O uso de esteróide anabolizante é cada vez maior por pessoas que praticam exercícios como forma de lazer, sem se importarem com os possíveis efeitos colaterais, o que vem se tornando um importante problema de saúde pública. Dentre os seus principais efeitos colaterais, destacamos a hipertrofia cardíaca, que parece ser ainda mais pronunciada quando associado ao treinamento físico, sendo esta relacionada a maior atividade da enzima conversora de angiotensina cardíaca. Tendo em vista esse cenário, o presente trabalho visa verificar a participação do sistema renina angiotensina aldosterona sobre a hipertrofia cardíaca e síntese de colágeno induzida pelo esteróide anabolizante, associado ao treinamento físico por natação em ratos, por meio do bloqueio de receptores AT1 com Losartan e dos receptores de mineralocorticóides com Espironolactona. Resultados mostram que a administração de esteróide anabolizante aumenta a ativação do sistema renina angiotensina aldosterona cardíaco, o qual está diretamente relacionado aos seus efeitos colaterais, visto que o bloqueio dos receptores AT1 ou dos RM inibiu esses efeitos. Sendo mostrado pela primeira vez, os efeitos do esteróide anabolizante sobre o aumento na expressão do gene da aldosterona sintase e da enzima 11-HSD2, sugerindo os efeitos dos esteróides anabolizantes sobre o aumento da síntese e atividade da aldosterona cardíaca / The anabolic steroid use is growing by recreational exercise practitioners, without worried about the possible collateral effects, becoming an important problem of public health. Among its deleterious effects we detach the cardiac hypertrophy, that looks to be still bigger when the swimming training was associated, being is related to bigger activity of the cardiac angiotensin converter enzime. With that, the present work is going to verify the renin angiotensin aldosteron system participation about the cardiac hypertrophy and collagen synthesis prompted by the anabolic steroid and the association with the swimming training in rat by means of the AT1 receivers blockade with Losartan and of the mineralocorticoids receivers blockade with Espironolacton. Our results show that the anabolic steroid administration increased the cardiac rennin angiotensin aldosteron system activity, that is straightly related to its deleterious effects, seen that the AT1 receivers blockade or the mineracorticoids receivers blockade inhibited those effects. Being shown by the first time the anabolic steroids effects about the increase of the aldosterone sintase gene expression and of the 11-HSD2 enzyme, suggesting the anabolic steroids effects about the cardiac aldosterone synthesis and activity increase

O treinamento físico aeróbio (TF) e a hipertensão arterial (HA) induzem hipertrofia cardíaca (HC) com características diferentes, e entre as diferenças moleculares podem estar a elucidação de abordagens terapêuticas como os microRNAs (miRNAs). Selecionamos de dados de miRNAarray, 15 miRNAs cardíacos induzidos por dois protocolos de treinamento físico de natação (TF) e comparamos com o miRNAarray em modelo de hipertensão arterial (animais espontaneamente hipertensos, SHR). Foram selecionados 4 miRNAs de interesse (miRNA-27a, 27b, 126 e 29c) que seguiram para a confirmação de sua expressão por qRT-PCR. Destes, selecionamos o miRNA-29c para que fosse realizada a modulação in vivo em SHR jovens. Foi realizada injeção cardíaca intramuscular de partículas de vetor lentiviral para a superexpressão do miRNA-29c. Foram testadas duas doses: baixa (B), 0,6x109 pv/animal e alta (A), 3x109 pv/animal; e por dois períodos de tratamento: 7 e 14 dias. Foi avaliada a expressão de GFP em fígado e coração por western blott para observar a eficiência da transdução viral in vivo. Os efeitos do tratamento na pressão arterial (PA) foram analisados por pletismografia de cauda; na HC pela razão VE/PC (peso do ventrículo esquerdo/peso corporal), peso do coração/PC e (cor/PC), e pelo diâmetro de cardiomiócitos (dCMO) por histologia. qRT-PCR foi utilizado para investigar a expressão do miRNA-29c e seus alvos, colágeno do tipo I e do tipo III (COLIAI e COLIIIAI). O conteúdo de colágeno também foi medido por análise histológica (picrossírius), pela fração volumétrica de colágeno (% col), e pela concentração de OHprolina no VE. Os grupos que receberam baixa dose das partículas lentivirais foram positivos para GFP em coração e fígado, tendo sido assumida a dose baixa como eficiente para futuras transduções. Todos os grupos tratados apresentaram aumento da expressão do miRNA-29c. A expressão gênica do COLIAI diminuiu para os grupos tratados o que não ocorreu para o COLIIIAI. A fração volumétrica foi menor em todos os grupos tratados o que mostra evidência que o tratamento foi eficaz para diminuir a concentração de colágeno cardíaco. Houve diminuição no cor/PC de 7-11% para os grupos SHR7A e SHR7B, que foi concatenada com um aumento no dCMO, com diminuição da fibrose. Nossos resultados sugerem, portanto, que o tratamento com o miRNA29c induz remodelamento cardíaco benéfico, abrindo perspectivas para investigações adicionais sobre terapias antifibróticas para doenças cardiovasculares / Both aerobic exercise training (ET) and Hypertension (HY) induce different cardiac hypertrophy (CH) phenotypes which molecular differences and may lead to new targets for therapies in cardiovascular disease, as microRNAs (miRNAs). We selected 15 miRNAS that were changed by ET from miRNAarray data and compared them with other from HY miRNAarray data. Four miRNAs were selected for qRT-PCR confirmation: miRNA-27a, 27b, 126 e 29c. Among then, miRNA 29c was choosen to be modulated by lentiviral vector due its role in fibrosis regulation. Intramuscular cardiac injection of the lentiviral vector particles was performed following two doses; low-dose , 0,6x109 vp/rat and high 3x109 vp/rat; and for two different times (7 and 14 days). The transduction efficiency was assessed by GFP expression by western blot. Blood pressure (BP) was measured by caudal pletysmography, CH was analysed by ratio LVw/BW (left ventricle weight/body weight), heartw/BW (heart weight/body weight) and by cardiomyocyte diameter (dCMO). qRT-PCR was used to assess miRNA-29c expression and its targets COLIAI and COLIIIAI gene expression. The LV collagen content was assessed by histology (Picrossirius red), by collagen volume fraction, and by Hydroxiproline concentration. Both groups that received the lowe doses were GFP positive in the heart and liver tissue,We assumed that low doses were better for future in vivo transduction. BP did not increase to SHR14A and SHR14B, what did not occurred to the 7 days groups. The miRNA-29c expression increased in all treated groups versus their control (CSI). COLIAI expression decreased in treated groups, while COLIIIAI did not change. Collagen volume fraction decreased in all treated groups, which shows that the treatment was efficient to decrease the cardiac collagen. Heart/BW decreased 7-11% in SHR14B and SHR14A and there were an increase in dCMO in all treated groups, that shows that cardiac remodeling of treated SHR included an increase in size of CMO and a decrease in cardiac fibrosis Our data suggests that there is a beneficial cardiac remodeling after treatment with miRNA-29c, which opens perspective for further investigation of antifibrotic therapies for cardiovascular disease

A hipótese do presente estudo é que as ações cardioprotetoras da Angiotensina1-7 (Ang1-7) consigam atenuar os efeitos cardíacos dos hormônios tireoidianos. Ratos selvagens ou transgênicos que superexpressam Ang1-7 foram induzidos ao hipertireoidismo por injeções intraperitoneais de triiodotironina (T3) (7µg/100g/dia). Parâmetros de morfologia e função cardíaca foram avaliados, bem como os componentes do Sistema-Renina-Angiotensina no coração. Os elevados níveis de Ang1-7 nos animais transgênicos atenuaram os efeitos tróficos do T3 e o hiperdinamismo cardíaco, além de promover a melhora da função cardíaca. Os animais hipertireoideos apresentaram aumento da Angiotensina II, do receptor AT1 e da atividade da ECA2 no coração. In vitro, o tratamento com Ang1-7 (1000nM) foi capaz de impedir o aumento da área de superfície celular em cultura primária de cardiomiócitos neonatos tratados com T3 (10nM) por 24 horas. Sendo assim, estes dados demonstram, pela primeira vez na literatura, ações cardioprotetoras da Ang1-7 frente às ações cardíacas e hipertróficas do T3. / The aim of this study is to verify if Angiotensin1-7 (Ang1-7) may influence the cardiovascular effects induced by thyroid hormone. Wild or Transgenic rats that constitutively overexpress Ang1-7 (TGR-L3292) received intraperitoneal injections of T3 (7µg/100g/day) for 14 days in order to develop hyperthyroidism. Cardiac morphology, function parameters and RAS components were evaluated in the heart. High levels of Ang1-7 attenuated cardiac hypertrophy and hemodynamic parameters induced by T3. Ang1-7 treatment improved the cardiac function of hyperthyroid animals. Moreover, high levels of T3 increased Angiotensin II levels, AT1 receptor and ACE2 activity in the heart. Anti-hypertrophic effects of Ang1-7 were also observed in vitro. Ang1-7 treatment (1000nM) prevented the increase in cell surface area in primary cultures of neonatal cardiomyocytes treated with T3 (10nM) for 24 hours. For the first time, cardioprotective actions of Ang1-7 were observed in the heart of hyperthyroid animals.

Recentes estudos têm revelado o envolvimento de microRNAs (miRNAs) no controle da hipertrofia cardíaca e na função do miocárdio. Ainda, várias pesquisas têm demonstrado que o consumo de dieta rica em gordura pode induzir hipertrofia e remodelamento cardíaco. No presente estudo, investigou-se o efeito de dietas contendo diferentes porcentagens de gordura na expressão do miRNA-22, um miRNA que está diretamente envolvido na regulação da morfologia e da função cardíaca e um importante mediador da hipertrofia e falência cardíaca deflagradas por diferentes estímulos. Para isso, camundongos C57BL/6 machos, com idade entre 4 e 5 semanas, foram alimentados com uma dieta controle (10% das calorias provenientes de lipídeos) ou dietas hiperlipídicas (HF) contendo 45% de kcal de gordura (HF45%) e 60% de kcal de gordura (HF60%) por 10 ou 20 semanas. A dieta HF60% promoveu um aumento do peso corpóreo, aumento dos níveis de glicose, insulina, leptina, colesterol total e triglicérides e induziu intolerância a glicose. As dietas HF promoveram remodelamento cardíaco, conforme evidenciado pelo aumento no diâmetro transverso dos cardiomiócitos e deposição de colágeno. A análise de sequenciamento de RNAs demonstrou que as dietas ricas em gordura induziram padrões distintos de expressão de miRNAs no coração, incluindo o miRNA-22. Análise de bioinformática identificou a caveolina-1 como potencial alvo do miRNA-22 e seus níveis encontraram-se aumentados no grupo HF60% tratado por 20 semanas. Considerando que o miRNA-22 está envolvido no desenvolvimento da hipertrofia cardíaca e falência do coração, é possível que algumas destas alterações estruturais e funcionais cardíacas induzidas pela dieta rica em gordura sejam, ao menos em parte, influenciadas pelo aumento da expressão deste miRNA. Entretanto estudos funcionais são necessários para determinar a contribuição do miRNA-22 para os efeitos promovidos pela dieta rica em gordura no coração. / Recent studies have revealed the involvement of microRNAs (miRNAs) in the control of cardiac hypertrophy and myocardial function. In addition, several reports have demonstrated that high fat (HF) diet induces cardiac hypertrophy and remodeling. In the current study, we investigated the effect of diets containing different percentages of fat on the miRNA-22 expression, which is a miRNA involved in the control of the cardiac morphology and function and an important mediator of cardiac hypertrophy and heart failure triggered by different stimuli. To address this question, 4-week-old male C57Bl/6 mice were fed with a low fat diet (10 kcal% fat) or high fat diets (HF), containing 45 kcal% fat (HF45%) and 60 kcal% fat (HF60%) for 10 and 20 weeks. HF60% diet promoted an increase on body weight, fasting glycemia, insulin, leptin, total cholesterol, triglycerides and induced glucose intolerance. HF feeding promoted cardiac remodeling, as evidenced by increased cardiomyocyte transverse diameter and interstitial fibrosis. RNA sequencing analysis demonstrated that HF feeding induced distinct miRNA expression patterns in the heart, including miRNA-22. Bioinformatics analysis identified caveolin-1 as a potential target of miRNA-22 and its levels were increased in HF60% group treated for 20 weeks. Considering that miRNA-22 is involved in the development of cardiac hypertrophy and heart failure, it is possible that some of the cardiac structural and functional alterations induced by high fat diet are, at least in part, influenced by the increased expression of this miRNA. However functional studies are needed to determine the contribution of miRNA-22 in the effects promoted by high fat diet in the heart.

O presente estudo avaliou aspectos relacionados ao contexto inflamatório na hipertrofia cardíaca induzida pelos hormônios tiroideanos (HT) e o possível envolvimento do sistema renina-angiotensina (SRA) nesse processo, utilizando análises in vivo e com enfoque maior na abordagem in vitro. Os resultados mostraram algumas alterações em citocinas circulantes e cardíacas de animais tratados com HT. Além disso, as expressões de S100A8 e MyD88 foram aumentadas no coração de ratos submetidos ao hipertiroidismo e em cardiomiócitos em cultura estimulados com HT. S100A8 e MyD88 mediaram a ativação do fator nuclear NF-κB pelos HT, tendo papel crucial para o crescimento hipertrófico de cardiomiócitos tratados com HT. Por fim, a ação dos HT modulando a expressão de S100A8 e NF-κB foi mediada pelo SRA. Estes dados contribuem com o entendimento das bases moleculares da ação dos HT e da relação deste com o SRA na hipertrofia cardíaca. / The present study evaluated inflammation related aspects in cardiac hypertrophy induced by thyroid hormones (TH) and the possible involvement of the renin-angiotensin system (RAS) in this process, by using in vivo and in vitro analysis. The results showed alterations in circulating and cardiac cytokines from TH treated animals. The expression of S100A8 and MyD88 were increased in the heart of hyperthyroid rats and in cultured cardiomyocytes stimulated with TH. S100A8 and MyD88 mediated the nuclear factor NF-κB activation by TH and these factors presented crucial role to the hypertrophic growth of TH-treated cardiomyocytes. Finally, the action of TH on S100A8 and NF-κB expression was mediated by RAS. These data contribute to the knowledge of molecular basis of TH action and the relationship between TH and RAS in cardiac hypertrophy.

The mammalian Na+/H+ exchanger isoform 1 (NHE1) is a ubiquitously expressed membrane protein that regulates intracellular pH. In the myocardium, NHE1 has been implicated in ischemia/reperfusion (I/R) and cardiac hypertrophy (CH). Hormonal, autocrine and paracrine stimuli, acidosis, cardiotoxic metabolites released during I/R and CH increases NHE1 protein expression and activity. The involvement of NHE1 in CH and I/R has been further supported with the use of NHE1 inhibitors, which have been beneficial in the prevention/regression of several models of CH and I/R injury. Despite the fact that elevation of NHE1 expression and activity have been demonstrated in several models of heart disease, it was unclear whether elevation of NHE1 protein expression was sufficient to induce a specific cardiac pathology, or whether activation of the protein was required. To understand the direct role of NHE1 in CH and I/R, an in vivo and in vitro gain-of-function model, expressing varying levels and activities of NHE1 were examined. In vivo, our N-line mice expressed wild type NHE1 and our K-line mice expressed constitutively active NHE1. In vitro, neonatal rat ventricular cardiomyocytes were infected with the IRM adenovirus containing wild type NHE1 or the K-IRM adenovirus containing active NHE1. We demonstrated that expression of constitutively active NHE1 promotes CH to a much greater degree than expression of wild type NHE1 alone, both in vivo and in vitro. This NHE1-dependent hypertrophic response occurred independent of signaling pathways involved in CH including, mitogen activated protein kinases, p90 ribosomal S6 kinase, calcineurin and glycogen synthase kinase. The NHE1-dependent hypertrophic effect also occurred independent of gender. In addition, the expression of active NHE1 increased the susceptibility of intact mice to neurohormonal stimulation and progressed the hypertrophic response. When these hearts expressing active NHE1 were subjected to I/R using the ex vivo working heart perfusion model, fatty acid (FA) oxidation and glycolysis rates increased, thus generating greater ATP production rates. This was associated with cardioprotective effects in the myocardium, as well as a more energetically efficient myocardium. Expression of the endoplasmic reticulum (ER) stress response proteins, calreticulin and PDI were also shown to be increased relative to controls, and may contribute to the cardioprotection observed. We demonstrate that active NHE1 induces cardioprotection and alters cardiac metabolism in working hearts subjected to I/R. Overall, our results suggest that expression of active NHE1 has a double edged sword effect, on one side it induces CH while on the other side, it protects the heart against I/R injury.

The endocrine heart synthesises and secretes two polypeptide hormones: the natriuretic peptides (NP) atrial natriuretic factor (ANF) and B-type natriuretic peptide (BNP). The biological actions of these hormones serve both acutely and chronically to reduce systemic blood pressure and hemodynamic load to the heart, thus contributing to the maintenance of cardiorenal homeostasis. Considerable effort has been focused on the elucidation of the mechanistic underlying ANF and BNP gene expression and secretion but much remains to be determined regarding specific molecular events involved in the cardiocyte secretory function. These hormones are produced by the atrial muscle cells (cardiocytes), which display a dual secretory/muscle phenotype. In contrast, ventricular cardiocytes display mainly a muscle phenotype. Comparatively little information is available regarding the genetic background for this important phenotypic difference with particular reference to the endocrine function of the heart. We postulated that comparison of gene expression profiles between atrial and ventricular muscles would help identify transcripts that underlie the phenotypic differences associated with the endocrine function of the heart as well as identify signaling pathways involved in its regulation. The cardiac atrial and ventricular transcriptomes were analyzed using oligonucleotide microarrays under normal or chronically induced aortocaval shunt volume-overload conditions. Transcriptional differences were validated by RT-PCR and transcripts of interest were knocked-down by RNAi. Comparison of gene expression profiles in the rat heart revealed a total of 1415 differentially expressed genes between normal atrial and ventricular tissues. Functional classification and pathway analysis identified numerous transcripts involved in mechanosensing, vesicle trafficking, hormone secretion, and G protein signaling. Volume-overloaded animals exhibited a progressive increase in cardiac mass over the four-week time course, an increase in expression of known hypertrophic genes, as well as the differential expression of 700 genes within the atria. Volume-overload specifically downregulated the accessory protein for heterotrimeric G protein signaling RASD1 in the atria. In vitro, knockdown of RASD1 in the atrial-derived HL-1 cells, significantly increased ANF secretion, demonstrating a previously unknown negative modulator role for RASD1. The data developed in this investigation provides insight into the expression profiles of genes particularly centered on the secretory function of the heart under normal and chronic hemodynamic overload conditions. Genome-wide expression profile analysis identified RASD1 as being differentially expressed between cardiac tissues as well as being modulated by chronic volume overload. RASD1 emerges as a tonic inhibitor of ANF secretion. The novel function identified herein for RASD1 in the atria is of considerable interest given the fact that secretory impairment of the cardiac natriuretic hormones can negatively impact cardiovascular homeostasis.

The endocrine heart synthesises and secretes two polypeptide hormones: the natriuretic peptides (NP) atrial natriuretic factor (ANF) and B-type natriuretic peptide (BNP). The biological actions of these hormones serve both acutely and chronically to reduce systemic blood pressure and hemodynamic load to the heart, thus contributing to the maintenance of cardiorenal homeostasis. Considerable effort has been focused on the elucidation of the mechanistic underlying ANF and BNP gene expression and secretion but much remains to be determined regarding specific molecular events involved in the cardiocyte secretory function. These hormones are produced by the atrial muscle cells (cardiocytes), which display a dual secretory/muscle phenotype. In contrast, ventricular cardiocytes display mainly a muscle phenotype. Comparatively little information is available regarding the genetic background for this important phenotypic difference with particular reference to the endocrine function of the heart. We postulated that comparison of gene expression profiles between atrial and ventricular muscles would help identify transcripts that underlie the phenotypic differences associated with the endocrine function of the heart as well as identify signaling pathways involved in its regulation. The cardiac atrial and ventricular transcriptomes were analyzed using oligonucleotide microarrays under normal or chronically induced aortocaval shunt volume-overload conditions. Transcriptional differences were validated by RT-PCR and transcripts of interest were knocked-down by RNAi. Comparison of gene expression profiles in the rat heart revealed a total of 1415 differentially expressed genes between normal atrial and ventricular tissues. Functional classification and pathway analysis identified numerous transcripts involved in mechanosensing, vesicle trafficking, hormone secretion, and G protein signaling. Volume-overloaded animals exhibited a progressive increase in cardiac mass over the four-week time course, an increase in expression of known hypertrophic genes, as well as the differential expression of 700 genes within the atria. Volume-overload specifically downregulated the accessory protein for heterotrimeric G protein signaling RASD1 in the atria. In vitro, knockdown of RASD1 in the atrial-derived HL-1 cells, significantly increased ANF secretion, demonstrating a previously unknown negative modulator role for RASD1. The data developed in this investigation provides insight into the expression profiles of genes particularly centered on the secretory function of the heart under normal and chronic hemodynamic overload conditions. Genome-wide expression profile analysis identified RASD1 as being differentially expressed between cardiac tissues as well as being modulated by chronic volume overload. RASD1 emerges as a tonic inhibitor of ANF secretion. The novel function identified herein for RASD1 in the atria is of considerable interest given the fact that secretory impairment of the cardiac natriuretic hormones can negatively impact cardiovascular homeostasis.

No description available.

Il y a 4 isoforme de p38 : α, β, δ, and γ. MK5, à l'origine identifié comme étant un régulateur de PRAK (Regulated/Activated Protein Kinase), est maintenant connu pour être activée par la protéine kinase p38 (qui est un mitogène activé par la protéine kinase, MAPK). Cette dernière est impliquée dans les mécanismes de fibrose et d'apoptose pendant l'hypertrophie cardiaque. De plus, MK5 est également activée par les MAPKs atypiques; ERK3 et ERK4. Bien qu’elles soient fortement exprimées dans le coeur, le rôle physiologique de MK5 et ERK3 demeure inconnu. Par conséquent, nous avons étudié l'effet de la constriction aortique transversale (TAC) – induisant un surcharge chronique de pression chez les souris hétèrozygotes knockout pour MK5 (MK5+/-) ou ERK3 (ERK3+/-) et pour leurs types sauvages (MK5+/+ et ERK3+/+). Deux sem post-TAC; le ratio de poids du coeur/poids corporel a été augmenté chez les 2 souris MK5+/- et MK5+/+. L'échocardiographie de la trans-thoracique démontre que la surcharge de pression a altéré la fonction diastolique du ventricule gauche chez MK5+/+, mais pas chez la souris MK5+/-. De plus, nous avons observé moins de dépôt de collagène, évalué par une coloration au trichrome de Masson, 2 et 3 sem post-TAC chez les souris MK5+/-. Parallèlement, le niveau de l’ARNm de collagène type1 alpha-1 a été significativement diminué dans les coeurs des souris MK5+/-, 2 et 3 sem post-TAC. De même, ERK3, mais pas ERK5 ni p38α, co-IP avec MK5 dans les 2 modèles des coeurs TAC; aigus ou chroniques. En revanche, l’ajout exogénique de GST-MK5 a abaissé ERK4 et p38α, mais pas ERK3 dans les lysâtes de coeur de souris. Par contre, GST-ERK3 et GST-p38α ne démontrent aucune co-IP avec MK5. Ces données suggèrent que dans le coeur seul ERK3, et non ERK4 ou p38α, est capable d’interagir avec, et réguler MK5. A niveau physiologique MK5 interagit entièrement avec ERK3 et par conséquent MK5 n’est pas disponible pour lier les protéines exogéniques. Les souris hétérozygotes pour ERK3 (ERK3+/-) ont également démontré une réduction ou une absence de collagène et une faible expression d’ARNm du collagène type1 alpha1, 3 sem post-TAC. Ces résultats démontrent un important rôle pro-fibrotique de la signalisation MK5-ERK3 pendant une surcharge chronique de pression.Nous avons également démontré 5 variant d'épissage de (MK5.1-5), y compris la forme originale (MK5.1). MK5.2 et MK5.5 subissent une délétion de 6 paires de base dans l’exon 12 : MK5.3 manque l'exon 12 : MK5.4 et MK5.5 manquent les exons 2-6. L'expression des ARNm des différents variant d'épissage a été vérifiée par PCR en temps réel (qPCR). Bien que l’expression est ubiquitaire, l'abondance relative de chaque variant était tissu-spécifique (coeur, rein, pancréas, muscle squelettique, poumon, foie, et cerveau). En plus, l'abondance relative des variant d’épissage varie pendant la surcharge de pression et le développement postnatal du coeur. En outre, l'immunofluorescence a indiqué que MK5.1-5.3 se localise au noyau alors que MK5.4-5.5 est situé au niveau cytoplasmic dans les cellules HEK 293 non stimulées. Suite à une stimulation avec l'anisomycin, un activateur de p38 MAPK, MK5.1-5.3 se translocalise du noyau au cytoplasme alors qu’une petite fraction de MK5.4-5.5 translocalise vers le noyau. Ces variant d'épissage peuvent diversifier la signalisation de MK5-ERK3 dans coeur, mais leur rôle exact oblige des recherches supplémentaires. Excepté l’isoforme δ, toutes les isoformes de p38 sont exprimées dans le coeur et la forme α est considérée comme étant l'isoforme dominante. L’analyse par qPCR et immunobuvardage de type western ont démontré que p38α et p38γ sont les deux isoformes prédominantes alors que p38β et p38δ sont exprimées aux mêmes niveaux dans le coeur de rat adulte. L'immunofluorescence a démontré que p38α et p38γ se trouvent dans le cytoplasme et le noyau. Cependant, suite à la surcharge par TAC, p38γ s'est accumulé dans noyau tandis que la distribution de p38α est demeurée inchangée. Ainsi, l'abondance de p38γ et sa translocalisation nucléaire suite à la surcharge de pression indique un rôle potentiel dans l'expression génique pendant le remodelage cardiaque. En conclusion, nous avons mis en évidence pour la première fois un rôle pro-fibrotique pour la signalisation MK5-ERK3 pendant une surcharge chronique de pression. D'ailleurs, les niveaux comparables d'expression de p38γ avec p38α, et la localisation différentielle de p38γ pendant la surcharge aiguë ou chronique de pression suggèrent différents rôles possibles pour ces isoformes pendant le remodelage hypertrophique cardiaque. / There are 4 isoforms of p38 MAP kinase: α, β, γ, and δ. p38 signaling has been implicated in fibrosis and apoptosis during cardiac hypertrophy. MK5, originally identified as a p38 Regulated/Activated Protein Kinase (PRAK), is known to be downstream of p38 mitogen activated protein kinase (MAPK). Although highly expressed in the heart, the physiological roles of MK5 remain unknown. To determine if MK5 plays a role in mediating detrimental effects downstream of p38, we studied the effect of transverse aortic constriction (TAC)-induced chronic pressure overload in mice heterozygous for a knockout of MK5 (MK5+/-). Moreover, as MK5 is also activated by the atypical MAPKs, ERK3 and ERK4, the effects of TAC were also studied in ERK3+/- mice. Wild-type (MK5+/+; ERK3+/+) littermates were used as controls. Two wks post-TAC, heart weight/body weight ratios were significantly and similarly increased in both MK5+/- and MK5+/+ hearts. Trans-thoracic echocardiography revealed that pressure overload impaired left ventricular diastolic function in MK5+/+, but not in MK5+/- hearts. In addition, less collagen deposition, assessed by Masson trichrome staining, was observed in MK5+/- hearts 2 and 3 wks post-TAC. Furthermore, TAC-induced increases in collagen alpha1 type1 mRNA levels were significantly lower in MK5+/- hearts at both 2 and 3 wks post-TAC. Immunoprecipitation of MK5 resulted in co-immunoprecipitation of ERK3 but not ERK4 or p38α in either acute or chronic sham-operated and TAC hearts. In contrast, exogenous GST-MK5 pulled down endogenous ERK4 and p38α, but not ERK3, from mouse heart lysates. Neither exogenous GST-ERK3 nor GST-p38α pulled down MK5. These results suggest that MK5 associates with, and is regulated by ERK3, but not ERK4 or p38α in heart. At physiological expressional levels, all MK5 was bound to ERK3 and hence not available to bind exogenous protein. Along similar lines, mice heterozygous for an ERK3 knockout (ERK3+/-) also showed reduced or absent collagen deposition and lower collagen alpha1 type1 mRNA levels 3 wks post-TAC. This data suggests an important pro-fibrotic role of MK5-ERK3 signaling during chronic pressure overload.We also demonstrated the existence of 5 splice variants of (MK5.1-5), including the originally published form (MK5.1). MK5.2 and MK5.5 had a 6 base pair deletion in exon 12: MK5.3 lacked exon 12: and MK5.4 and MK5.5 lacked exons 2-6. Subsequently, expression of the splice variants at the mRNA level was quantified by real time qPCR. Although ubiquitously expressed, the relative abundance of each variant was tissue-specific (heart, kidney, pancreas, skeletal muscle, lung, liver, and brain). Additionally, the relative abundance of MK5 splice variants changed in the heart during pressure overload and post-natal development. Furthermore, immunofluorescence revealed MK5.1-5.3 localized to the nucleus and MK5.4-5.5 to the cytoplasm in unstimulated HEK 293 cells. Upon stimulation with anisomycin, which activates p38 MAPK, MK5.1-5.3 translocated from the nucleus to the cytoplasm and small amounts of MK5.4-5.5 relocated to the nucleus. These splice variants may further diversify MK5-ERK3 signaling in the heart, but their exact role awaits further investigation. With the exception of p38δ, all p38 isoforms are expressed in the heart and α is considered to be the prominent isoform in this tissue. qPCR and western blot analysis revealed p38α and p38γ to be the predominant isoforms and p38β and p38δ are expressed at comparable levels in the adult heart. Confocal immunofluorescence studies revealed p38α and p38γ in both the cytoplasm and nucleus. However, in response to TAC, p38γ accumulated in the nucleus whereas the distribution of p38α remained unaffected. The high abundance of p38γ and its nuclear accumulation during chronic pressure overload suggest that this isoform may play a role in gene expression during pathological cardiac remodeling. In conclusion, we have shown for the first time a pro-fibrotic role for MK5-ERK3 signaling during chronic pressure overload. Moreover, comparable expression levels of p38γ with p38α, and differential localization of p38γ during acute or chronic pressure overload, suggest these isoforms play different roles during cardiac remodeling.