytes. The NF-kB activation inhibitor, CAPE, blocked H9c2 cell differentiation into cardiomyocytes. Fast nitric oxide releasing donors (SIN-1 and NOC-5) markedly delayed the onset of differentiation of H9c2 cells into cardiomyocytes while slow nitric oxide releasing donors (SNAP and NOC-18) were less effective in delaying the onset of differentiation or long term differentiation of H9c2 cells into cardiomyocytes. Akt (protein kinase B) is the key downstream target of PI3K. Our cross-talk data also showed that PKC inhibition and p38 MAPK inhibition respectively enhanced and reduced the activation of Akt, as determined by the phosphorylation of Akt at serine residue 473. In conclusion, PKC, PI3K, p38 MAPK and NF-kB are relevant for the differentiation of stem cells into cardiomyocytes. Our data also show that the PKC, PI3K and p38 MAPK signalling pathways are activated as very early events during the differentiation of stem cells into cardiomyocytes. Our data also suggest that PKC may negatively regulate Akt activation while p38 MAPK inhibition inhibits Akt activation. Our fast NO releasing donor data suggest that nitric oxide may negatively regulate H9c2 cell differentiation.
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Kolker, Ljudmila. "Differentation and maturation of pluripotent stem cell-derived cardiomyocytes." Thesis, Imperial College London, 2013. http://hdl.handle.net/10044/1/26149.
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Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) may serve as tools in pharmacology for drug screening and safety evaluations and in future cardiac regenerative therapies. However, the use of hPSC-CMs is hampered by their immature foetal-like structural and functional characteristics. This project aimed to evaluate two pro-maturation stimuli to enhance the maturation of hPSC-CMs: administration of triiodothyronine (T3), a hypertrophic stimulus known to be crucial for cardiac development, and overexpression of Bin1(-EGFP), which may be implicated in the formation of t-tubules (TTs). T3-signalling may be suboptimal during in vitro culture and TTs, a hallmark of mature ventricular cardiomyocytes, are absent in hPSC-CMs. To investigate the effect of the pro-maturation stimuli initially cardiomyocytes were derived from the human embryonic stem cell line Shef3 via spontaneous or directed differentiation methods but due to low differentiation efficiency all maturation studies were conducted on iCell-CMs, a commercially produced line of human induced pluripotent stem cell-derived cardiomyocytes. To assess their initial state of maturity and subsequently to evaluate the impact of the pro-maturation stimuli on iCell-CMs the molecular, structural and electrophysiological properties of the cells were extensively characterised using qRT-PCR, immunostaining, bright field and transmission electron microscopy, micro electrode array and the whole-cell patch-clamping technique. Characterisation of iCell-CMs revealed an overall immature phenotype: iCell-CMs were smaller than adult cardiomyocytes; exhibited relatively low expression of key ion channels (Kir2.1 and Kv4.3), major Ca2+ handling components (e.g. RyR2, SERCA2a and CSQ2) and TT-associated proteins (e.g. Bin1 and caveolin 3); showed poor ultrastructural organisation and lacked TTs. However, the sarcoplasmic reticulum (SR) Ca2+ contributed to cell contraction similarly to adult cardiomyocytes. Administration of T3 increased the expression of the hypertrophic marker ANP and enhanced the maturation of iCell-CMs as measured by a shift in the expression levels of SERCA2a, Kv4.3 and Cav3.1 closer to the levels of adult heart controls. Overexpression of Bin1(-EGFP) increased the expression of CSQ2, Kir2.1 and Kv4.3 and induced formation of TT-like sarcolemmal invaginations. However, the tubular structures were not aligned to myofibrils and may have been connected to the SR at only few foci. Localisation of L-type Ca2+ channels on the induced tubules could not be definitely confirmed but the increased Ca2+ current density in transfected cells suggested that the channels may have been recruited to the tubules. Also, overall the expression levels of most analysed genes in T3-treated or Bin1-EGFP-transfected cells remained markedly below adult heart controls including RyR2, CSQ2, Kir.2.1, Kv4.3 and caveolin 3. In conclusion, both T3-treatment and overexpression of Bin1(-EGFP) promoted the maturation of iCell-CMs to some measure but did not induce a fully adult-like phenotype. Thus the ultimate maturation strategy for hPSC-CMs still remains to be found.
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Emond-Boisjoly, Marc-Alexandre. "Rôle de la protéine DUSP5 dans l’autophagie des cardiomyocytes." Mémoire, Université de Sherbrooke, 2016. http://hdl.handle.net/11143/8908.
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Résumé: L’autophagie est un processus essentiel au maintien de l’homéostasie cellulaire. Elle permet de dégrader et recycler aussi bien des organelles entières que des composants cytoplasmiques non fonctionnels. De plus, l’augmentation d’autophagie en condition de stress constitue une réponse adaptative favorisant la survie cellulaire. Chez les cardiomyocytes, l’autophagie en condition basale est indispensable au renouvellement, entre autres, des mitochondries et des protéines formant les sarcomères. De plus, les stress tels l’ischémie cardiaque ou la carence en nutriments induisent une augmentation de l’autophagie protectrice. Dans certaines conditions extrêmes, il a été suggéré qu’un surcroît d’autophagie puisse toutefois exacerber la pathologie cardiaque en provoquant la mort des cardiomyocytes. Considérant l’importance de ce processus dans la physiopathologie cardiaque, l’identification des mécanismes signalétiques régulant l’autophagie chez les cardiomyocytes a été le sujet de recherches intenses. À cet effet, l’activation des Mitogen-Activated Protein Kinase (MAPK) a été démontrée pour réguler, avec d’autres voies signalétiques, l’autophagie et l’apoptose des cardiomyocytes. Il est donc probable que les Dual-Specificity Phosphatase (DUSP), enzymes clés contrôlant l’activité des MAPK, participent aussi à la régulation de l’autophagie. Afin de vérifier cette hypothèse, nous avons induit l’autophagie chez des cardiomyocytes isolés de rats nouveau-nés en culture. L’analyse de marqueurs d’autophagie par immunobuvardage démontre que l’activation des MAPK ERK1/2 et p38 corrèle avec l’activité autophagique chez les cardiomyocytes. Dans ces conditions, la diminution d’expression de la majorité des ARNm encodant les différentes DUSP retrouvées chez les cardiomyocytes contraste de façon marquée avec l’augmentation d’expression de l’ARNm Dusp5. De plus, nous avons démontré par une étude de gain de fonction que l’activation soutenue de p38 par surexpression d’un mutant MKK6 constitutivement actif stimule l’autophagie chez les cardiomyocytes. De façon surprenante, la perte de fonction de p38 obtenue par surexpression d’un mutant p38 dominant négatif n’altère en rien la réponse autophagique initiatrice dans notre modèle in vitro. Nos résultats suggèrent que les DUSP puissent réguler, via leurs actions sur les MAPK, d’importantes étapes du processus autophagique chez les cardiomyocytes.Abstract: Autophagy is a process essential to the maintenance of cellular homeostasis. It helps degrade and recycle whole organelles and nonfunctional cytoplasmic components. In addition, the adaptative up regulation of autophagy in stress condition promotes cell survival. In cardiomyocytes basal autophagy is essential to the renewal of, among others, mitochondria and proteins forming sarcomeres. In addition, stresses such as ischemic heart or nutrient deficiency induce an increase in protective autophagy. In extreme conditions, it has been suggested that autophagy may exacerbate cardiac disease causing the death of cardiomyocytes. Considering the importance of this process in cardiac pathophysiology, identify ing safety mechanisms regulating autophagy in cardiomyocytes has been the subject of intense research. To this end, activation of mitogen-activated protein kinase (MAPK) has been demonstrated to regulate, with other signaling pathways, autophagy and cardiomyocyte apoptosis. It is therefore likely that Dual-Specificity Phosphatases (DUSPs), key enzymes that control the activity of MAPKs, also participate in the regulation of autophagy. To test this hypothesis, we have induced autophagy in isolated cardiomyocytes of newborn rats in culture. Analysis of autophagy markers by immunoblotting demonstrated that the activation of MAPKs ERK1/2 and p38 correlates with autophagic activity in cardiomyocytes. Under these conditions, the decrease in expression of the majority of mRNAs encoding different DUSPs found in cardiomyocytes contrast sharply with the increase mRNA expression of Dusp5. Furthermore, we demonstrated by again of function study that sustained activation of p38 by overexpression of a constitutively active MKK6 mutant stimulates autophagy in cardiomyocytes. Surprisingly, the loss of p38 function obtained by overexpression of a dominant negative p38 mutant does not affect the autophagic response in our in vitro model, but increases the lipidation of autophagosomes marker LC3. Our results suggest that DUSPs can regulate, through their actions on MAPKs, important stages of autophagy in cardiomyocytes.
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Góes, Maria Elisa Almeida. "Kinin B2-Receptor in human iPSC differentiation into cardiomyocytes." Universidade de São Paulo, 2018. http://www.teses.usp.br/teses/disponiveis/46/46131/tde-24092018-144907/.
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Cardiovascular diseases are responsible for almost one third of all global deaths yearly, and therefore are largely studied. Cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CM) have emerged as an exciting technology for cardiac disease modelling and personalised therapy. Nevertheless, issues concerning functional and molecular maturation are still faced. In addition to this, differentiation protocols generally yield a heterogeneous mixed population comprised of nodal, atrial and ventricular-like subtypes, being unsuitable for therapeutic purposes. Bradykinin (BK) is a vasoactive peptide which exerts important physiological roles in the cardiovascular system, having been previously described as important for cellular, keratinocyte and skeletal muscle differentiation. This project performed in cooperation with PluriCell Biotech, a startup specialized in the production and differentiation of hiPSC-CM, has sought (1) characterizing gene and protein expression of molecular markers of maturation and of subtype specification throughout of differentiation; (2) Assessing the electrical functionality of hiPSC-CM through the characterization of subtype-specific action potentials (APs) and (3) Investigating whether the progress of hiPSCCM maturation is regulated by BK through kinin-B2 receptors (B2R). Our results have validated the model that proposes a developmental-dependent switch between skeletal (ssTnI) and cardiac (cTnI) isoforms of troponin I as differentiation progresses, at least to some extent. Furthermore, prolonged time in culture has resulted in higher levels of expression of the ventricular marker MLC2v and in increased rates of ventricular-like action APs. Electrophysiological analysis of hiPSC-CM reveals a mixed population with AP morphologies correspondent to nodal, atrial and ventricular subtypes, all showing pronounced automaticity as well as other features of immature cardiomyocytes, such as low amplitude and depolarization velocity. Such findings are coherent with those from other groups who have attempted to differentiate mature native-like cardiac cells from pluripotent stem cells sources, without fully succeeding. After showing that differentiating hiPSC-CM express a functional and responsive B2R, the receptor was subjected to chronic activation with 10µM BK and 1µM BK or inhibition with 5µM Firazyr+BK. Even though B2R modulation has not interfered negatively with differentiation yields nor cell morphology, analysis of gene andprotein expression of ssTnI or cTnI and of the ventricular marker MLC2v, have revealed no significant results in comparison to untreated controls. This suggests that BK does not interfere on hiPSC-CM maturation nor subtype specification, although we cannot rule out that it could be leading to other unexplored effects. We recommend a closer look into which intracellular signalling pathways become active upon B2R stimulation in hiPSC-CM, in order to narrow down cellular processes for further investigation.Doenças cardiovasculares são responsáveis por quase um terço de todas as mortes globais anualmente, e por isto o sistema cardiovascular é amplamente estudado. Cardiomiócitos derivados a partir de células-tronco pluripotentes induzidas humanas (hiPSCCM) emergiram como uma promissora tecnologia para modelagem de doenças cardíacas e terapia personalizada. No entanto, desafios acerca de sua maturação funcional e molecular ainda são enfrentados. Além disso, protocolos de diferenciação geralmente levam à obtenção de populações heterogêneas contendo células com fenótipos similares aos de cardiomiócitos nodais, atriais e ventriculares sendo, portanto, inapropriadas para fins terapêuticos. A bradicinina (BK) é um peptídio vasoativo que exerce importantes papeis fisiológicos no sistema cardiovascular, além de ter sido previamente descrita como importante para a diferenciação neuronal, de queratinócitos e de músculo esquelético. Este projeto foi realizado em colaboração com a empresa PluriCell Biotech, uma startup especializada na produção e diferenciação de hiPSC-CM, e buscou (1) caracterizar a expressão gênica e proteíca de marcadores moleculares de maturação e de especificação de subtipos cardíacos durante a diferenciação; (2) avaliar a funcionalidade elétrica de hiPSC-CM por meio da caracterização de seus potenciais de ação (PAs) e (3) Investigar se o progresso da diferenciação de hiPSCCM é regulado por bradicinina por meio do receptor B2 (B2R). Nossos resultados validaram o modelo que propõe um switch na expressão das isoformas funcionais de troponina I esquelética (ssTnI) e cardíaca (cTnI), durante o desenvolvimento e diferenciação celular, pelo menos parcialmente. Além disso, tempo prolongado em cultura resultou em maiores níveis de expressão do marcador ventricular MLC2v, assim como maiores frequências de PAs com morfologias similares a de cardiomiócitos ventriculares. Análise eletrofisiológica de hiPSCCM revelam a existência de uma população mista contendo PAs correspondentes aos subtipos nodais, atriais e ventriculares, assim como pronunciada automaticidade e outros atributos típicos de cardiomiócitos imaturos, como baixa amplitude e devagar velocidade de despolarização. Estes resultados são coerentes com os de outros grupos que ainda não foram totalmente bem-sucedidos em diferenciar células cardíacas maduras similares acardiomiócitos nativos a partir de células-troncos pluripotentes. Após mostrar que as hiPSCCM expressam receptores B2 funcionais e responsivos, submetemos o receptor a uma ativação crônica com BK 10µM e BK 1µM ou inibição crônica com Firazyr 5µM + BK. Apesar da modulação do B2R não ter interferido de forma negativa no rendimento da diferenciação ou na morfologia celular, análise de expressão gênica e proteica de ssTnI e cTnI e do marcador ventricular MLC2v não revelou resultados significativos em comparação aos controles não-tratados. Isto sugere que a BK não interfere na maturação e especificação de subtipos cardíacos em hiPSC-CM, apesar de não podermos ignorar o fato de que ela poderia estar desencadeando outros efeitos inexplorados. Nós recomendamos um estudo mais aprofundado acerca de quais vias de sinalização se tornam ativas após estimulação do receptor B2 em hiPSC-CM, com o objetivo de afunilar quais processos celulares poderiam ser investigados em uma próxima etapa deste estudo.
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Chan, Sai-yen Victor. "Effect of homocysteine on nitric oxide production in cardiomyocytes." Hong Kong : University of Hong Kong, 2001. http://sunzi.lib.hku.hk/hkuto/record.jsp?B23476552.
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Sun, Haipeng. "Regulation of Cyclooxygenase Gene Expression by Glucocorticoids in Cardiomyocytes." Diss., The University of Arizona, 2007. http://hdl.handle.net/10150/194896.
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Glucocorticoids (GCs) are endogenous steroid hormones that regulate a number of critical physiological processes. Psychological stress increases the level of GCs in the circulating system. The biological effect of elevated GCs on the heart is not well understood. We found that GCs induced Cyclooxygenase-1 (COX-1) and COX-2 gene expression in cardiomyocytes. COX-1 or COX-2 encodes the rate-limiting enzyme in the biosynthesis of prostanoids, which modulate crucial physiological and pathophysiological responses. The present studies aim to elucidate the signaling transduction pathway and the mechanism underlying GC induced COX expression.Our data demonstrate that GCs activate COX-1 gene expression through transcriptional regulation. COX-1 gene promoter studies support a role of Sp binding site in CT induced COX-1 gene expression. The nuclear protein binding to this site appears to be Sp3 transcription factor. Co-immunoprecipitation assays indicated a physical interaction between GR and Sp3 protein. Silencing of Sp3 transcription factor with small interfering RNA suppressed CT-induced COX-1 promoter activation. These data suggest that the activated GR interacts with Sp3 transcription factor that binds to COX-1 promoter to up-regulate COX-1 gene expression in cardiomyocytes.We also found that administration of GC in adult mice increased the level of COX-2 in the ventricles. With isolated neonatal cardiomyocytes, corticosterone (CT) induces the transcription of COX-2 gene. This response appears to be cardiomyocyte cell type specific and GC receptor (GR)-dependent. CT causes activation of p38 MAPK and subsequently CREB phosphorylation that mediates COX-2 gene expression. Mifepristone, a GR antagonist, failed to inhibit p38 and CREB activation and p38 inhibition failed to prevent activation of GR. These data suggest that two parallel signaling pathways, GR and p38 MAPK, act in concert to regulate the expression of COX-2 gene in cardiomyocytes.In addition to the investigation of mechanism and signaling transduction pathway, I have explored pharmacological agents that modulate COX expression. LY294002, a commonly used PI3K inhibitor, inhibited COX-2 gene expression via a PI3K-independent mechanism. Whereas GSK-3 inhibitors, such as lithium chloride, upregulated COX-2 gene expression, but suppressed GC-induced COX-1 expression. These data have paved the foundation for pharmacological manipulation of COX-1 and COX-2 gene expression in the heart.
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Aguilar, David Christopher. "GILZ: A Novel Glucocorticoid Induced Cytoprotective Protein in Cardiomyocytes." Diss., The University of Arizona, 2012. http://hdl.handle.net/10150/228177.
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Glucocorticoids (GCs) are frequently prescribed pharmacological agents most notably for their immunosuppressant effects. Endogenous GCs mediate biological processes such as energy metabolism and tissue development. At the cellular level, GCs bind to the Glucocorticoid Receptor (GR), a cytosolic receptor that translocates to the nuclei upon ligand binding and alters gene transcription. Among a long list of genes activated by GCs is the Glucocorticoid Induced Leucine Zipper (GILZ). Although GC induced GILZ expression has been well established in lymphocytes, little is known whether cardiomyocytes respond to GCs by inducing GILZ. Unlike lymphocytes, in which GCs induce apoptosis and GILZ mediates GC induced apoptosis, cardiomyocytes respond to GCs by gaining resistance against apoptosis. We determined GILZ expression pattern in cardiomyocytes in vivo and in vitro. Our data demonstrate GILZ induction in cardiomyocytes both in vivo and in vitro by GCs and point to H9C2 cell line as a valid model for studying the biological function of GILZ in cardiomyocytes. I have also determined GILZ functions as GC induced cytoprotective protein against the known cardiac toxicant Doxorubicin. Finally I have determined GILZ stabilizes Bcl-xL pro-survival protein, providing a possible mechanism of cytoprotection in cardiomyocytes.
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Jahangiri, Anisa. "n-3 PUFAs and reperfusion injury in isolated cardiomyocytes." Title page, table of contents and abstract only, 2002. http://web4.library.adelaide.edu.au/theses/09PH/09phj251.pdf.
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"September 2002" Bibliography: leaves 207-230. Ch. 1. Literature review -- Ch. 2. General methods -- Ch. 3. Dietary n-3 PUFAs and reperfusion injury in isolated cardiomyocytes -- Ch. 4. The effect of dietary n-3 PUFAs on cardiomyocyte membrane fluidity, intracellular ROS and Ca 2+ levels during oxidative stress -- Ch. 5. The effect of dietary fish oil supplementation on antioxidant enzyme gene expression in rat myocardium -- Ch. 6. The effect of dietary lipids on ischaemia-reperfusion injury in rat myocardium -- Ch. 7. General discussion -- Ch. 8. Appendices. The broad aims of this thesis were to develop a cellular model for studying reperfusion injury, in order to investigate the reported protective effects of n-3 PUFAs, and to examine the underlying mechanisms associated with such protection.
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Cohen-Aubart, Fleur. "Rôle de la protéine STIM1 sur la fonction des cellules musculaires vaculaires et cardiaques par modulation de la voie de signalisationcalcineurine-NFAT : implications physiopathologiques et pharmacologiques." Paris 6, 2012. http://www.theses.fr/2012PA066169.
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Le calcium est un second messager universel régulant de multiples fonctions intra-cellulaires grâce à des mouvements compartimentés dans l'espace et de dynamique variable. Les mouvements rapides localisés régulent les fonctions nécessitant des réponses rapides (contraction, exocytose) alors que les mouvements répétés ou larges dans le temps ou l'espace régulent des fonctions plus lentes (régulation de l'expression génique). Les mouvements calciques sont altérés de façon adaptative ou pathologique dans les situations pathologiques.
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Gonzalez, Granillo Marcela Alejandra. "La bioénergétique systémique moléculaire des cellules cardiaques : la relation structure-fonction dans la régulation du métabolisme énergétique compartmentalisé." Thesis, Grenoble, 2012. http://www.theses.fr/2012GRENV078/document.
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An important element of metabolic regulation of cardiac and skeletal muscle energetics is the interaction of mitochondria with cytoskeleton. Mitochondria are in charge of supplying the cells with energy, adjusting its functional activity under conditions of stress or other aspects of life. Mitochondria display a tissue-specific distribution. In adult rat cardiomyocytes, mitochondria are arranged regularly in a longitudinal lattice at the level of A band between the myofibrils and located within the limits of the sarcomeres. In interaction with cytoskeleton, sarcomeres and sarcoplasmic reticulum they form the functional complexes, the intracellular energetic units (ICEUs). The ICEUs have specialized pathways of energy transfer and metabolic feedback regulation between mitochondria and ATPases, mediated by CK and AK. The central structure of ICEUs is the mitochondrial interactosome (MI) containing ATP Synthasome, respiratory chain, mitochondrial creatine kinase and VDAC, regulated by tubulins. The main role of MI is the regulation of respiration and the intracellular energy fluxes via phosophotransfer networks. The regulation of ICEUs is associated with structural proteins. The association of mitochondria with several cytoskeletal proteins described by several groups has brought to light the importance of structure-function relationship in the metabolic regulation of adult rat cardiomyocytes. To purvey a better understanding of these findings, the present work investigated the mechanism of energy fluxes control and the role of structure-function relationship in the metabolic regulation of adult rat cardiomyocytes. To show these complex associations in adult cardiac cells several proteins were visualized by confocal microscopy: α-actinin and β-tubulin isotypes. For the first time, it was showed the existence of the specific distribution of β-tubulin isotypes in adult cardiac cells. Respiratory measurements were performed to study the role of tubulins in the regulation of oxygen consumption. These results together confirmed the crucial role of cytoskeletal proteins -i.e. tubulins, α-actinin, plectin, desmin, and others- for the normal shape of cardiac cells as well as mitochondrial arrangement and regulation. In addition, in vivo - in situ mitochondrial dynamics were studied by the transfection of GFP-α-actinin, finding that fusion phenomenon does not occur as often as it is believed in healthy adult cardiac cellsUn élément important de la régulation du métabolisme énergétique des muscles cardiaque et squelettiques est l'interaction des mitochondries avec le cytosquelette. Les mitochondries sont responsables de l'approvisionnement des cellules en énergie, elles sont capables d'ajuster leur activité fonctionnelle en fonction des conditions de stress ou d'autres aspects de la vie. Les mitochondries ont une distribution spécifique selon les tissus. Dans les cardiomyocytes de rats adultes, les mitochondries sont disposées régulièrement dans un entrelacement longitudinal au niveau des bandes A, entre les myofibrilles et dans les limites des sarcomères. En interaction avec le cytosquelette, le sarcomère et le réticulum sarcoplasmique, elles forment des complexes fonctionnels appelés unités énergétiques intracellulaires (ICEUs). Les ICEUs ont des voies spécialisées de transfert d'énergie et de régulation des feedback métaboliques entre les mitochondries et les ATPases, médiée par la CK et l'AK. La structure centrale des ICEUs est l'interactosome mitochondrial (MI) qui confient l'ATP synthasome, la chaîne respiratoire, la créatine kinase mitochondriale et VDAC, qui pourrait être régulé par les tubulines. Le rôle principal du MI est la régulation de la respiration et des flux d'énergie intracellulaires via les réseaux de phosphotransfert. La régulation des ICEUs est liée aux protéines structurales. L'association des mitochondries avec plusieurs protéines du cytosquelette, décrite par plusieurs groupes, a mis en évidence l'importance de la relation structure-fonction dans la régulation métabolique des cardiomyocytes de rats adultes. Pour fournir une meilleure compréhension de ces résultats, le présent travail étudie le mécanisme de contrôle des flux d'énergie et le rôle des relations structure-fonction dans la régulation métabolique de cardiomyocytes de rats adultes. Pour montrer ces associations complexes dans les cellules cardiaques adultes, plusieurs protéines ont été visualisées par microscopie confocale: l'α-actinine et les isoformes des β-tubulines. Pour la première fois, l'existence d'une distribution spécifique des isoformes de β-tubuline dans les cellules cardiaques adultes a été montré. Des mesures respiratoires ont été réalisées pour étudier le rôle des tubulines dans la régulation de la consommation d'oxygène. Ces résultats ont confirmé le rôle déterminant des protéines du cytosquelette -tubulines, α-actinine, plectine, desmine, et autres- pour le maintien de la forme normale des cellules cardiaques, ainsi que de l'arrangement et de la régulation mitochondrial. En outre, la dynamique mitochondriale a été étudiée in vivo et in situ par la transfection de la GFP-α-actinine, ceci permettant la mise en évidence du fait que le phénomène de fusion ne se produit pas aussi souvent qu'on ne le croit pour des cellules cardiaques adultes en bonne santé
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David, Florian. "Hétérogénéité ribosomique et régulation de la traduction des ARNm des facteurs de croissance (lymph)angiogéniques dans les cardiomyocytes stressés." Thesis, Toulouse 3, 2022. http://www.theses.fr/2022TOU30054.
Full textAbstract:
L'ischémie cardiaque, définie comme la baisse de perfusion sanguine d'une partie du cœur, soumet les cellules à différents stress induits par la baisse de l'apport en oxygène et nutriments. S'ils perdurent, ces stress induisent la mort cellulaire et par la suite un infarctus du myocarde. Afin de rétablir l'homéostasie tissulaire et de revasculariser le tissu ischémique, les cellules activent différents mécanismes tels que l'angiogenèse et la lymphangiogenèse. Mon projet de thèse a porté sur l'étude de ces voies et leur régulation au niveau traductionnel dans des cardiomyocytes soumis à différents stress. L'analyse semi-globale du transcriptome et du traductome en condition hypoxique nous a montré que, dans les cardiomyocytes, les gènes (lymph)angiogéniques sont majoritairement régulés au niveau traductionnel. Parmi ces gènes, ceux possédant des structures IRES (Internal Ribosome Entry Site) sont recrutés de manière plus efficace dans les polysomes. Or, la régulation de la traduction via ces structures représente un mécanisme-clé dans la réponse à l'hypoxie. Nous avons identifié une protéine, la vasohibine 1, liée à l'ARN et présentant lors de l'hypoxie une fonction d'ITAF (IRES Trans-Acting Factor). Dans un deuxième volet de cette thèse, nous avons caractérisé le rôle d'un long ARN non codant, Neat1, dans la régulation de la traduction IRES dépendante en réponse à l'hypoxie. Neat1 est le composant principal d'un corps nucléaire, le paraspeckle, qui se forme en réponse au stress. Le paraspeckle est formé de Neat1 et de plusieurs protéines qui interagissent avec ce long ARNnc, dont certaines présentent aussi une fonction d'ITAF, suggérant un rôle du paraspeckle dans le contrôle de la traduction. Ainsi nous avons identifié par des expériences de déplétion que Neat1, plus particulièrement sa grande isoforme Neat1-2, possède un rôle d'ITAF qui régule tous les IRES des facteurs (lymph)angiogéniques. D'autres composants du paraspeckle, p54nrb et pSPC1, régulent différents sous-groupes d'ARNm à IRES. L'analyse de l'intéractome de p54nrb a permis d'identifier de nouveaux partenaires nucléaires et cytoplasmiques de cette protéine, spécifiques de l'hypoxie, dont la protéine ribosomique uS5 (RPS2) et la nucléoline, qui présentent elles aussi une fonction d'ITAF. Ces résultats suggèrent que le paraspeckle pourrait être une plateforme d'assemblage de l'IRESome, complexe responsable de la traduction IRES-dépendante, dont Neat1 est un acteur majeur. Le troisième volet de ma thèse porte sur l'identification de ribosomes spécialisés responsables de la traduction IRES-dépendante lors du stress. L'analyse de la composition des polysomes de cardiomyocytes humains soumis à un stress du RE nous a permis de découvrir plusieurs protéines ribosomiques mitochondriales associées aux polysomes. Pour certaines d'entre elles cette association augmente significativement (MRPS15, MRPS12) alors que pour d'autres elle diminue lors du stress (MRPS35, MRPL52), appuyant le concept de ribosomes spécialisés. Nous avons par la suite confirmé l'interaction de MRPS15 avec le ribosome en réalisant des expériences de PLA (proximity ligation assay) et d'immunoprécipitations. De plus MRPS15 comporte une fraction cytoplasmique qui augmente en réponse au stress. [...]Cardiac ischemia, defined as a blood perfusion diminution in a part of the heart, subjects cells to various stresses caused by oxygen and nutrient supply diminution. If they persist, these stresses induce cell death and subsequently myocardial infarction. In order to restore tissue homeostasis as well as the vascularization of ischemic tissue, cells activate various mechanisms such as angiogenesis and lymphangiogenesis. My thesis project focused on the study of these pathways and their regulation at the translational level in cardiomyocytes subjected to different stresses. The semi-global analysis of the transcriptome and the translatome in hypoxic condition showed us that angiogenic and lymphangiogenic genes are not drastically regulated at the transcriptional level while the majority of them are induced at the translational level in murine cardiomyocytes. Among these genes, those having IRES (Internal Ribosome Entry Site) structures are recruited more efficiently into polysomes. Regulation of translation through the presence of these structures is a key mechanism in the response to hypoxia. We have identified a protein, vasohibin 1, bound to FGF1 IRES and presenting during hypoxia an ITAF (IRES Trans-Acting Factor) function. In a second part of this thesis work, we characterized the key role of a long non-coding RNA, Neat1, in the regulation of IRES-dependent translation in response to hypoxia. Neat1 is the main component of a nuclear body, the paraspeckle, which forms in response to stress. The paraspeckle is made up of Neat1 and several proteins, that interact with this long ncRNA, and are also known to exhibit ITAF function, hence the hypothesis of a role of the paraspeckle in the control of translation. Thus we have identified by depletion experiments, that Neat1, more particularly the long isoform of this ncRNA, Neat1-2, has a role of ITAF promoting the IRES-dependent translation of angiogenic and lymphangiogenic factors. Other components of the paraspeckle, p54nrb and pSPC1, regulate different subgroups of IRESs. The analysis of the interactome of p54nrb by mass spectrometry allowed to identify new nuclear and cytoplasmic partner specific of hypoxia, among them the ribosomal protein uS5 (RPS2) and nucleolin, which both present an ITAF function. These results suggest that the paraspeckle could be an assembly platform for IRESome, a complex responsible for IRES-dependent translation, and that Neat1 is a key regulator of this mechanism. The third part of my thesis concerns the identification of specialized ribosomes involved in IRES-dependent translation during stress. Analysis of the composition of polysomes of human cardiomyocytes under endoplasmic reticulum (ER) stress allowed us to discover several mitochondrial ribosomal proteins associated with the polysomes. Cellular stresses induced a switch in polysome composition, inducing an increase of the association with some mitochondrial ribosomal proteins (MRPS12 and MRPS15) while others were decreased (MRPS35 and MRPL52). The rest of the study focused on MRPS15. First, experiments with PLA (proximity ligation assay) and immunoprecipitations from cytosolic and polysomal fractions confirmed the interaction of this mitochondrial protein with the ribosome. In addition, a cytoplasmic fraction of MRPS15 increases in response to stress. [...]
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Aguilar, Sanchez Cristina. "Epigenetic transitions in cardiovascular development and cell reprogramming." Thesis, University of Edinburgh, 2017. http://hdl.handle.net/1842/28787.
Full textAbstract:
Epigenetic modifications are alterations in the cell nucleus that affect gene expression and can occur in chromatin at the level of DNA methylation or histone modifications. Such ‘epigenetic marks’ can be heritable through cell division but leave the DNA sequence unchanged. Post-translational modifications can be found on the histone proteins associated with DNA; the majority of histone modifications are found on the lysine-rich N-‐terminal amino acid “tails”. Histone acetylation and methylation influence the chromatin structure by loosening or tightening the packaging of DNA, respectively, in association with other chromatin modifiers. Condensed chromatin is linked to transcriptional silencing and genetic imprinting and also occurs at chromosomal centromeres, where it is linked to kinetochore binding. Heart development is well studied, but the epigenetic processes involved are not yet completely understood. While active chromatin mechanisms such as histone acetylation and chromatin remodelling have been described in the heart, the role of gene repressive epigenetic mechanisms has been poorly investigated. Cardiomyocytes are post-mitotic cells that do not divide to regenerate a damaged heart. The regeneration of cardiomyocytes after myocardial infarction is an important topic of interest in cardiovascular science. There are various approaches to heart repair after infarction, including activating cardiomyocytes so they become mitotic once again, or growing cardiomyocytes in vitro to attach to a lesion site. An important factor in these approaches is understanding the epigenetic mechanisms controlling cell division. In this thesis, we aim to advance the current knowledge of the epigenetic repressive mechanisms involved in cardiomyocyte formation and heart development to explain their lack of regenerative capacities. We studied the epigenetic changes that occur during cardiac development leading to a non-‐regenerative state to pinpoint the moment at which these changes arise. We found that the epigenetic process is independent of whether cardiac lineage differentiation occurs during embryogenesis or during differentiation in vitro. We discovered that cardiac heterochromatin displays a singular epigenetic signature during development as compared to brain, another post-mitotic tissue, or liver, an actively regenerative tissue. We observed an epigenetic change in the repressive histone modification histone H3 lysine 9 trimethylation that was specific to heart development. This change involved a nuclear reorganisation of heterochromatin and a reduction of the levels of this mark in E13.5 and E14.5 embryos, as compared to E10.5 embryos. This was consistent with our observations of the histone lysine methyltransferase SUV39H1, the levels of which were lower after stage E10.5 of development. However, contradictorily, in differentiated cardiomyocytes in vitro, SUV39H1 was increased but showed low levels of H3K9me3, compared to ES cells, which had low levels of SUV39H1 and high levels of H3K9me3. We detected extremely low levels of the H3K9me3 in adult heart tissue. We observed that in adult hearts, the myocardium had maintained these major changes in H3K9me3, while this effect was not observed in the epicardium. Genomic studies were carried out to determine changes at a genomic level between the two key epigenetic stages in heart development we identified at E10.5 and E13.5. Methylated DNA immunoprecipitation sequencing and chromatin immunoprecipitation sequencing for H3K9me3 analyses were carried out to find overall changes in methylation patterns. No global changes in DNA methylation were detected between these developmental stages. These results imply that the differences observed in H3K9me3 are due to remodelling of the heterochromatin during heart development and cardiomyocyte formation, rather than quantitative changes.
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Ghaffari, Nader. "Block of Na+ current in isolated rat cardiomyocytes by protamine /." Title page and abstract only, 2004. http://web4.library.adelaide.edu.au/theses/09SB/09sbg4111.pdf.
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