Naringin is a flavonoid glycoside (C27H32O14) found in citrus fruits and grapes, recognized for exercising antioxidant, antiatherogenic, hypoglycemic activity, among others. Contractile and electrical effects of naringin were characterized on the heart muscle of rats. The experiments were performed in the left atrium isolated rat (tub with 8 ml, Krebs-Hanseilet, 29 ± 0.1 ° C; Stimulation: 1 gf, 1 Hz, 100 V, 1.5 ms). The force data were obtained by isometric transducer (Grass FT03), amplified (Grass P11T), digitized (DATAQ DI710) and stored in a computer for analysis. The naringin (0.003 to 6 mM) was added cumulatively to the bath to determine their influence on the contractile parameters. Curves concentration-effect of naringin were obtained after atrial preincubation with 1 uM propranolol or 1 uM nifedipine or 1 uM ryanodine or still, using atria of animals with depletion of catecholamine. The effect of naringin on electrocardiograms were obtained by Langendorff technique (10 ml / min - Milan peristaltic pump; Krebs solution at 34 ± 0.1 °C aerated with carbogen), with measurement of the left intraventricular pressure (PVE) by inserting the balloon. Data were expressed as mean ± standard error of the mean and the results were evaluated by one-way analysis of variance (ANOVA) with Tukey post test or Student t test. P values ≤ 0.05 were considered significant and statistical analyzes were performed with the GraphPad Prism© version 5.0 (GraphPad Software Inc., San Diego CA, USA). Naringin (0.03-2 mM) induced a positive inotropic effect on atrium (147%; EC50 of 0.32 ± 0.01 mM, n=5) dependent on concentration. From 3 mM, naringin reduced the contractile force. At maximum positive inotropic effect of naringin there was a decrease of systole duration of 0.11 ± 0.006 sec to 0.09 ± 0.002 sec (p <0.05) and increase in the diastole duration of 0.84 ± 0.019 sec to 0.88 s ± 0.0024 sec (p <0.05). Observed increase dT/dt (+) of 6.16 ± 1.76 gf/sec to 19.74 ± 2.64 gf/sec (p <0.01) and dT/dt (-) of 6.21 ± 1.14 gf/sec to 13.52 ± 1.78 gf /sec (p <0.01). Naringin also promoted diastolic relaxation (44%). Preincubation of the atria with propranolol (Non-selective β-adrenoceptor antagonist) or nifedipine (L- type calcium channels antagonist) or ryanodine (ryanodine receptors antagonist) abolished the positive inotropic effect induced by naringin, as well as no detectable increase in contractile force in atria of animals with depletion of catecholamines. With regard to electrical parameters, naringin shortened PRi of 48.42 ± 0.81ms to 47.31 ± 0.89ms (n = 5, p <0.05) and reduced QT intervals (QTc) of 66, 75 ms ± 2.35 to 64.36 ± 2.24 ms. Conversely, the QRS complex increased (Control: 18.5 ± 1.0 ms and Naringin: 20.83 ± 1.24 ms). This flavonoid also influenced the activity of the cardiac pacemaker, increasing heart rate of 226.9 ± 1.12 bpm to 240.2 ± 3.94 bpm. No cardiac arrhythmia event was recorded during the perfusion of the heart with naringin. The PVE suffered increase of 6%. Naringin exerts positive inotropic and chronotropic effect on cardiac muscle by indirect activation of β-adrenergic receptors through endogenous catecholamines release and promotes significant electrocardiographic changes, reducing PRi, QTc and RRi, and slow down the speed of the QRS. / A naringina é um glicosídeo flavonoide (C27H32O14) encontrado em uvas e frutas cítricas, reconhecida por exercer atividades antioxidante, antiaterogênica, hipoglicemiante, dentre outras. Os efeitos contráteis e elétricos da naringina foram caracterizados sobre o músculo cardíaco de rato. Os experimentos foram realizados em átrio esquerdo isolado de rato (cuba com 8 mL, Krebs-Hanseilet, 29 ± 0,1 °C; Estimulação: 1 gf, 1 Hz; 100 V; 1,5 ms). Os dados de força foram captados por transdutor isométrico (Grass FT03), amplificados (Grass P11T), digitalizados (DATAQ DI710) e armazenados em computador para análise. A naringina (0,003 - 6 mM) foi adicionada cumulativamente ao banho para determinar sua influência sobre parâmetros contráteis. Curvas concentração-efeito da naringina foram obtidas após a pré-incubação do átrio com 1 μM de propranolol ou 1 μM nifedipina ou 1 μM de rianodina ou ainda, usando átrios de animais com depleção de catecolaminas. Os efeitos da naringina sobre o eletrocardiograma foram obtidos através da técnica de Langendorff (10 mL/min - Bomba peristáltica Milan; Solução de Krebs a 34 ± 0,1 °C aerada com carbogênio), com mensuração da pressão intraventricular esquerda (PVE) por inserção de balonete. Os dados foram expressos pela média ± erro padrão da média e os resultados foram avaliados pela análise de variância de uma via (ANOVA) com pós-teste de Tukey ou pelo teste t de Student. Valores de p ≤ 0,05 foram considerados significativos e as análises estatísticas foram realizadas com o GraphPad Prism© versão 5.0 (GraphPad Software Inc., San Diego CA, USA). A Naringina (0,03 - 2 mM) promoveu efeito inotrópico positivo em átrio (147%; CE50 de 0,32 ± 0,01 mM; n = 5) dependente de concentração. A partir de 3 mM, a naringina reduziu a força contrátil. No efeito inotrópico positivo máximo da naringina, houve redução da duração da sístole de 0,11 ± 0,006 s para 0,09 ± 0,002 s (p < 0,05) e aumento na duração da diástole de 0,84 ± 0,019 s para 0,88 ± 0,0024 s (p < 0,05). Observado aumento da dT/dt(+) de 6,16 ± 1,76 gf/s para 19,74 ± 2,64 gf/s (p < 0,01) e da dT/dt(-) de 6,21 ± 1,14 gf/s para 13,52 ± 1,78 gf/s (p < 0,01). A naringina também promoveu relaxamento diastólico (44 %). A pré-incubação dos átrios com propranolol (antagonista β-adrenérgico não-seletivo) ou nifedipina (antagonista de canais de cálcio tipo-L) ou rianodina (antagonista de receptores de rianodina) aboliu o efeito inotrópico positivo induzido pela naringina, assim como, não se evidenciou aumento de força contrátil em átrios obtidos de animais previamente reserpinizados. Quanto aos parâmetros elétricos, a naringina encurtou o PRi de 48,42 ± 0,81 ms para 47,31 ± 0,89 ms (n = 5, p < 0,05) e reduziu o intervalo QT(QTc) de 66,75 ± 2,35 ms para 64,36 ± 2,24 ms. Por outro lado, a duração do complexo QRS aumentou (Controle: 18,5 ± 1,0 ms e Naringina: 20,83 ± 1,24 ms). Este flavonoide também influenciou a atividade do marcapasso cardíaco, aumentando a frequência cardíaca 226,9 ± 1,12 bpm para 240,2 ± 3,94 bpm. Nenhum evento de arritmia cardíaca foi registrado durante a perfusão do coração com a naringina. A PVE sofreu aumento de 6%. A naringina exerce efeito cronotrópico e inotrópico positivos em coração de rato por ativação indireta de receptores β-adrenérgicos através da liberação catecolaminas endógenas e promove alterações eletrocardiográficas significativas, ao reduzir PRi, QTc e RRi, além de lentificar a velocidade do QRS.

Dados mostram que a estimulação do Sistema Nervoso Simpático (SNS) induz osteopenia via receptor &beta;2-adrenérgico, que é expresso no osso. Porém, um estudo recente demonstrou que camundongos knockouts (KO) para os receptores adrenérgicos &alpha;2A e &alpha;2C (&alpha;2A/&alpha;2C-AR-/-) apresentam alta massa óssea, mesmo com hiperatividade simpática. Além disso, esses camundongos são resistentes à osteopenia induzida pelo excesso de hormônio tireoidiano. Esses achados sugerem que o &alpha;2A e/ou &alpha;2C-AR também possam mediar as ações do SNS no esqueleto e que esses receptores estão envolvidos na interação do HT com o SNS para regular o metabolismo ósseo. Neste estudo, tivemos como objetivo: avaliar se o &alpha;2CAR interfere na fisiologia óssea e se a ação do HT no tecido ósseo depende do &alpha;2CAR, avaliando o efeito do HT na fisiologia óssea dos camundongos &alpha;2CAR-/- tratados com dose suprafisiológica de T3. A microtomografia computadorizada mostrou que o volume de osso trabecular foi menor e maior nos animais &alpha;2CAR-/-, no fêmur e na vértebra respectivamente. Os animais KO também apresentaram diminuição da resistência óssea quando comparados com os selvagens. Além disso, vimos que os animais KOs foram resistentes aos efeitos deletérios da tirotoxicose no osso, tanto no fêmur quanto na vértebra. Esses achados sugerem que o &alpha;2C-AR apresenta um papel na mediação dos efeitos da ativação do SNS no osso e que o HT interage via &alpha;2C-AR, para regular massa e resistência óssea. / Data demonstrates that sympathetic nervous system (SNS) activation causes osteopenia via &beta;2-adrenoceptor signaling. A recent study showed that mice with gene inactivation of the adrenergic receptor &alpha;2A and &alpha;2C (&alpha;2A/&alpha;2C-AR-/-) have a high bone mass phenotype, even presenting SNS hyperactivity. Also, these knockout (KO) mice are resistant to the thyroid hormone-induced osteopenia. These findings suggest that SNS interacts with thyroid hormone (TH) to regulate bone mass and &alpha;2A and/or &alpha;2C adrenoceptors may have an important role in mediating the actions of the SNS in the skeleton. In this study, we had the following objectives: (i) to evaluate whether the isolated inactivation of &alpha;2CAR interferes with the bone metabolism and to evaluate whether the action of HT on bone tissue depends on &alpha;2CAR, treated with 20 times the physiological dose of T3. The microtomography analysis showed that the trabecular bone volume of the femur and of the sixth lumbar vertebra (L6) were, respectively, lower and higher in &alpha;2C-AR-/- mice, when compared with WT animals. Furthermore, we showed a resistance of KO animals on the deleterious effects of TH on bone. These findings suggest: (i) &alpha;2C-AR is involved with bone longitudinal growth; (ii) &alpha;2C-AR may mediates the effects of the SNS in the bone in a skeletal site specific manner, (iii) the actions of thyroid hormone on bone metabolism involves interactions with the SNS via &alpha;2C adrenergic receptors.

Müller cells are major glial cells in the retina and have a broad range of functions that are vital for the retinal neurons. During retinal injury gliotic response either leads to Müller cell dedifferentiation and formation of a retinal progenitor or to maintenance of mature Müller cell functions. The overall aim of this thesis was to investigate the intra- and extracellular signaling of Müller cells, to understand how Müller cells communicate during an injury and how their properties can be regulated after injury. Focus has been on the α2-adrenergic receptor (α2-ADR) and endothelin receptor (EDNR)-induced modulation of Müller cell-properties after injury. The results show that α2-ADR stimulation by brimonidine (BMD) triggers Src-kinase mediated ligand-dependent and ligand-independent transactivation of epidermal growth factor receptor (EGFR) in both chicken and human Müller cells. The effects of this transactivation in injured retina attenuate injury-induced activation and dedifferentiation of Müller cells by attenuating injury-induced ERK signaling. The attenuation was concomitant with a synergistic up-regulation of negative ERK- and RTK-feedback regulators during injury. The data suggest that adrenergic stress-signals modulate glial responses during retinal injury and that α2-ADR pharmacology can be used to modulate glial injury-response. We studied the effects of this attenuation of Müller cell dedifferentiation on injured retina from the perspective of neuroprotection. We analyzed retinal ganglion cell (RGC) survival after α2-ADR stimulation of excitotoxically injured chicken retina and our results show that α2-ADR stimulation protects RGCs against the excitotoxic injury. We propose that α2-ADR-induced protection of RGCs in injured retina is due to enhancing the attenuation of the glial injury response and to sustaining mature glial functions. Moreover, we studied endothelin-induced intracellular signaling in Müller cells and our results show that stimulation of EDNRB transactivates EGFR in Müller cells in a similar way as seen after α2-ADR stimulation. These results outline a mechanism of how injury-induced endothelins may modulate the gliotic responses of Müller cells. The results obtained in this thesis are pivotal and provide new insights into glial functions, thereby uncovering possibilities to target Müller cells by designing neuroprotective treatments of retinal degenerative diseases or acute retinal injury.

A síndrome isquêmica miocárdica instável (SIMI) leva a importantes alterações neurovasculares, tais como à hiperativação simpática e a diminuição do fluxo sanguíneo muscular (FSM) tanto em repouso como durante manobras fisiológicas como o exercício. A presença de alguns polimorfismos na genética humana, como o dos receptores \'beta\'2-adrenérgicos Gln27Glu, apresenta importante associação com a funcionalidade cardiovascular em indivíduos saudáveis. Contudo, não é conhecido se em pacientes com SIMI, a presença dos polimorfismos do receptor \'beta\'2-adrenérgico leva a respostas neurovasculares distintas durante o exercício, e, ainda, se o treinamento físico poderá modificar essa resposta. OBJETIVOS: Estudar a influência do polimorfismo do receptor \'beta\'2-adrenérgico Gln27Glu no controle neurovascular da atividade nervosa simpática muscular (ANSM) e do FSM em repouso e durante o exercício físico de preensão de mão, em pacientes com SIMI e, num segundo momento, avaliar o efeito do treinamento físico nas respostas neurovasculares durante o exercício nestes pacientes. MÉTODOS: Inicialmente, foram selecionados para o estudo, 78 pacientes com SIMI com fração de ejeção >= 45%, no momento da hospitalização. Um mês após o evento isquêmico, 61 pacientes retornaram para as avaliações iniciais. Os pacientes foram genotipados e posteriormente divididos em dois grupos de acordo com o polimorfismo Gln27Glu do receptor \'beta\'2-adrenérgico: 1- Gln27Gln (CC, n=35) e 2- Gln27Glu+Glu27Glu (CG+GG, n=26). Destes, 29 pacientes concordaram em participar de um protocolo de treinamento físico por um período de 8 semanas, sendo que, 25 finalizaram o protocolo (CC, n=17; CG+GG, n=8). A avaliação do controle neurovascular foi realizada em repouso e durante o exercício físico de preensão de mãos em 30% da contração voluntária máxima. Foram avaliados a ANSM, medida pela técnica direta de microneurografia, o FSM, medido pelo método de pletismografia de oclusão venosa, a pressão arterial, medida pelo método oscilométrico indireto e a frequência cardíaca, pelo eletrocardiograma. Todas as avaliações foram realizadas nos pacientes um mês após o evento isquêmico e, para aqueles que participaram do protocolo de treinamento físico, foram repetidas após 8 semanas de intervenção. RESULTADOS: Um mês após o evento isquêmico, a ANSM (P=0,26) e a pressão arterial média (PAM, P=0,14) de repouso foram semelhantes entre os grupos com genótipo CC e CG+GG. Entretanto, durante o exercício, a resposta da ANSM foi maior no grupo CC quando comparado com o grupo com CG+GG (\'delta\'=11±2 vs. 4±2 disparos/100batimentos, P=0,02). Adicionalmente, a resposta de PAM foi maior no grupo CC quando comparado ao grupo CG+GG (\'delta\'=24±2 vs. 18±2 mmHg, P=0,04). A resposta de condutância vascular no antebraço (CVA) durante o exercício foi semelhante entre ambos os grupos. Após treinamento físico a ANSM basal diminuiu no grupo com genótipo CC (63±3 vs. 48±5 disparos/100batimentos, P <0,001), mas não no grupo com genótipo CG+GG (70±4 vs. 55±5 disparos/100batimentos, P =0,06). De forma semelhante, durante o exercício, o treinamento físico diminuiu o nível (P= 0,007) e a resposta da ANSM (\'delta\'=12±2 vs. 5±2 disparos/100batimentos, P=0,02) no grupo com genótipo CC, mas não no grupo com genótipo CG+GG (P=0,10) e (\'delta\'=7±3 vs. 7±3 disparos/100batimentos, P=0,96), respectivamente. O treinamento físico não modificou os níveis de PAM e CVA, durante o exercício, em ambos os grupos. Contudo, analisando-se o período pós-treinamento físico, o grupo CG+GG apresentou resposta de PAM menor (P=0,01) e CVA maior (P=0,03) durante o exercício quando comparado ao grupo CC. CONCLUSÃO: Pacientes com SIMI, portadores do genótipo CC do receptor \'beta\'-adrenérgico apresentam resposta aumentada da ANSM durante a manobra fisiológica de exercício, quando comparados aos pacientes com genótipo CG+GG. O treinamento físico reverte esta resposta exacerbada de ANSM nos pacientes com genótipo CC. Esses resultados sugerem um risco cardiovascular aumentado nos pacientes com SIMI com genótipo CC do receptor \'beta\'-adrenérgico. Por outro lado, o treinamento físico deve ser fortemente recomendado para esses pacientes, sobretudo naqueles com o genótipo CC. / Acute coronary syndrome (ACS) leads to important neurovascular abnormalities such as sympathetic hyperactivity and decreased forearm blood flow (FBF) at rest and during physiological maneuvers as exercise. The presence of some polymorphisms in human genetics, as the &#946;2-adrenoceptor Gln27Glu, presents a significant association with cardiovascular functionality in healthy subjects. However, it is not known whether in patients with ACS, the presence of polymorphisms of the &#946;2-adrenoceptor leads to distinct neurovascular responses during exercise, and if the exercise training can modify this response. OBJECTIVES: To study the influence of Gln27Glu \'beta\'2-adrenoceptor polymorphisms on neurovascular control of muscle sympathetic nerve activity (MSNA) and FBF at rest and during handgrip exercise, in patients with ACS; and to evaluate the effect of exercise training on neurovascular responses during exercise in these patients. METHODS: Initially, were selected 78 patients with ACS with ejection fraction >= 45% at the time of hospitalization. One month after the ischemic event, 61 patients returned for the initial assessments. Patients were genotyped and then divided into two groups according to the Gln27Glu \'beta\'2-adrenoceptor polymorphisms: 1-Gln27Gln (CC, n=35) and 2-Gln27Glu + Glu27Glu (CG +GG, n=26). Of these, 29 patients agreed to participate in an exercise training protocol for a period of 8 weeks, but only 25 patients completed the protocol (CC, n=17; CG+GG, n=8). The evaluation of neurovascular control was performed at rest and during a handgrip exercise at 30% of the maximum voluntary contraction. We evaluated the MSNA, by the direct technique of microneurography, the FBF, by venous occlusion plethysmography technique, blood pressure (BP), by indirect oscillometric device and heart rate, by electrocardiogram. All evaluations were performed one month after the ischemic event and, for those patients subjected to the exercise training protocol the same evaluations were repeated after 8 weeks of intervention. RESULTS: One month after the ischemic event, the MSNA (P=0.26) and mean arterial pressure (MAP, P=0.14) at rest were similar between groups with genotype CC and CG+GG. However, during exercise, the response of MSNA was higher in the CC group compared with the CG+GG group (\'delta\'=11±2 vs. 4±2 bursts/100HB, P=0.02). In addition, BP response during exercise was higher in the CC group compared to the CG + GG group (\'delta\' =24 ±2 vs. 18±2 mmHg, P=0.04). The forearm vascular conductance (FVC) response during exercise was similar in both groups. After exercise training, baseline MSNA decreased in the group with CC genotype (63± 3vs. 48±5 bursts/100HB, P <0.001) but not in the group with CG+GG genotypes (70±4 vs. 55±5 bursts/100HB, P =0.06). Similarly, exercise training decreases the level (P= 0.007) and the response of MSNA (\'delta\'= 12±2 vs. 5±2 bursts/100HB, P= 0.02) during exercise in the group with CC genotype but not in the CG+GG group (P= 0.10) and (\'delta\'=7±3 vs 7±3 bursts/100HB, P= 0.96), respectively. Exercise training did not change the levels of MAP and FVC, during exercise in both groups. However, in the post exercise training period, the CG+GG group had lower MAP response (P =0.01) and higher FVC (P =0.03) during exercise compared to the CC group. CONCLUSION: Patients with ACS, carrying the CC genotype of the \'beta\'2-adrenoceptor have increased MSNA response during physiological maneuver as exercise when compared with patients carrying the CG+GG genotype. Exercise training reverses this exacerbated response of MSNA in patients with CC genotype. These results suggest an increased cardiovascular risk in patients with ACS with CC genotype of the \'beta\'2-adrenoceptor. Furthermore, exercise training should be strongly recommended for patients with ACS, especially for those with the CC genotype

Η στεφανιαία νόσος αποτελεί ένα από τα πιο συχνά αίτια νοσηρότητας και θνητότητας στο γενικό πληθυσμό. Ένα μεγάλο ποσοστό από τους ασθενείς με στεφανιαία νόσο οδηγείται σε αγγειοπλαστική στεφανιαίων αρτηριών (PTCA) με ή χωρίς εμφύτευση stent, όταν η στένωση στο αγγείο είναι ≥ 70-75%. Παρά την πρόοδο στον τομέα της αγγειοπλαστικής, με τη χρήση των drug-eluting stents και την ελάττωση της επαναστένωσης σε ποσοστό <5-10%, το υψηλό ποσοστό (20-25%) επαναστένωσης παραμένει η Αχίλλειος πτέρνα στα συμβατικά, μεταλλικά(bare)stents. Η χρήση των drug-eluting stents περιορίζεται σε περιπτώσεις με επαναστένωση, σε σακχαροδιαβητικούς και σε υψηλού κινδύνου βλάβες για επαναστένωση. Τα μεγάλα ποσοστά όψιμης επαναστένωσης(≥ 9-10%) και το υψηλό κόστος τους, κάνουν ακόμα πιο επιτακτική την ανάγκη εντατικοποίησης της έρευνας προς την κατεύθυνση εντοπισμού παραγόντων σχετιζόμενων με την επαναστένωση. Σκοπός της εργασίας μας ήταν να διερευνήσει τον πιθανό ρόλο πολυμορφισμών γονιδίων στην υποτροπή ισχαιμίας μετά από αγγειοπλαστική στεφανιαίων αρτηριών και εμφύτευση μεταλλικών stents. Συγκεκριμένα, εξετάσθηκε αναλυτικά ο γενετικός πολυμορφισμός των γονιδίων α2Β-αδρενεργικού υποδοχέα και της CETP(πρωτεΐνη μεταφοράς εστέρων χοληστερόλης). Η υπόθεση στηρίχτηκε στο γεγονός ότι σε μια σημαντική μελέτη 912 αρρένων Φινλανδών μέσης ηλικίας, αποδείχτηκε ότι ο D/D γονότυπος σε σχέση με τον I/D γονότυπο και τον I/I γονότυπο, εμφανίζει 2,5 φορές μεγαλύτερο κίνδυνο για οξέα στεφανιαία επεισόδια, συμπεριλαμβανομένου του εμφράγματος μυοκαρδίου. Η εκσεσημασμένη αγγειοσύσπαση, τόσο στην περιφέρεια, όσο και στις στεφανιαίες αρτηρίες μέσω του πολυμορφισμού του γονιδίου α2Β που θεωρείται πιθανό αίτιο για τα οξέα στεφανιαία επεισόδια, μαζί με το σημαντικό ρόλο του α2Β αδρενεργικού υποδοχέα στην υπερπλασία και μετανάστευση των λείων μυϊκών κυττάρων, πιθανόν να έχει μεγάλη συμβολή στη διεργασία της υποτροπής ισχαιμίας. Μελετήσαμε προοπτικά 96 Έλληνες που υπέστησαν επιτυχή PTCA και εμφύτευση stents, εκ των οποίων 81 ήταν άνδρες και 15 γυναίκες(μέση ηλικία ± σταθερά απόκλιση=57,7± 10,1 ετών, με όρια 37-76 ετών) που προσήλθαν με συμπτωματική στεφανιαία νόσο. Όλοι οι παραπάνω ασθενείς συμμετείχαν στη μελέτη μεταξύ των ετών 2001 και 2003 και παρακολουθήθηκαν κλινικά για 6-8 μήνες μετά από μια επιτυχή τεχνική διάνοιξης του αποφραγμένου αγγείου. Αμέσως μετά την PTCA και για ένα(1) μήνα οι ασθενείς έλαβαν ασπιρίνη(100-325mg/day) και κλοπιδογρέλη 75mg/day. Η εκτίμηση της υποτροπής ισχαιμίας βασίστηκε σε στατικό και δυναμικό σπινθηρoγράφημα θαλλίου στους 3 και 6-8 μήνες μετά την PTCA. Αιμοδυναμικά, σε όσους υπέστησαν νέα στεφανιογραφία μέχρι και τους 6-8 μήνες, η επαναστένωση ορίσθηκε ως ≥ 50% στένωση του αυλού του αγγείου στο σημείο όπου έγινε η αγγειοπλαστική. Εκτός από την ομάδα των ασθενών και μια ομάδα υγιών μαρτύρων 83 ατόμων, συμμετείχε στη μελέτη για σύγκριση της συχνότητας του γονότυπου. Το τελικό καταληκτικό σημείο για την παραπάνω μελέτη, ήταν η συχνότητα της υποτροπής ισχαιμίας στους 8 μήνες κλινικής παρακολούθησης. Υποτροπή ισχαιμίας και της επαναστένωσης ≥ 50% σε όσους υπεβλήθησαν σε νέα στεφανιογραφία, συνέβη σε 15 από τους 96 ασθενείς. Ας σημειωθεί ότι οι περισσότεροι ασθενείς (70/96) είχαν το φυσιολογικό γονότυπο με το αλληλόμορφο I, λιγότεροι ασθενείς (23/96) είχαν το Insertion/Deletion και μόλις 3/96 είχαν το Deletion/ Deletion γονότυπο. Από το γονοτυπικό group, υποτροπή ισχαιμίας παρουσιάσθηκε σε 11/70 για τον I/I, 3/23 για τον I/D γονότυπο και 1/3 για τον D/D γονότυπο. Δε βρέθηκε συσχέτιση μεταξύ πολυμορφισμού γονιδίου και υποτροπής ισχαιμίας στους ασθενείς μετά από αγγειοπλαστική στεφανιαίων αρτηριών. Προηγούμενες μελέτες έχουν ερευνήσει τη συσχέτιση των πολυμορφισμών των γονιδίων της ACE, του AT1 υποδοχέα της αγγειοτενσίνης II και της CETP με την επαναστένωση μετά από αγγειοπλαστική. Εντούτοις, καμιά μελέτη δεν πραγματοποιήθηκε που να συγκρίνει τον α2Β-AR πολυμορφισμό και την υποτροπή ισχαιμίας μετά από αγγειοπλαστική στεφανιαίων αρτηριών. Όπως αρχικά αναφέρθηκε, ο γονότυπος α2Β ευνοεί τη μετανάστευση των αγγειακών SMCs, επηρεάζει τη λειτουργία του Α.Ν.Σ. και συσχετίζει το α2Β-AR αλληλόμορφο D deletion με οξέα στεφανιαία επεισόδια. Όλα τα παραπάνω στοιχεία μπορεί να δικαιολογούν το ρόλο του α2Β-AR πολυμορφισμού στην υποτροπή ισχαιμίας και πιθανόν την επαναστένωση μετά από αγγειοπλαστική στεφανιαίων αρτηριών. Βέβαια, η αρνητική συσχέτιση των πολυμορφισμών του α2Β-AR και της CETP ΤaqIB με την υποτροπή ισχαιμίας μετά από αγγειοπλαστική, μπορεί να θεωρηθεί προκαταρκτική, δεδομένου ότι συμμετείχε σχετικά μικρός αριθμός ασθενών συγκριτικά με μεγάλες πληθυσμιακές μελέτες και επειδή ο D/D γονότυπος δεν είναι ιδιαίτερα συχνός(για τη μελέτη των α2Β-AR). Όσον αφορά τη μελέτη με τη CETP, διερευνήσαμε τον πολυμορφισμό ΤaqIB που είναι μια σιωπηρή μετάλλαξη βάσης στο 277 νουκλεοτίδιο της CETP(η οποία μπορεί να αναγνωρισθεί με την περιοριστική ενδονουκλεάση ΤaqI), με την πιθανότητα υποτροπής ισχαιμίας μετά από αγγειοπλαστική στεφανιαίων αρτηριών. Οι όροι Β1 και Β2 αντίστοιχα χρησιμοποιήθηκαν για να δηλώσουν την ύπαρξη ή μη της περιοριστικής περιοχής (site) της ΤaqIB. Το Β2 αλληλόμορφο σχετίζεται με αυξημένα επίπεδα HDL και ελαττωμένα επίπεδα CETP, τόσο σε υγιείς όσο και σε άτομα με στεφανιαία νόσο(μοιάζει με ήπιας μορφής ανεπάρκεια CETP). Αντίθετα το Β1 αλληλόμορφο σχετίζεται με ελαττωμένα επίπεδα HDL και με αυξημένα επίπεδα και δραστηριότητα CETP. Επειδή η ΤaqIB σχετίζεται με χαμηλά επίπεδα HDL και αυξημένο κίνδυνο για CHD(επηρεάζοντας το μεταβολισμό των λιποπρωτεϊνών), μπορεί να συμμετέχει στην παθοφυσιολογία της υποτροπής ισχαιμίας μετά από αγγειοπλαστική. Μελετήσαμε 204 ασθενείς από το έτος 2001 έως και το 2003 με την προοπτική να διερευνηθεί η συσχέτιση ΤaqIB στον Ελλαδικό πληθυσμό με την πιθανότητα υποτροπής ισχαιμίας μετά από αγγειοπλαστική σε άτομα που φέρουν τον παραπάνω γονότυπο. Η συχνότητα της ΤaqIB(54%) ήταν παρόμοια με τη συχνότητα του πολυμορφισμού σε μια ομάδα 35 υγιών μαρτύρων. Το αποτέλεσμα από αυτή τη μελέτη δεν αποδεικνύει ότι ο ΤaqIB πολυμορφισμός στο γονίδιο της CETP είναι σημαντικός προγνωστικός παράγων για εκτίμηση του κινδύνου υποτροπής ισχαιμίας μετά από αγγειοπλαστική στεφανιαίων αρτηριών. Συμπερασματικά, η υποτροπή ισχαιμίας μετά από αγγειοπλαστική στεφανιαίων αρτηριών οφείλεται σε έναν πολύπλοκο μηχανισμό και σε ένα πολυπαραγοντικό φαινόμενο. Μπορεί οι πολυμορφισμοί του α2Β και της CETP, να μην αναδείχθηκαν ως ανεξάρτητοι παράγοντες υποτροπής ισχαιμίας μετά από αγγειοπλαστική στεφανιαίων αρτηριών, αλλά σε συνδυασμό με άλλους πολυμορφισμούς γονιδίων και υπό την επίδραση συγκεκριμένων περιβαλλοντικών παραγόντων, είναι πολύ πιθανόν να συμμετέχουν στην παραπάνω διεργασία της υποτροπής ισχαιμίας και κατ’επέκταση της επαναστένωσης μετά από PTCA. / Coronary heart disease is one of the most common causes of morbidity and mortality in the population. A great percent of the patients with coronary heart disease may undergo percutaneous coronary angioplasty (PTCA) with or without the implantation of stents, mainly when the stenosis of the vessel is ≥ 70-75%. Despite the progress made with the introduction of drug-eluting stents and the reduction of the restenosis rate up to 5%-10%, the Achillean heel of the angioplasty using bare metal stents, is the restenosis rate which is 20%-25% of all cases. The use of drug-eluting stents is limited in cases with restenosis, in patients with diabetes mellitus and in high-risk for restenosis lesions. The great percent of late restenosis(≥ 9-10%) and the high price of drug-eluting stents, make more urgent the necessity for more intense research on the identification of the exact factors involved in the pathophysiology of restenosis. The primary objective of our study is to define the role of gene polymorphisms in the recurrence of ischaemia after PTCA and the implantation of the stents. Virtually, we scrutinely examined the role of the genetic polymorhisms of α2Badrenergic receptor and the CETP(Cholesteryl Ester Transfer Protein)-TaqIB polymorphism. Our assumption was based on the conclusion drawn by a study conducted in Finnish patients, which showed that D/D genotype confers 2,5 increase in the risk for acute coronary events(including acute myocardial infarction). The intense vasoconstriction properties of the α2Badrenergic polymorphism both on the coronary arteries and the periphery is considered to be the primary cause of the acute coronary events. The aforementioned statement with the significant role of α2B adrenergic receptor α2B-AR on the hyperplasia and mainly the migration of smooth muscle cells, probably correlates well with the pathophysiology of the recurrence of ischaemia after PTCA. We conducted a genetic association/prospective follow-up study in 96 Greek coronary artery disease patients undergoing coronary angioplasty and stent implantation. 81 patients were men and 15 women(mean age ± standard deviation=57,7± 10,1 years, ranges 37-76 years old) who presented with symptomatic CAD. All patients were enrolled in the study between 2001 and 2003 and were followed-up clinically for 6-8 months after an initially successful procedure. Post-angioplasty and for one (1) month following the procedure, all the patients received aspirin(100-325mg/day) and clopidogrel(75mg/day). Assessment of recurrence of ischaemia was based on positive thallium stress testing(at least moderate defect to the distribution of the culprit lesion of the vessel which was revascularised). Hemodynamically, restenosis was defined as ≥50% narrowing of the vessel at the point where angioplasty was performed. In addition to the patient group, a control group of totally 83 asymptomatic individuals were included in the study for comparison of the frequency of the genotype. The final end-point of the current study was the incidence of restenosis at 8 months of clinical follow-up. Recurrence of ischaemia (including restenosis rate ≥50% to the patients who underwent coronary angiography) occurred in 15 of the 96 patients. In note, the majotiy of patients (70/96) had the Insertion/Insertion genotype, fewer patients (23/96) had the Insertion/Deletion genotype and only 3/96 had the Deletion /Deletion genotype. With regard to to the genotype groups , restenosis was found in 11/70 for I/I, 3/23 for I/D and 1/3 for the D/D genotype. No association between gene polymorphisms and recurrence of ischaemia was detected to the patients who underwent coronary angioplasty. Previous studies have investigated the association between gene polymorhisms of angiotensin-converting enzyme(ACE), the AT1 receptor for angiotensin II and cholesteryl ester transfer protein (CETP) with restenosis in patients after coronary angioplasty. However, no study has been performed to involve the α2B-AR polymorphism with recurrence of ischaemia after percutaneous angioplasty of coronary vessels. As it was initially mentioned, α2B genotype promotes the migration of vascular SMCs, influences the function of autonomic nervous system and the α2B-AR deletion variant is associated with acute coronary events. All these data might correlate the role of α2B-AR polymorphism with the recurrence of ischaemia and probably with the restenosis after an angioplasty of coronary vessels. Nevertheless, the negative findings of our study might be considered preliminary, taking into account the small number of patients that were studied and the rarity of the Deletion/Deletion(D/D) genotype. As far as the CETP study, we investigated the TaqIB polymorphism, which is a silent base change affecting the 277th nucleotide and can be identified by the restriction endonuclease TaqI, with the chance of recurrence of ischaemia after PTCA. The terms B1 and B2 are used to denote the presence and absence, respectively, of the TaqI restriction site. The B2 allele has been associated with increased HDL levels and decreased CETP levels and activity in both patients with CHD and healthy subjects(resembling a mild form of CETP deficiency). On the other hand, the B1 allele has been associated with decreased HDL levels and increased CETP levels and activity. Due to the fact that TaqIB is associated with decreased HDL levels and increased risk for CHD, affecting the lipoprotein metabolism might be involved in the pathophysiology of reccurence of ischaemia after PTCA. We studied 204 patients between 2001 and 2003 with the primary objective to investigate the frequency of TaqIB and possible association with reccurence of ischaemia after PTCA in the patients who have this genotype. The frequency of TaqIB was 54% similar to the frequency of the polymorphism in a group of 35 healthy controls. The results from this study does not indicate that the TaqIB polymorphism at the CETP gene locus is a significant predictor for assessing the risk of reccurence of ischaemia after PTCA. As a conclusion, reccurence of ischaemia after PTCA is due to a complicated mechanism and to a multifactoral phenomenon. Virtually, we didn’t find any correlation of α2ΒAR polymorphism and CETP TaqIB with reccurence of ischaemia, especially as causitive factors, but based on their role in the pathophysiology, under certain circumstances, especially with the cooperation of other genes, these polymorphisms can not be definitely excluded in the reccurence of ischaemia and the restenosis after PTCA.

Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro / O estado nutricional e hormonal em fases iniciais de desenvolvimento (gestação e lactação) está relacionado a alterações epigenéticas, que podem levar ao desenvolvimento de doenças. A obesidade infantil está relacionada com a ocorrência da obesidade na idade adulta, resistência à insulina e maior risco cardiometabólico. Em estudos experimentais, a superalimentação neonatal causa obesidade e aumenta o risco de doenças cardiovasculares. Estes animais apresentam obesidade visceral, hiperfagia, hiperleptinemia e hipertensão na idade adulta. Previamente, demonstramos que a hiperleptinemia neonatal causa hiperfunção da medula adrenal e microesteatose na idade adulta. No presente estudo avaliamos a função adrenal de ratos adultos obesos no modelo de superalimentação neonatal por redução do tamanho da ninhada e a sensibilidade as catecolaminas no tecido adiposo visceral (TAV) e no fígado. Ao nascimento todas as ninhadas tiveram seu número de filhotes ajustados para 10. Para induzir a superalimentação neonatal, o tamanho da ninhada foi reduzido de dez para três filhotes machos no terceiro dia de lactação até o desmame (SA), enquanto que o grupo controle permaneceu com 10 filhotes durante toda a lactação. Após o desmame, os ratos tiveram livre acesso à dieta padrão e água até 180 dias (1 animal de cada ninhada, n = 7). O TAV e as glândulas adrenais foram pesadas. As contrações hormonais séricas, o conteúdo hepático de glicogênio e triglicerídeos foram avaliados por kits comerciais. O conteúdo e a secreção de catecolaminas adrenais foram avaliados utilizando o método do trihidroxindol. O conteúdo dos hormônios eixo hipotálamo-hipófise-córtex adrenal, das enzimas da via de síntese das catecolaminas na glândula adrenal, ADRB2 no fígado e ADRB3 no TAV foram determinados por Western blotting ou imunohistoquímica. As diferenças foram consideradas significativas quando p <0,05. Aos 180 dias de vida, o grupo SA apresentou maior massa corporal (+15%), maior consumo alimentar (+15%) e maior adiposidade visceral (+79%). Os hormônios do eixo hipotálamo-hipófise-córtex-adrenal não foram alterados. O grupo SA apresentou maior expressão de tirosina hidroxilase e de DOPA descarboxilase (+31% e 90%, respectivamente); conteúdo de catecolaminas adrenais (absoluta: 35% e relativa: 40%), e secreção de catecolaminas, tanto basal quanto estimulada por cafeína (+35% e 43%, respectivamente). O conteúdo ADRB3 no TAV não foi alterado nos grupo SA, entretanto o ADRB2 no fígado apresentou-se menor (-45%). O grupo SA apresentou maior conteúdo de glicogênio e triglicerídeos no fígado (+79% e +49%, respectivamente), além de microesteatose. A superalimentação neonatal resulta em hiperativação adrenomedular e aparentemente está associada a preservação da sensibilidade às catecolaminas no VAT. Adicionalmente sugerimos que o maior conteúdo de glicogênio e triglicerídeos hepático seja devido a menor sensibilidade as catecolaminas. Tal perfil pode contribuir para a disfunção metabólica hepática e hipertensão arterial que são características deste modelo de obesidade programada. / Nutritional and hormonal status at early phases of development are related to epigenetic changes, promoting disease development. Childhood overweight is related with late obesity, insulin resistance and higher cardiometabolic risk. Rats overfed during lactation show higher visceral adiposity, hyperphagia, leptin resistance and hypertension in adulthood. Previously, we demonstrated that neonatal hyperleptinemia is associated with adrenal medullary hyperfunction and liver steatosis at adulthood. Here, we evaluated the adrenal function and liver tissue of adult obese rats that were overfed during lactation. To induce early overfeeding, the litter size was reduced from ten to three male pups at the third day of lactation until weaning (SL). Control group had ten rats per litter (NL). After weaning, rats had free access to standard diet and water until 180 days old (1 animal from each litter, n=7). Significant differences had p<0.05. The SL group presented higher adrenal catecholamine content (absolute: +35% and relative: +40%), tyrosine hydroxylase (+31%), DOPA decarboxylase (+90%) protein contents, basal and caffeine-induced catecholamine in vitro secretion (+35% and +43%, respectively). However, hormones of the hypothalamic-pituitary-adrenal cortex axis were unchanged. The &#946;3-adrenergic receptor content in visceral adipose tissue was unchanged in SL rats, but the &#946;2-adrenergic receptor in the liver was lower (-45%). SL group showed higher glycogen and triglycerides contents in liver (+79% and +49%, respectively), which showed microesteatosis. Although the neonatal overfeeding leads to higher adrenomedullary function, adult obese SL rats have a dysfunction in hepatic &#946;2-adrenergic receptor, which can contribute for the hepatic dysfunction characteristic of liver obesity complications.

The purpose of this investigation was to examine selected biochemical mechanisms known to influence contractility and energy metabolism in the myocardium, with particular emphasis placed on the regulatory role of protein phosphorylation in the ventricular myocardium. The investigation was conducted in three phases; initially the cardiac contraction cycle was examined to determine whether reported fluctuations in myocardial cAMP levels were associated with other biochemical events known to be cAMP-dependent. The second phase involved the determination of specific kinase activities and endogenous substrates in a highly purified cardiac sarcolemmal preparation. In the final phase, ventricular myocytes were utilized to examine the ability of adenosinergic and muscarinic agonists to influence the isoproterenol-induced increases in protein phosphorylation. Studies in the first phase examined cyclic AMP levels and selected kinase activities in hearts frozen at various stages of the cardiac cycle. An automated clamping device, capable of freezing a perfused rat heart in less than 50 msec, was utilized to separate the cardiac cycle into various phases. Three different timing schemes were employed to divide the cycle into 2 to 4 segments. These different timing schemes revealed no significant differences in cAMP during the cardiac cycle. Myocardial cAMP values ranged from 2.5 to 4.1 pmol/min/mg protein in all phases. However, in one scheme there was a tendency for cAMP to be elevated in early systole, with minimal values occurring diastole. There were also no significant differences seen for either glycogen phosphorylase or cAMP-dependent protein kinase (PKA) activity between various phases of the cardiac cycle. Since no significant fluctuations were observed in the levels of cAMP or the activities of PKA or glycogen phosphorylase during a single cardiac contraction cycle, it would appear that these agents do not exert their effects on cardiac function on a beat to beat basis. The second phase of study examined the nature and function of individual protein kinases in the myocardium. Using a highly purified cardiac sarcolemmal preparation, kinase specific, synthetic substrates were employed to quantify the activities of cAMP-dependent (PKA), calcium/calmodulin-dependent (PKCM), calcium/phospholipid-dependent (PKC) and cGMP-dependent (PKG) protein kinases. Additionally, endogenous protein substrates were examined in this preparation to provide possible insight as to the function of these kinases in the heart. The activities of PKA, PKG, PKCM, and PKC in nmol 32P/min/μg protein were as follows: PKA, 1606; PKG, 35.7; PKCM, 353; and PKC, 13.2. Three endogenous protein substrates of apparent molecular weights of 15kD, 28kD and 92kD were phosphorylated. While no endogenous protein phosphorylation was detectable as a result of cG-PK activity, all of the substrates were phosphorylated, to varying degrees, by both PKA and CACM-PK. PKC phosphorylated only the 15kD substrate. Even though several endogenous kinases are evident in the sarcolemmal preparation, cAMP-dependent protein kinase demonstrates the greatest degree of activity. This kinase also appeared to be the most abundant; however, there is some concern as to the source of these kinases in the membrane preparation since endothelial membranes as well as cardiac membranes appeared to be present. Evidence for endothelial contamination was provided by the finding that the membrane preparation contained appreciable amounts of angiotensin converting enzyme (ACE) activity, an enzyme felt to reside in the vascular endothelium. Since studies with this preparation could not exclude contribution of nonmuscle cell membranes a model consisting solely of dispersed ventricular myocytes was developed. The third phase of these studies examined protein phosphorylation in primary cultures of ventricular myocytes. Specifically, these studies examined protein phosphorylation induced by exposure to isoproterenol (ISO), a catecholamine known to effect changes in the phosphorylation state of proteins in the heart by means of a β-adrenergic-mediated/cAMP-dependent mechanism was examined. Additionally, the effects of phenylisopropy-ladenosine (PIA) and carbamyl choline chloride (CARB) were examined with regard to their anti-adrenergic role(s) in this process. Adherent, collagenase-dispersed, radiolabelled (32p) ventricular myocytes exposed to ISO demonstrated a dose and time dependent increase in 32p incorporation into several endogenous protein substrates. When the myocytes were exposed (60 sec) to either PIA or CARB prior to the exposure to ISO, ISO-induced 32p incorporation into protein substrates of apparent molecular weight of 6kD, 31kD and 155kD was reduced up to 67% when compared to the effects of ISO alone. Additionally, both PIA and CARB attenuated the ISO-induced increase in PKA activity in the myocyte, yet only CARB was seen to produce an inhibitory effect on the ISO-induced increase in cAMP levels in the myocytes. The effects of CARB were dose-dependent and inhibited the effects of ISO on 32p incorporation at all doses tested. PIA elicited biphasic effects: lower PIA concentrations were inhibitory in nature, while higher concentrations of PIA appeared to potentiate the increase in 32p incorporation induced by ISO. Based on electrophoretic mobilities (SDS/PAGE) of the 6kD and the 155kD substrates, these substrates have been tentatively identified as the monomeric form of the sarcoplasmic reticulum-associated protein, phospholamban, and the contractile filament-associated protein, C protein, respectively. The 31kD substrate has been identified, by means of immunoblot, as the contractile filament-associated protein, troponin I. The role of protein phosphorylation in the myocardium involves complex, inter-related mechanisms that encompass extracellular, transmembranal and cytoplasmic elements in the heart. It is well understood that certain mechanisms of the contraction cycle known to vary on a beat to beat basis, such as myosin ATPase, involve changes in protein phosphorylation. However, the nature of the various kinases and substrates examined in this study appear to influence longer-term events of myocardial contractility. Mechanisms coupled with hormone action, modulation of second messenger-dependent components, and factors associated with changes in contractility seen with aging and disease are more likely to exhibit changes similar to those described herein. A better understanding of the underlying biochemistry may provide greater insight into the importance of these metabolic changes.

Made available in DSpace on 2015-03-26T13:22:04Z (GMT). No. of bitstreams: 1 text completo.pdf: 757671 bytes, checksum: 435de7db4aaa7b1bdaf1bc5260f57dd9 (MD5) Previous issue date: 2014-03-11 / O objetivo do presente estudo foi investigar os efeitos do treinamento físico sobre as propriedades morfológicas e mecânicas de cardiomiócitos de camundongos knockout (KO) para receptores os β1- e β2 adrenérgicos. Camundongos C57BL/6J, KO para os receptores β1-adrenérgicos, FVB/N e KO para os receptores β2-adrenérgicos com 4 meses de idade, foram inicialmente separados aleatoriamente em 8 grupos: C57c, C57t, KO β1c, KO β1t, FVBc, FVBt, KO β2c e KO β2t. Os animais dos grupos treinados (C57t, KO β1t, FVBt, KO β2t) foram submetidos a um protocolo de treinamento aeróbico de 8 semanas, 5 dias por semana, 1hora por dia, com intensidade de 60% da velocidade máxima de corrida. 3 a 4 animais de cada grupo foram separados para a análise de contração celular com perfusão do agonista β-adrenérgico isoproterenol (ISO) [100nM]. Após a eutanásia, os cardiomiócitos do ventrículo esquerdo foram isolados por dispersão enzimática. O comprimento e a largura dos cardiomiócitos foram medidos utilizando um sistema de captação de imagens e o volume celular foi calculado. As contrações celulares foram medidas através da técnica de alteração do comprimento dos cardiomiócitos, após estimulação elétrica a 1 Hz, em temperatura controlada à 37oC, usando-se um sistema de detecção de bordas. Os resultados mostraram que os camundongos KO β1 apresentaram menor comprimento celular comparados aos C57. O grupo KO β2t apresentou maior comprimento celular comparado ao grupo KO β2c. O protocolo de treinamento aumentou a amplitude de contração, a velocidade máxima de contração e a velocidade máxima de relaxamento dos cardiomiócitos dos animais KO β1t e KO β2t. O ISO não alterou a amplitude de contração, a velocidade máxima de contração e de relaxamento nos cardiomiócitos dos camundongos KO β1+ISO, já os animais KO β2+ISO apresentaram aumento nas mesmas propriedades mecânicas. Conclui-se que o protocolo de treinamento aeróbico: a) aumentou as propriedades mecânicas dos cardiomiócitos dos animais KO β1 e KO β2; b) aumentou o comprimento dos cardiomiócitos dos camundongos KO β2; c) associado ao ISO não alterou as propriedades mecânicas nos cardiomiócitos dos camundongos KO β1 e reduziu nos cardiomiócitos dos camundongos KO β2. / The aim of this study was to investigate the effects of endurance training on cardiovascular parameters and morphological and mechanical properties of isolated cardiomyocytes from the left ventricle of β1-and β2 adrenergic receptors knockout mice. 4 months old C57BL/6J, β1- adrenergic receptor knockout (KO 1), FVB/N and β2-adrenergic receptor knockout (KO 2) mice were randomly allocated into one of the 8 groups: C57c, C57t, KO β1c, KO β1t, FVBc, FVBt, KO β2c e KO β2t. The animals of the trained groups (C57t, KO β1t, FVBt, KO β2t) underwent a protocol of aerobic training 8 weeks, 5 days/week, 1 hour/day, intensity of 60% of maximal speed. 3 to 4 animals in each group were divided for the analysis of cellular contraction with infusion of -adrenergic agonist isoproterenol (ISO) [100nM]. At sacrifice, the heart was removed and left ventricular myocytes were enzymatically dispersed. Cell length and width were measured using an image capture system and cell volume was calculated. Myocytes were stimulated at 1 Hz at controlled temperature (37oC) and cell contractility was measured by using an edge detection system. The results showed that KO 1 mice showed lower cell length compared to C57. The KO β2t group showed higher cell length compared to the KO β2c group. The training protocol increased the contraction amplitude, maximum contraction velocity and maximum relaxation velocity of cardiomyocytes of KO β1t and KO β2t animals. The ISO did not alter the amplitude of contraction, maximum contraction velocity and maximum relaxation velocity of cardiomyocytes from KO β1+ISO animals, while the KO β2+ISO animals showed an increase in the same mechanical properties. It is concluded that the aerobic training protocol: a) increased the mechanical properties of cardiomyocytes from KO β1t and KO β2t animals; b) increased the length of cardiomyocytes from β2 KO mice; c) associated with the ISO did not alter the mechanical properties of cardiomyocytes from β2 KO 1mice and reduced of cardiomyocytes from KO β2 mice.

Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro / O estado nutricional e hormonal em fases iniciais de desenvolvimento (gestação e lactação) está relacionado a alterações epigenéticas, que podem levar ao desenvolvimento de doenças. A obesidade infantil está relacionada com a ocorrência da obesidade na idade adulta, resistência à insulina e maior risco cardiometabólico. Em estudos experimentais, a superalimentação neonatal causa obesidade e aumenta o risco de doenças cardiovasculares. Estes animais apresentam obesidade visceral, hiperfagia, hiperleptinemia e hipertensão na idade adulta. Previamente, demonstramos que a hiperleptinemia neonatal causa hiperfunção da medula adrenal e microesteatose na idade adulta. No presente estudo avaliamos a função adrenal de ratos adultos obesos no modelo de superalimentação neonatal por redução do tamanho da ninhada e a sensibilidade as catecolaminas no tecido adiposo visceral (TAV) e no fígado. Ao nascimento todas as ninhadas tiveram seu número de filhotes ajustados para 10. Para induzir a superalimentação neonatal, o tamanho da ninhada foi reduzido de dez para três filhotes machos no terceiro dia de lactação até o desmame (SA), enquanto que o grupo controle permaneceu com 10 filhotes durante toda a lactação. Após o desmame, os ratos tiveram livre acesso à dieta padrão e água até 180 dias (1 animal de cada ninhada, n = 7). O TAV e as glândulas adrenais foram pesadas. As contrações hormonais séricas, o conteúdo hepático de glicogênio e triglicerídeos foram avaliados por kits comerciais. O conteúdo e a secreção de catecolaminas adrenais foram avaliados utilizando o método do trihidroxindol. O conteúdo dos hormônios eixo hipotálamo-hipófise-córtex adrenal, das enzimas da via de síntese das catecolaminas na glândula adrenal, ADRB2 no fígado e ADRB3 no TAV foram determinados por Western blotting ou imunohistoquímica. As diferenças foram consideradas significativas quando p <0,05. Aos 180 dias de vida, o grupo SA apresentou maior massa corporal (+15%), maior consumo alimentar (+15%) e maior adiposidade visceral (+79%). Os hormônios do eixo hipotálamo-hipófise-córtex-adrenal não foram alterados. O grupo SA apresentou maior expressão de tirosina hidroxilase e de DOPA descarboxilase (+31% e 90%, respectivamente); conteúdo de catecolaminas adrenais (absoluta: 35% e relativa: 40%), e secreção de catecolaminas, tanto basal quanto estimulada por cafeína (+35% e 43%, respectivamente). O conteúdo ADRB3 no TAV não foi alterado nos grupo SA, entretanto o ADRB2 no fígado apresentou-se menor (-45%). O grupo SA apresentou maior conteúdo de glicogênio e triglicerídeos no fígado (+79% e +49%, respectivamente), além de microesteatose. A superalimentação neonatal resulta em hiperativação adrenomedular e aparentemente está associada a preservação da sensibilidade às catecolaminas no VAT. Adicionalmente sugerimos que o maior conteúdo de glicogênio e triglicerídeos hepático seja devido a menor sensibilidade as catecolaminas. Tal perfil pode contribuir para a disfunção metabólica hepática e hipertensão arterial que são características deste modelo de obesidade programada. / Nutritional and hormonal status at early phases of development are related to epigenetic changes, promoting disease development. Childhood overweight is related with late obesity, insulin resistance and higher cardiometabolic risk. Rats overfed during lactation show higher visceral adiposity, hyperphagia, leptin resistance and hypertension in adulthood. Previously, we demonstrated that neonatal hyperleptinemia is associated with adrenal medullary hyperfunction and liver steatosis at adulthood. Here, we evaluated the adrenal function and liver tissue of adult obese rats that were overfed during lactation. To induce early overfeeding, the litter size was reduced from ten to three male pups at the third day of lactation until weaning (SL). Control group had ten rats per litter (NL). After weaning, rats had free access to standard diet and water until 180 days old (1 animal from each litter, n=7). Significant differences had p<0.05. The SL group presented higher adrenal catecholamine content (absolute: +35% and relative: +40%), tyrosine hydroxylase (+31%), DOPA decarboxylase (+90%) protein contents, basal and caffeine-induced catecholamine in vitro secretion (+35% and +43%, respectively). However, hormones of the hypothalamic-pituitary-adrenal cortex axis were unchanged. The &#946;3-adrenergic receptor content in visceral adipose tissue was unchanged in SL rats, but the &#946;2-adrenergic receptor in the liver was lower (-45%). SL group showed higher glycogen and triglycerides contents in liver (+79% and +49%, respectively), which showed microesteatosis. Although the neonatal overfeeding leads to higher adrenomedullary function, adult obese SL rats have a dysfunction in hepatic &#946;2-adrenergic receptor, which can contribute for the hepatic dysfunction characteristic of liver obesity complications.

Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro / As doenças cardiovasculares possuem a maior taxa de óbitos no mundo, e notavelmente nos últimos anos as pesquisas genéticas sobre as mesmas estão baseadas em estudos de associação, no qual o gene suspeito que esteja em maior frequência entre os pacientes passa a ser considerado um possível fator causal. Os polimorfismos genéticos que ocorrem no receptor beta-adrenérgico podem resultar em mudanças significativas na função do receptor, podendo acarretar fisiopatologias. Neste trabalho, o objetivo foi estimar a diversidade e a frequência do polimorfismo Ser49Gly do gene do receptor beta-adrenérgico 1 a partir de uma amostra de 188 indivíduos da população do Estado do Rio de Janeiro. As frequências também foram analisadas a partir da estratificação da amostra por critério fenotípico em função do padrão de cor da pele em (negros e não negros) ou ancestralidade genética em (afrodescendente e não afrodescendente), definida através da informação dos marcadores de ancestralidade Indels e SNP de cromossomo Y, para avaliar se os padrões de ancestralidade ou cor da pele são fundamentais para a diferenciação e distanciamento genético. Fragmentos de interesse foram amplificados por PCR (reação de cadeia de polimerase) com primers específicos para o marcador Ser49Gly e as reações de genotipagem foram realizadas com enzimas de restrição Eco0109I. Os valores da heterozigosidade variaram entre 0,25-0,50 e 0,20-0,41 nos grupos estratificados por ancestralidade e cor da pele, respectivamente. No que diz respeito à análise do equilíbrio de Hardy-Weinberg, não houve um desvio significativo na distribuição do marcador nas amostras gerais do Estado do Rio de Janeiro, ou mesmo nas amostras estratificadas. A distribuição dos alelos na amostra dos 188 indivíduos da população geral do Rio de Janeiro (AC_RJ) mostrou uma frequência de 80,30% e 19,70% para o alelo selvagem e mutado Ser49Gly, respectivamente. A comparação das análises sobre a distribuição das frequências alélicas para este marcador mostrou a ocorrência de diferenças significativas na distribuição das frequências alélicas entre negros e não negros e afrodescendentes e não afrodescendentes. A diferença significativa observada entre os negros e afrodescendentes, foi em menor grau de distanciamento. A informação obtida em relação à ancestralidade foi crucial para a obtenção dos dados sobre o aumento da variável mutada do polimorfismo Ser49Gly nas populações negras e afrodescendentes do Estado Rio de Janeiro. Tal evidência, em combinação com estudos clínicos podem contribuir para uma análise pormenorizada do padrão de susceptibilidade à doença em questão, em falhas do mecanismo deste receptor. / Cardiovascular diseases have the highest death rate in the world, and notably in recent years genetic research about them are based on association studies, in which the gene suspected to be at a higher frequency among patients is now considered a possible causal factor. Genetic polymorphisms that occur in the beta adrenergic receptor can result in significant changes in the receptor function that may trigger physiopathologies. The main aim of this study was to estimate the diversity and the frequency of Ser49Gly polymorphism of &#914;eta adrenergic 1 receptor gene in a sample of 188 individuals of the population of Rio de Janeiro. The frequencies were also analyzed from the sample stratification by phenotypic criteria due to skin color pattern (blacks and non-blacks) or by genetic ancestry (African descent and non-African descent), defined by ancestry information SNP and Indels markers from Y chromosome, to evaluate whether the ancestry criteria and/or skin color are crucial to the pattern of differentiation and genetic distance. Fragments of interest were amplified by PCR (polymerase chain reaction) with specific primers for the marker Ser49Gly and genotyping reactions performed by restriction with the enzyme Eco0109I. The values of heterozygosity ranged from 0.25 to 0.50 and 0.20 to 0.41 in the groups stratified by ancestry and skin color, respectively. Regarding the analysis of EHW, there was no significant deviation from this marker genotype distribution in Rio de Janeiro sample or even the stratified sample. The distribution of alleles in the sample of 188 individuals from the general population of Rio de Janeiro (AC_RJ) shows a frequency of 80.30% and 19.70% for the wild-type allele mutated Ser49 and Gly49, respectively. The comparison analysis showed the occurrence of significant differences in the distribution of allele frequencies of this marker between blacks and non-blacks and African descent and non-African descent. A significant difference was also observed between blacks and African descent, with a lesser degree of detachment. The information obtained in relation to ancestry was crucial for obtaining data on the increase in variable mutated polymorphism Ser49Gly in the black populations and African descent in Rio de Janeiro State. Such evidence, in combination with clinical studies may contribute to a detailed analysis of the pattern of susceptibility to disease involved in mechanism crashes of this receptor.