The regulation of cardiac-specific genes such as GATA-4 and its co-factor FOG-2 is paramount for normal heart development and function. Indeed, those mechanisms that regulate GATA-4 and FOG-2 function, such as nuclear transport and the post-translational modification of SUMOylation, are of critical importance for cardiogenesis. Therefore the aims of this study were to: i) elucidate the nuclear transport mechanisms of GATA-4; ii) determine the function of SUMOylation on the biological activity of both GATA-4 and FOG-2; and iii) examine how these mechanisms impact on the role of GATA-4 and FOG-2 in cardiac hypertrophy. Firstly, we characterised a non-classical nuclear localisation signal that mediates active import of GATA-4 in both HeLa cells and cardiac myocytes. Fine mapping studies revealed four crucial residues within this region that interacted with importin ?? to mediate GATA-4 import via the non-classical import pathway. In addition, a cardiac myocyte-specific CRM1-dependent nuclear export signal, which consists of three essential leucine residues, was identified. We also investigated the residues of GATA-4 that are responsible for its DNAbinding activity and therefore transcriptional control of cardiac-specific genes. Secondly, we demonstrated that SUMOylation of both GATA-4 and FOG-2 is exclusively carried out by SUMO-2/3. Moreover, SUMOylation is involved in the nuclear localisation of both GATA-4 and FOG-2 in cardiac myocytes as well as the transcriptional regulation of cardiac-specific genes, such as cardiac troponin I. Finally, and perhaps most biologically significant, we showed that nuclear transport as well as SUMOylation of GATA-4 is imperative for the ability of GATA-4 to induce cardiac hypertrophy. Moreover, it was determined that FOG-2 SUMOylation is involved in the ability of FOG-2 to protect against cardiac hypertrophy. In conclusion, the current study provides detailed information on the nuclear transport pathways of GATA-4 as well as the SUMOylation of both GATA-4 and FOG-2 and the role these two mechanisms play in gene transcription and cardiac hypertrophy.

Le muscle cardiaque est un organe qui s'adapte à différents stress hémodynamiques en activant la synthèse protéique et en augmentant la taille des cardiomyocytes, résultant sur le développement d'une hypertrophie cardiaque. L'objectif de cette thèse est d'étudier le rôle potentiel du canal TRPM4 dans différents types d'hypertrophie cardiaque. Une altération du Ca2+ diastolique est à l'origine du signal initial activant les voies de signalisation d'une hypertrophie cardiaque délétère de type pathologique telle que la voie de la calcineurine-NFAT et la ré-expression de gènes fœtaux. Cette hypertrophie est alors compensatrice et vise à préserver la fonction de pompe du myocarde. Cette altération peut être conduite par divers stimulis tels qu'une augmentation de l'angiotensine II ou par des pathologies cardiovasculaires telles que l'infarctus du myocarde et l'hypertension. Cependant, une hypertrophie cardiaque bénéfique est également décrite dans la littérature, notamment lors des stades de développement du myocarde lors de l'embryogénèse ou en encore en réponse à une activité physique modérée régulière. Elle se caractérise par l'activation d'une toute autre voie de signalisation qu'est la voie de l'IGF-1-PI3K-Akt engendrée par une augmentation du taux de facteur de croissance qu'est l'insulin growth factor-1. Ces voies de signalisation ont été largement décrites dans la littérature et s'entrecroisent. Le canal TRPM4 est un canal cationique non sélectif perméable de manière égale aux ions Na+ et au K+, imperméables au Ca2+, mais activé par le Ca2+ intracellulaire. Dans le système immunitaire, il régule négativement l'entrée de Ca2+ et ce canal apparaît donc impliqué dans de nombreuses fonctions cellulaires dépendantes du Ca2+ dans différents types cellulaires. Par l'utilisation de deux modèles d'hypertrophie cardiaque, un physiologique généré par quatre semaines d'entraînement en endurance et un pathologique suite à un infarctus du myocarde induit par la ligature de l'artère coronaire gauche sur des souris wild-type et knock-out (KO) pour le canal TRPM4, nous avons mis en évidence une augmentation d'expression fonctionnelle du canal TRPM4 au sein du ventricule gauche associée à une régulation négative d'entrée de Ca2+. Le canal TRPM4 étant un régulateur de l'homéostasie calcique des cardiomyocytes, son expression fonctionelle après l'infarctus du myocarde ainsi que l'entraînement favorise l'activation de la voie de l'IGF-1-PI3K-Akt et prévient partiellement l'activation de la voie de la Calcineurine-NFAT et le développement d'une hypertrophie cardiaque pathologique, notamment dans le modèle d'infarctus du myocarde. En effet, en absence d'expression du canal, l'entrée de Ca2+ n'étant plus régulée, la voie de la Calcineurin-NFAT est favorisée. Mots clés : TRPM4, hypertrophie cardiaque, entraînement, IGF-1-PI3K-Akt, Calcineurine / Abstract: Cardiac muscle is an organ that adapts to different hemodynamic stress by activating protein synthesis and increasing cardiomyocytes size, resulting in cardiac hypertrophy. The objective of this PhD is to study the potential role of TRPM4 channel in different types of cardiac hypertrophy. Impaired diastolic Ca2+ is responsible for the initial signal activating signaling pathways in a deleterious cardiac hypertrophy pathological type such as Calcineurin-NFAT pathway and the re-expression of fetal genes. This hypertrophy is first compensatory and preserves the myocardial pump function. This alteration can be carried out by various stimuli such as increased angiotensin II or by cardiovascular diseases such as myocardial infarction and hypertension.However, a beneficial cardiac hypertrophy is also described in the literature, especially during development stages during embryogenesis or even in response to regular moderate physical activity. It is characterized by the activation one different signaling pathway, the IGF-1 - PI3K –Akt, generated by an increase in growth factor levels that is the insulin growth factor -1. These signaling pathways have been widely described in the literature and cross-talking. TRPM4 channel is a nonselective cation channel permeable equally to Na+ and K+, impermeable to Ca2+ but activated by the intracellular Ca2+. In the immune system, it downregulates Ca2+ entry and therefore appears to be involve in many Ca2+-dependent cellular functions in different cell types. By the use of two models of cardiac hypertrophy, a physiological generated by four weeks of training in endurance and pathological after myocardial infarction induced by ligation of the left coronary artery on wild-type and knockout mice -out (KO) for TRPM4 channel, we have demonstrated a functional expression increased TRPM4 channel within the left ventricle associated with down-regulation of Ca2 + entry. TRPM4 the channel being a regulator of calcium homeostasis in cardiomyocytes functional expression after myocardial infarction as well as the drive promotes the activation of the pathway of IGF-1-PI3K-Akt and partially prevents the pathway activation of the NFAT-calcineurin and the development of pathological cardiac hypertrophy, in particular myocardial infarction model. Indeed, in the absence of expression of the channel, the Ca2 + is not regulated, the path of Calcineurin-NFAT is favored. Keywords: TRPM4, cardiac hypertrophy, training, IGF-1-PI3K-Akt, calcineurin

Heart failure induced by cardiac hypertrophy is a cause of high mortality in the world and has been the fastest growing cardiovascular disease over the past decade. Cardiac hypertrophy is characterised as a reactive increase in cardiac mass growth with a complex of ventricular remodelling. It occurs initially as a compensatory response to an increased workload but eventually leads to cardiac dysfunction. An in-depth understanding of cardiac hypertrophy and the capacity to regulate it has profound clinical implications. The MAPK pathways provide an important connection between external stimuli and intracellular signals for cardiac hypertrophic response. At least four MAPK subfamilies have been identified: extracellular-regulated protein kinases 1 and 2 (ERK1/2), ERK5, c-Jun NH2-terminal protein kinases (JNKs) and p38 MAPKs. Mitogen-activated protein kinase kinase 4 (MKK4), a vital activator of JNK and p38 is implicated as an important mediator of hypertrophy. ERK5, an atypical MAPK, is also involved in both hypertrophic growth and cardiomyocyte survival. However, conflicting data have been yielded from previously-published studies, since the results are based entirely on experiments conducted in cultured cardiomyocytes or transgenic and conventional knockout mouse models. To elucidate their biological roles and underlying signalling mechanisms in hypertrophy, mice with a cardiomyocyte-specific deletion of MKK4 or ERK5 (MKK4cko and ERK5cko mice) were generated in the present study. In response to pathological hypertrophic stresses including pressure overload or isoprenaline stimulation, MKK4cko mice developed exacerbated pathological hypertrophy with increased cardiomyocyte apoptosis, impaired cardiac function and remarkably upregulated NFAT (nuclear factor of T-cell) transcriptional activity. However, MKK4cko mice exhibited a similar extent of swimming exercise-induced physiological hypertrophy compared with the controls. In response to pathological hypertrophic stimuli, ERK5cko mice were resistant to hypertrophic growth, foetal gene induction and ventricular fibrosis, which is associated with repressed activation of MEF2 (myocyte enhancer factor 2). ERK5 deficiency also caused a profound increase in cardiomyocyte apoptosis which accounted for the impaired cardiac function. In conclusion, the present study provides biological evidence that clarifies in vivo functions of MKK4 and ERK5 in hypertrophy. MKK4 acts a protective role against pathological hypertrophy through inhibiting NFAT signalling, but it is not necessary for the regulation of physiological hypertrophy. ERK5 is essential for pathological hypertrophic remodelling and cardiomyocyte survival and its function in hypertrophic remodelling is mediated through regulation of MEF2 activity. Taken together, these data presented in my thesis advances knowledge about biological functions of MAPK pathways in the heart.

Abstract Cardiac loading induces left ventricular hypertrophy and cardiac remodeling which when prolonged, leads to heart failure, a complex syndrome affecting approximately 1-2% of the adult population of the Western world with a prevalence increasing with age. Pathological remodeling involves functional and structural changes that are associated with fetal gene expression, sarcomeric re-organization, hypertrophy of cardiomyocytes, fibrosis, inflammation, oxidative stress and impairment of metabolism. The aim of this study was to investigate the role of three novel factors during the cardiac remodeling process with different experimental models of cardiac overload. Phosphatase and actin regulator 1 (Phacr1) expression was rapidly downregulated due to myocardial infarction (MI). Adenovirus-mediated Phactr1 overexpression changed the skeletal α-actin to cardiac α-actin ratio in both healthy and infarcted rat hearts and cultured cardiomyocytes. Phactr1 could regulate the actin isoform switch via the serum response factor (SRF). The expression of transforming growth factor (TGF)- β-stimulated clone 22 (TSC-22) was rapidly induced by multiple hypertrophic stimuli and was also evident post-MI. In addition, TSC-22 could regulate collagen 3a1 expression in the heart. The expression of retinal degeneration 3-like (Rd3l) was downregulated in response to pressure overload and also downregulated post-MI. Rd3l knockout mice expressed increased myocyte hypertrophy and cardiac dysfunction in response to a transverse aortic constriction (TAC) induced pressure overload. This thesis provides novel information about Phactr1, TSC-22 and Rd3l in load-induced cardiac hypertrophy and remodeling. Collectively these studies increase our understanding of the regulatory mechanisms underlying the progression of heart failure. / Tiivistelmä Sydämen kuormitus saa aikaan vasemman kammion liikakasvun eli hypertrofian ja sydämen uudelleenmuovautumisen, mikä pitkittyessään johtaa sydämen vajaatoimintaan. Sydämen vajaatoiminta on monimutkainen oireyhtymä, josta länsimaissa kärsii noin 1-2 % aikuisväestöstä, ja esiintyvyys nousee iän myötä. Patologisessa uudelleenmuovautumisessa tapahtuu toiminnallisia ja rakenteellisia muutoksia, joihin liittyy muutoksia geenien ilmentymisessä, sarkomeerin uudelleen järjestäytymistä, sydänlihassolujen koon kasvua, fibroosia, tulehdusta, oksidatiivista stressiä ja aineenvaihdunnan huonontumista. Tämän työn tarkoituksena oli tutkia kolmen uuden tekijän roolia sydämen uudelleenmuovautumisessa erilaisissa kokeellisissa sydämen kuormituksen malleissa. Fosfataasin ja aktiinin säätelijä 1:n (Phactr1) ilmentyminen väheni nopeasti infarktin seurauksena. Adenovirusvälitteinen Phactr1:n ylituotanto muutti luusto- ja sydänlihasaktiinien isomuotojen suhdetta sekä terveessä että infarktisydämessä, samoin viljellyissä sydänlihassoluissa. Phactr1 saattaa säädellä isomuotojen suhdetta seerumiresponsiivisen tekijän (SRF) avulla. Transformoituvan kasvutekijä β1:n stimuloima proteiini 22:n (TSC-22) ilmentyminen nousi nopeasti usean hypertrofisen stimuluksen seurauksena sekä infarktin jälkeen. Lisäksi TSC-22 voisi säädellä kollageeni 3a1:n ilmentymistä sydämessä. Retinan degeneroituvan proteiinin 3 kaltaisen tekijän (Rd3l) ilmentyminen väheni sekä painekuormituksen että infarktin seurauksena. Rd3l-poistogeenisillä hiirillä aortan ahtauman aiheuttama painekuormitus sai aikaan lisääntynyttä sydänlihassolujen hypertrofiaa ja sydämen toimintahäiriöitä. Tämä väitöskirjatutkimus tuo uutta tietoa Phactr1-, TSC-22- ja Rd3l-geeneistä kuormituksen aiheuttamassa sydämen hypertrofiassa ja uudelleenmuovautumisessa. Nämä tulokset auttavat osaltaan ymmärtämään monimutkaisia molekyylitason mekanismeja, jotka johtavat sydämen vaajatoiminnan kehittymiseen.

A hipertrofia cardíaca é um mecanismo de adaptação do coração ao aumento de demanda. De acordo com o estímulo, fisiológico ou patológico, a hipertrofia apresenta diferentes características morfológicas e moleculares. Compreender os mecanismos comuns e distintos entre os dois tipos de hipertrofia é um passo importante para o desenvolvimento de estratégias de prevenção e tratamento da IC. Dentre os mecanismos distintos cabe ressaltar a participação das espécies reativas do oxigênio (EROs) que parecem estar presentes em altos níveis na hipertrofia cardíaca patológica e em baixos na fisiológica. Além disso, o papel regulatório dos microRNAs (miRs) tem sido demonstrado nas doenças cardiovasculares. No entanto, a influência das EROs no desenvolvimento da hipertrofia e nas adaptações decorrentes a ela ainda não está estabelecido. Assim, nosso objetivo foi avaliar as diferenças morfológicas e moleculares da hipertrofia cardíaca fisiológica, induzida pelo exercício, e da patológica, induzida por bandeamento aórtico (TAC), e sua modulação pela vitamina E. Os modelos de exercício e TAC desenvolveram hipertrofia cardíaca de forma compatível com o estímulo recebido. Essas adaptações ocorreram conjuntamente com alterações na expressão dos miRs-21, -26b, -150, -210 e -499. A vitamina E inibiu o estímulo angiogênicos, no modelo fisiológico, assim como a expressão dos miRs-21, -150 e -210. No entanto, esses efeitos não alteraram o fenótipo final da hipertrofia cardíaca fisiológica. No modelo patológico, por outro lado, a vitamina E reduziu a fibrose e o dano oxidativo, além de alterar a expressão de miRs já descritos no desenvolvimento da hipertrofia cardíaca patológica. Novamente, esse efeito não foi suficiente para reduzir a hipertrofia cardíaca. Em conjunto, os dados desse estudo sugerem que a vitamina E e/ou sua capacidade antioxidante têm a capacidade de influenciar de forma benéfica a hipertrofia patológica; no entanto, seus efeitos podem ser desfavoráveis no estímulo fisiológico. / Cardiac hypertrophy is an adaptive mechanism of the heart to the increased demand. According to the stimulus, physiological or pathological, cardiac hypertrophy present different morphological and molecular features. Understanding both the unique and the shared features in each type of hypertrophy is an important step to the development of novel approaches in the HF management. Among the unique mechanisms, the participation of reactive oxygen species (ROS) seems to be present at high levels in pathological and at low levels in physiological cardiac hypertrophy. Furthermore, the regulatory role of microRNAs (miRs) have been shown in cardiovascular diseases. However, ROS influence in cardiac hypertrophy development and their adaptations were not established yet. Thus, our objective was to evaluate morphological and molecular differences between physiological cardiac hypertrophy (physical exerciceinduced) and pathological cardiac hypertrophy (transverse aortic constrictioninduced), and its modulation by vitamin E. Exercise and TAC models developed cardiac hypertrophy in a manner consistent with the received stimulus. These adaptations occurred along with changes in miR-21, -26b, -150, -210 and -499 expression. Vitamin E inhibited angiogenic adaptations, as well as miR-21, -150 and -210 expression in physiological model. However, these effects did not change the final physiological cardiac hypertrophy phenotype. On the other hand, in the pathological model, vitamin E reduced oxidative damage and fibrosis, and altered the expression of miRs described in pathological cardiac hypertrophy development. Again, this effect was not sufficient to reduce cardiac hypertrophy. In conclusion, vitamin E and/or its antioxidant capacity have the capacity to influence the pathological hypertrophy in a beneficial way, but its effects can be unfavorable in the physiological stimulus.

O exercício físico modula o sistema renina angiotensina (SRA), que tem um importante papel na fisiologia cardíaca, especialmente na promoção da hipertrofia cardíaca. O SRA pode ser dividido em dois eixos principais: o eixo clássico – representado pelo receptor AT1 (AT1R) ativado pela angiotensina II (ANG II) – e o eixo alternativo – que é ativado pela interação da angiotensina 1- 7 (ANG 1-7) com o receptor MAS (MASR). O balanço entre os eixos do SRA pode determinar um remodelamento cardíaco fisiológico ou patológico. Estudos têm demonstrado que altos volumes de exercício físico podem desencadear possíveis efeitos deletérios ao sistema cardiovascular. Neste contexto, nosso objetivo foi avaliar a hipertrofia cardíaca, o eixo clássico e o eixo alternativo do SRA no miocárdio de camundongos submetidos a variados volumes e intensidades de treinamento em natação. Para tal, camundongos balb/c foram divididos em três grupos: (I) sedentário (SED), (II) treinados 2x ao dia (T2) sem sobrecarga e (III) treinado 3x ao dia com sobrecarga de 2% do peso corporal (T3), totalizando 6 semanas de treinamento efetivo. Ambos os grupos treinados desenvolveram hipertrofia cardíaca, sem diferença nos níveis de fibrose. Bioquimicamente, observamos um aumento nos níveis do receptor MASR somente no grupo T2, enquanto que os níveis de AT1R aumentaram somente no grupo T3. Contudo, não foi observada alteração na concentração dos peptídeos ANGI, ANGII e ANG 1-7 no tecido cardíaco entre os grupos. Além do mais, o grupo T3 demonstrou um aumento na expressão de miosina de cadeia pesada- β em comparação ao grupo SED e redução da expressão da isoforma- @ em relação ao grupo T2. Em conclusão, nossos resultados sugerem que ambos os protocolos de exercício promoveram uma hipertrofia cardíaca semelhante, mas o protocolo com maior volume e intensidade promoveu uma ativação diferencial dos receptores do SRA e reativação de genes fetais. Estudos que avaliem protocolos com maior duração são necessários para esclarecer se estas mudanças representam uma ativação precoce dos mecanismos relatados para o desenvolvimento de um fenótipo com características patológicas. / Exercise promotes physiological cardiac hypertrophy and induces the activation of the renin angiotensin system (RAS), which plays an important role in cardiac physiology, both through the classical axis – represented by the angiotensin II receptor type 1 (AT1) activated by angiotensin II (ANG II) – and the alternative axis – which is activated by the angiotensin 1-7 interaction with the MAS receptor. However, very intense exercise protocols could have deleterious effects on the cardiovascular system. In this context, we aimed to analyze the cardiac hypertrophy phenotype, as well as the classical (ANGII/AT1) and alternative (ANG1-7/MAS) RAS axes, in the myocardium of mice submitted to varying volume and intensity swimming exercises for the development of cardiac hypertrophy. To this end, male balb/c mice were divided into three groups: (I) sedentary (SED), (II) swimming twice a day (T2) without overload, and (III) swimming three times a day with a 2% body weight overload (T3), totaling six weeks of training. Both training groups developed cardiac hypertrophy. Interestingly, we observed an increase in MAS receptor levels only in group T2, while AT1 levels increased only in group T3. However, no change was observed regarding the levels of angiotensin peptides ANG-I, ANG-II, and ANG1-7, in either group. In addition, group T3 displayed a higher expression of myosin heavy chain-β (MHC-β) and lower expression levels of the alpha isoform (MHC-@). Fibrosis was not observed in any of the groups. In conclusion, our results suggest that both exercise protocols promoted a similar cardiac hypertrophy phenotype, but the protocol applying increased volume and intensity resulted in differential activation of RAS receptors and fetal gene reactivation. Studies applying longer duration protocols could elucidate if these changes represent early activation of mechanisms related to hypertrophy development with phenotypic pathological characteristics.

A hipertrofia cardíaca é uma adaptação do coração frente a estímulos de crescimento, sejam eles patológicos e irreversíveis como a sobrecarga de pressão ou de volume, ou fisiológicos e reversíveis como a gravidez e o exercício físico. A hipertrofia derivada de estímulos patológicos é conhecida como mal adaptativa enquanto que a hipertrofia proveniente de estímulos ditos fisiológicos é conhecida como benéfica ou adaptativa. Embora ambas hipertrofias tenham fatores em comum no que diz respeito ao crescimento do cardiomiócito e adaptações moleculares, elas acabam divergindo para desfechos completamente diferentes. A hipertrofia patológica evolui para um quadro de disfunção cardíaca ao passo que a hipertrofia fisiológica não acarreta nenhum dano funcional ao miocárdio. Essa linha tênue entre um fenótipo e outro envolve mecanismos celulares complexos que ainda precisam ser esclarecidos. Dentro deste cenário, os microRNAs aparecem como reguladores de diversos processos celulares, e têm sido associados ao crescimento miocárdico. Portanto, nosso objetivo foi comparar o padrão de expressão de microRNAs entre os modelos de hipertrofia fisiológica, induzido por natação (SWIM), e o modelo de hipertrofia patológica, induzida por bandeamento aórtico transtorácico (TAC). As análises foram realizadas após 28 dias para o modelo de natação, e 35 dias para o modelo de TAC. A comparação foi realizada através da técnica de microarranjo de microRNAs (Affymetrix). Interessantemente, apenas 20 microRNAs apresentaram níveis de expressão distinta entre os dois modelos de hipertrofia. Destes, 12 microRNAs apresentaram aumento de expressão (miR-193a-3p, miR-299a-5p, miR- 127-5p, miR-214-5p, miR-188-5p, miR-326-3p, miR-6395, miR-547-3p, miR-199a-5p, miR-381-3p, miR-223-3p e miR-199b-5p) e 8 estavam com seus níveis diminuídos (miR11 708-5p, miR-30c-1-3p, miR-22-5p, miR-6921-5p, miR-30a-3p, miR-30e-3p, miR-27a-5p and miR-6975-5p) no grupo TAC em relação ao grupo SWIM. Além disso, apenas 3 microRNAs, miR-21a-5p, miR-206-3p e miR-1983, apresentaram aumento de expressão tanto no grupo TAC quanto no grupo SWIM em comparação aos grupos SHAM e Sedentário, respectivamente. Após isso, foi realizada uma busca por possíveis alvos destes microRNAs na base de dados KEGG Pathway que identificou 4 rotas enriquecidas (665 genes) entre os alvos dos microRNAs reduzidos, e 80 rotas (3394 genes) fortemente associadas aos microRNAs que estavam aumentados no grupo TAC comparado ao SWIM. Conclui-se que existem microRNAs específicos para o desenvolvimento da hipertrofia cardíaca fisiológica, bem como patológica conforme os dados obtidos na análise de microarranjo. Além disso, os possíveis alvos destes microRNAs parecem estar envolvidos em rotas bastante envolvidas no crescimento celular, sobrevivência e adaptação cardíaca. / Cardiac hypertrophy is a heart adaptation in response to growth stimuli whether pathological and irreversible such as pressure overload or physiological and reversible as pregnancy and exercise. Hypertrophy because of pathological stimuli is known as mal adaptive while the one that comes from physiological triggers is known as beneficial or adaptive. Although both have similarities about cardiomyocyte growth and molecular adaptations, they diverge to distinct outcomes. The pathological hypertrophy evolves to a pattern of cardiac dysfunction while the physiological one does not cause any damage to the heart. This tenuous line between those phenotypes involves complex cellular mechanisms that need to be clarified. In this context, microRNAs are considered as regulators of many biological processes, and have been associated to myocardial growth. Therefore, our aim was to compare microRNA expression between physiological (swiminduced) and pathological (TAC-induced) hypertrophy. The analysis was performed after 28 days for SWIM protocol and 35 days for TAC model. The comparison was done using microRNA microarray technology (Affymetrix). Interestingly, only 20 microRNAs were differential expressed between both models. Out of those, 12 were up regulated (miR- 193a-3p, miR-299a-5p, miR-127-5p, miR-214-5p, miR-188-5p, miR-326-3p, miR-6395, miR-547-3p, miR-199a-5p, miR-381-3p, miR-223-3p and miR-199b-5p) while 8 were down regulated in TAC group compared to SWIM group. Besides, only 3 microRNAs, miR-21a-5p, miR-206-3p and miR-1983, were upregulated in TAC and SWIM model compared to SHAM and SED groups. After that, a search at KEGG Pathway database retrieved 4 pathways (665 genes) enriched with targets from microRNAs downregulated and 80 pathways (3394 genes) enriched with targets from up-regulated microRNAs in in 13 TAC group compared to SWIM group. In conclusion, there are microRNAs specific committed to the physiological cardiac hypertrophy development as well to the pathological cardiac growth as observed in our microarray data. Furthermore, the possible targets of those microRNAs could be involved in pathways associated with cellular growth, survival and cardiac adaptation.

Orientadora: Profa. Dra. Marcela Sorelli Carneiro Ramos / Tese (doutorado) - Universidade Federal do ABC, Programa de Pós-Graduação em Biossistemas, 2017. / As doencas cardiovasculares correspondem ao maior risco de morte subita em todo o mundo, a populacao mundial tem se tornado cada vez mais sedentaria e junto as doencas cardiovasculares outros problemas de saude publica aumentam como a obesidade e diabetes mellitus. Os gastos com a saude publica aumentam muito devido a maior incidencia de doencas associadas ao sedentarismo, obesidade, tabagismo, estresse, hipertensao arterial entre outros. Especificamente a obesidade e considerada uma epidemia mundial, que esta associada aos habitos alimentares e sedentarismo, e esta amplamente relacionada ao fator de risco cardiovascular como hipertensao arterial, resistencia insulinica, dislipidemia e diabetes mellitus, que favorecem o processo aterosclerotico e a ocorrencia de eventos cardiovasculares. Ha uma maior prevalencia de hipertensao arterial em individuos obesos e presenca de hipertrofia cardiaca, que e a maior condicao de morbimortalidade cardiovascular. Trata-se de uma doenca cronica relacionada a eventos pro-inflamatorios com acao de diversas citocinas e quimiocinas alem de hormonios envolvidos na resposta imunologica inata. Os receptores Toll-Like fazem parte da Resposta Imunologica Inata e estao presentes em diversos tecidos, incluindo o coracao. Quando ativados desencadeiam uma resposta intracelular, ativando fatores transcricionais, como NF-kB, que leva a transcricao genica de citocinas inflamatorias: IL-6, TNF-¿¿, INF-gama, IL-1. Algumas doencas cardiovasculares sao antecedidas por inflamacoes sistemicas ou locais, demonstrando que as citocinas sao responsaveis por modular o trofismo cardiaco. Diante do exposto, uma analise mais completa envolvendo obesidade e processos inflamatorios com o risco cardiovascular se faz necessaria para a compreensao de moleculas e vias de sinalizacao, reguladas nessa patologia. O presente estudo tem como objetivo avaliar a contribuicao dos fatores inflamatorios na hipertrofia cardiaca induzida por modelo experimental de obesidade. Para tanto, foram utilizados camundongos C57Bl/6 e camundongos FVB/NJ submetidos ao tratamento com dieta rica em gordura contendo 23% proteina, 35,5% carboidrato e 35,9% gordura e foram comparados aos grupos chow (controle) submetidos a dieta composta por 19% de proteina, 56% de carboidrato, 3,5% de gordura por 68 dias de tratamento. Os animais C57Bl/6 apresentaram maior ganho de peso em relacao aos FVB/NJ ao final dos 68 dias de dieta High Fat. O aumento de colesterol serico, insulina, glicemia, epiWAT (epididymal white adipose tissue) foi maior nos animais C57Bl/6. As duas linhagens apresentaram Heart Weight/Body Lenght e Heart Weight/Tibia Lenght maior em relacao ao grupo Chow, ou seja, grupo controle. Atraves da tecnica de Q-PCR os niveis de mRNA dos marcadores moleculares de hipertrofia cardiaca foram avaliados, e o marcador alfa-actina mostrou-se aumentado na linhagem C57Bl/6 enquanto o marcador ANF (atrial natriuretic factor) mostrou-se aumentado na linhagem FVB/NJ, quando comparados aos seus respectivos grupos chow. Os genes TLR2, TLR4, TNF-¿¿, IL-6, MyD88 e NF-kB tambem foram avaliados. A expressao genica de TLR2, MyD88 e NF-kB mostrou-se diminuida nos animais FVB/NJ submetidos a dieta rica em gordura quando comparados aos animais controle. Na linhagem C57Bl/6, observou-se aumento na expressao genica de TNF-¿¿ e NF-kB. A inducao da obesidade promoveu maior ganho de peso na linhagem C57Bl/6 acompanhado de maior adiposidade abdominal e no tecido epididimal, essa linhagem apresentou maior sensibilidade a insulina e maiores niveis de colesterol na dieta High Fat em relacao a linhagem FVB/JN. Houve remodelamento cardiaco em ambas linhagens e a expressao genica de citocinas inflamatorias foi mais evidente na linhagem C57Bl/6, tambem apresentando maior expressao genica de NF-kB. A linhagem FVB/NJ apresentou diminuicao na expressao de MyD88 e NF-kB na dieta High Fat. Com base nos resultados obtidos verificamos que os camundongos C57Bl/6 mostraram-se mais suscetiveis a dieta enquanto a linhagem FVB/JN mostrou-se mais resistente. / Cardiovascular diseases correspond to an increased risk of worldwide sudden death, the world population has become increasingly sedentary and with cardiovascular diseases other public health problems increase such as obesity and diabetes mellitus. Expenditures on public health increase greatly due to a higher incidence of diseases associated with sedentarism, obesity, smoking, stress, hypertension among others. Obesity specifically is considered a worldwide epidemic, which is associated with eating habits and sedentary lifestyle, and is largely related to cardiovascular risk such as hypertension, insulin resistance, dyslipidemia and diabetes mellitus, which favor the atherosclerotic process and occurrence of cardiovascular events. There is a higher prevalence of hypertension in obese and presence of cardiac hypertrophy, which is a higher cardiovascular morbidity and mortality condition. It is a chronic disease related to pro-inflammatory events with the action of various cytokines and chemokines in addition to hormones involved in the innate immune response. Toll-like receptors are part of the innate immune response and are present in several tissues including the heart. When activated trigger an intracellular response, activating transcriptional factors such as NF-kB, which leads to the genetic transcription of inflammatory cytokines: IL-6, TNF- á, IFN-gamma, IL-1. Some cardiovascular diseases are antecedents of systemic or local inflammations, demonstrating that cytokines are responsible for modulating cardiac trophism. In summary, a more complete analysis involving obesity and inflammatory processes with cardiovascular risk to understand molecules and signaling pathways regulated in this pathology are needed. The present study aims to evaluate the contribution of inflammatory factors in cardiac hypertrophy induced by the experimental model of obesity. For this, C57Bl/6 mice and FVB/NJ mice submitted to a high fat diet containing 23% protein, 35.5% carbohydrate and 35.9% fat were compared to the Chow (control) groups submitted to a chow diet containing 19% protein, 56% carbohydrate, 3.5% fat for 68 days of treatment. The C57Bl/6 animals presented greater weight gain in relation to FVB/NJ at the end of 68 days of high fat diet. Serum cholesterol, insulin, glycemia, epiWAT (epididymal white adipose tissue) increased in C57B1/6 animals. Both strains presented Heart Weight/Body Lenght and Heart Weight/Tibia Lenght higher than the Chow group. mRNA levels of cardiac hypertrophy molecular markers were evaluated through Q-PCR technique, and the alpha-actin marker was shown to be increased in the C57B1/6 lineage while the ANF (atrial natriuretic factor) marker was shown to be increased in the FVB/NJ lineage, when compared to their respective chow groups. TLR2, TLR4, TNF-á, IL-6, MyD88 and NF-kB genes were also evaluated. Gene expression of TLR2, MyD88 and NF-kB were decreased in FVB / NJ animals submitted to a high fat diet when compared to control animals. There was an increase in the gene expression of TNF-á and NF-kB in C57Bl/6 lineage. The induction of obesity promoted greater weight gain in the C57Bl/6 mice followed by a greater abdominal and epididymal adiposity, this lineage presented higher insulin sensitivity and higher cholesterol levels in the High Fat diet when compared to the FVB/JN lineage. There was cardiac remodeling in both lineages and inflammatory cytokines gene expression were more evident in the C57Bl/6 mice, also presenting a greater gene expression of NF-kB. The FVB/NJ mice showed decreased expression of MyD88 and NF-kB on the High Fat diet. Based on the results, we found that the C57Bl/6 mice were more susceptible to the High Fat diet than the FVB/JN strain.

Orientadora: Profa. Dra. Marcela Sorelli Carneiro Ramos / Tese (doutorado) - Universidade Federal do ABC, Programa de Pós-Graduação em Biossistemas, 2017. / hipertrofia cardiaca pode ser considerada uma adaptacao fisiologica que se desenvolve quando ha uma sobrecarga cronica imposta ao miocardio. A lesao renal e definida como uma diminuicao abrupta da funcao renal e pode ser considerada uma doenca inflamatoria que pode atingir outros orgaos como, por exemplo, pulmoes, figado e coracao. Evidencias sugerem um papel importante para interleucina-6 (IL-6) nas vias moleculares que iniciam a hipertrofia. O sistema nervoso simpatico (SNS) esta relacionado com a funcao imunologica inervando os orgaos imunes sendo responsavel pela liberacao de catecolaminas. Por sua vez, as catecolaminas quando liberadas no coracao modificam o debito cardiaco por alterarem a forca de contracao nas fibras miocardicas e a frequencia cardiaca. Alem de influenciar funcionalmente a circulacao, o aumento na atividade do SNS, pode exercer um efeito trofico sobre as celulas musculares lisas e miocardicas, podendo assim, contribuir para o trofismo do sistema cardiovascular. O presente estudo buscou avaliar a participacao do Sistema Nervoso Simpatico, no desenvolvimento da hipertrofia cardiaca induzida por lesao renal isquemica. Para tanto, foram utilizados camundongos machos C57BL/6 submetidos a oclusao do pediculo renal esquerdo, por 60 min, seguido de reperfusao por 12 dias, e submetidos ou nao ao tratamento com o ¿À-bloqueador atenolol. Os resultados demostraram que o modelo experimental de isquemia/reperfusao (I/R), foi capaz de gerar um quadro de falencia renal e induzir hipertrofia cardiaca, devido ao processo inflamatorio causado pela lesao. Alem disso, o tratamento com atenolol foi capaz de prevenir o crescimento hipertrofico observado apos a I/R, avaliado pelas relacoes Peso coracao/Peso Corporeo, pelos niveis de RNAm de ANF e BNP (marcadores moleculares de hipertrofia cardiaca) e pelos niveis das citocinas inflamatorias. Da mesma forma, o tratamento reduziu os niveis de expressao genica e proteica do receptor ¿À adrenergico assim como dos demais componentes da via, e tambem a atividade enzimatica de PKA. Os niveis de noradrenalina tambem se mostraram elevados apos isquemia e reperfusao, apresentando uma relacao com a hipertrofia observada, sugerindo um papel importante do SNS na regulacao do quadro hipertrofico. Diante do exposto, os dados apresentados demonstram uma sensibilidade do tecido cardiaco para as acoes do SNS, o qual pode estar presente na regulacao de mecanismos que levam a patogenese da hipertrofia cardiaca neste modelo. / Cardiac hypertrophy is a physiological adaptation that develops when a chronic overload is imposed on the myocardium. Kidney damage is defined as an abrupt decrease in kidney function and can be considered an inflammatory disease that can target other organs such as the lungs, the liver and the heart. Evidence suggests an important role for interleukin-6 (IL-6) in the molecular pathways that initiate hypertrophy. The sympathetic nervous system is related to the immune function by innervating the immune organs and also being responsible for the release of catecholamines. On the other hand, the catecholamines released in the heart modify the cardiac output because they change the contraction force of the myocardial fibers and the heart rate. An increase in the activity of the SNS, which functionality influencescirculation, can exert a trophic effect on the smooth and over myocardial muscle cells, thus contributing to the trophism of the cardiovascular system. The present study aimed to evaluate the participation of the Sympathetic Nervous System in thedevelopment of cardiac hypertrophy induced ischemia/reperfusion animal model. Therefore, C57BL / 6 mice were submitted to left renal pedicle occlusion for 60 min, followed by reperfusion for 12 days, with or without treatment with the â-blocker atenolol. The results demonstrated that the experimental model of ischemia / reperfusion (I / R) was able to generate renal failure and induce cardiac hypertrophy due to the inflammatory process caused by the lesion. In addition, atenolol treatment was able to prevent the hypertrophic development observed after I / R, performed by heart weight/ bodyweight ratios, ANF and BNP mRNA levels (molecular markers of cardiac hypertrophy), and cytokine levels. Similarly, the treatment reduced the levels of gene and protein expression of the â-adrenergic receptor as well as of the other components of the pathway and enzymatic activity of PKA. Noradrenaline levels were also elevated after ischemia / reperfusion, showing a relationship with the hypertrophy phenomenon and therefore suggesting an important role of SNS in the regulation of hypertrophic conditions. In conclusion, this data indicates a sensitivity of the cardiac tissue to the actions of the SNS, which may be present in the regulation of mechanisms that lead to the pathogenesis of cardiac hypertrophy in this model.

O exercício físico modula o sistema renina angiotensina (SRA), que tem um importante papel na fisiologia cardíaca, especialmente na promoção da hipertrofia cardíaca. O SRA pode ser dividido em dois eixos principais: o eixo clássico – representado pelo receptor AT1 (AT1R) ativado pela angiotensina II (ANG II) – e o eixo alternativo – que é ativado pela interação da angiotensina 1- 7 (ANG 1-7) com o receptor MAS (MASR). O balanço entre os eixos do SRA pode determinar um remodelamento cardíaco fisiológico ou patológico. Estudos têm demonstrado que altos volumes de exercício físico podem desencadear possíveis efeitos deletérios ao sistema cardiovascular. Neste contexto, nosso objetivo foi avaliar a hipertrofia cardíaca, o eixo clássico e o eixo alternativo do SRA no miocárdio de camundongos submetidos a variados volumes e intensidades de treinamento em natação. Para tal, camundongos balb/c foram divididos em três grupos: (I) sedentário (SED), (II) treinados 2x ao dia (T2) sem sobrecarga e (III) treinado 3x ao dia com sobrecarga de 2% do peso corporal (T3), totalizando 6 semanas de treinamento efetivo. Ambos os grupos treinados desenvolveram hipertrofia cardíaca, sem diferença nos níveis de fibrose. Bioquimicamente, observamos um aumento nos níveis do receptor MASR somente no grupo T2, enquanto que os níveis de AT1R aumentaram somente no grupo T3. Contudo, não foi observada alteração na concentração dos peptídeos ANGI, ANGII e ANG 1-7 no tecido cardíaco entre os grupos. Além do mais, o grupo T3 demonstrou um aumento na expressão de miosina de cadeia pesada- β em comparação ao grupo SED e redução da expressão da isoforma- @ em relação ao grupo T2. Em conclusão, nossos resultados sugerem que ambos os protocolos de exercício promoveram uma hipertrofia cardíaca semelhante, mas o protocolo com maior volume e intensidade promoveu uma ativação diferencial dos receptores do SRA e reativação de genes fetais. Estudos que avaliem protocolos com maior duração são necessários para esclarecer se estas mudanças representam uma ativação precoce dos mecanismos relatados para o desenvolvimento de um fenótipo com características patológicas. / Exercise promotes physiological cardiac hypertrophy and induces the activation of the renin angiotensin system (RAS), which plays an important role in cardiac physiology, both through the classical axis – represented by the angiotensin II receptor type 1 (AT1) activated by angiotensin II (ANG II) – and the alternative axis – which is activated by the angiotensin 1-7 interaction with the MAS receptor. However, very intense exercise protocols could have deleterious effects on the cardiovascular system. In this context, we aimed to analyze the cardiac hypertrophy phenotype, as well as the classical (ANGII/AT1) and alternative (ANG1-7/MAS) RAS axes, in the myocardium of mice submitted to varying volume and intensity swimming exercises for the development of cardiac hypertrophy. To this end, male balb/c mice were divided into three groups: (I) sedentary (SED), (II) swimming twice a day (T2) without overload, and (III) swimming three times a day with a 2% body weight overload (T3), totaling six weeks of training. Both training groups developed cardiac hypertrophy. Interestingly, we observed an increase in MAS receptor levels only in group T2, while AT1 levels increased only in group T3. However, no change was observed regarding the levels of angiotensin peptides ANG-I, ANG-II, and ANG1-7, in either group. In addition, group T3 displayed a higher expression of myosin heavy chain-β (MHC-β) and lower expression levels of the alpha isoform (MHC-@). Fibrosis was not observed in any of the groups. In conclusion, our results suggest that both exercise protocols promoted a similar cardiac hypertrophy phenotype, but the protocol applying increased volume and intensity resulted in differential activation of RAS receptors and fetal gene reactivation. Studies applying longer duration protocols could elucidate if these changes represent early activation of mechanisms related to hypertrophy development with phenotypic pathological characteristics.