Papel da adenosina na insuficiência cardíaca num modelo animal de hipertensão pulmonar
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2016-08-22
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A hipertensão arterial pulmonar (HAP) é uma doença progressiva, caraterizada pela
elevação da pressão arterial pulmonar e pela remodelação vascular pulmonar, conduzindo à
hipertrofia do ventrículo direito (VD), insuficiência cardíaca e morte. Apesar da disfunção do
VD estar intimamente relacionada com a sobrevida, pouco se sabe acerca da adaptação e
insuficiência do VD no contexto da HAP. A adenosina é um importante modulador da função
cardiovascular, atuando através da ativação dos seus recetores, A1, A2A, A2B e A3. Estudos
recentes sugerem o envolvimento da adenosina na HAP, devido ao seu papel na regulação do
tónus vascular pulmonar. Contudo, desconhece-se o papel que este nucleósido desempenha na
disfunção do VD que ocorre nesta doença. Neste contexto, o principal objetivo deste estudo
foi investigar o papel da sinalização da adenosina na disfunção ventricular associada à HAP.
Para tal, usou-se um modelo animal de HAP induzida pela monocrotalina.
Ratazanas Wistar machos receberam aleatoriamente uma injeção subcutânea de
monocrotalina (grupo MCT) ou veículo (grupo CTRL). Os estudos hemodinâmicos,
miográficos e a colheita de amostras para análise morfométrica, histológica e para os estudos
de imunofluorescência e moleculares foram realizados 21 a 25 dias após a indução do
modelo. A avaliação hemodinâmica confirmou a presença de HAP nos animais MCT,
traduzida no aumento da pressão sistólica do VD. Além disso, a morfometria cardíaca e
pulmonar, bem como a presença de hipertrofia dos cardiomiócitos ventriculares e da camada
média das arteríolas pulmonares nestes animais permitiram a validação do modelo.
Os estudos funcionais in vitro foram realizados em aurículas a contrair
espontaneamente e em tiras de ventrículo direito estimuladas eletricamente, de modo a avaliar
o papel dos recetores A1 e A2 da adenosina na função cardíaca. Os resultados obtidos com a
R-PIA (0,001-1 μM) agonista seletivo dos recetores A1, e com a NECA (0,01-100 μM),
agonista não seletivo, mostraram que os recetores A1 diminuem a frequência (cronotropismo
negativo) e a força de contração (inotropismo negativo) auricular, tanto em animais CTRL
como MCT. Tanto a ativação dos recetores A2A com o CGS 21680 (0,003-1 μM), como dos
recetores A2AB com o BAY 60-6583 (0,01-10 μM) não alteraram a função auricular.
No tecido ventricular a ativação dos recetores A1 pela R-PIA e pela NECA não
modificou a contratilidade ventricular, apesar de se ter comprovado a presença destes
recetores no miocárdio ventricular por experiências de imunofluorescência analisadas por
microscopia confocal. De igual forma, a ativação dos recetores A2A pelo CGS 21680 também
não alterou a contratilidade ventricular. Por outro lado, o bloqueio dos recetores A2B com o
PSB 603 (100 nM) evidenciou um efeito inotrópico positivo da NECA nos animais MCT,
sugerindo nestes animais o envolvimento destes recetores num efeito inotrópico negativo. As
experiências de imunofluorescência analisadas por microscopia confocal permitiram verificar
uma presença ligeira dos recetores A2B no miocárdio de animais CTRL e MCT. Contudo, nos
animais com HAP, verificou-se uma presença marcada do recetor A2B em agrupamentos de
células existentes no espaço intersticial do VD. Em conclusão, a adenosina parece modular a
atividade cardíaca na HAP por intermédio dos seus recetores A1 e A2B, sendo que no futuro
importa avaliar a relevância funcional das células intersticiais que expressam recetores A2B.
Pulmonary arterial hypertension (PAH) is a progressive disease characterized by increases in pulmonary arterial pressure and pulmonary vascular remodelling, leading to right ventricle (RV) hypertrophy, heart failure and death. Despite RV dysfunction is closely tied to survival in PAH, little is known about RV adaptation and failure within the context of PAH. Adenosine is an important modulator of cardiovascular function, via the activation of A1, A2A, A2B and A3 receptors. Recent studies have implicated adenosine in PAH, due to its role on the regulation of pulmonary vascular tonus. Nevertheless, the role of this nucleoside in RV dysfunction secondary to this pathology is still unknown. In this context, our main goal was to study the role of adenosine in ventricular dysfunction associated to PAH. To carry out the study, an animal model of monocrotaline-induced PAH was used. Male Wistar rats randomly received monocrotaline (MCT group) or vehicle (CTRL group). Hemodynamic, myographic and sample collection for morphometric, histological, immunofluorescence, and molecular studies were performed 21 to 25 days after monocrotaline administration. Hemodynamic evaluation confirmed the presence of PAH in MCT animals, as demonstrated by the elevation of systolic pressure of RV. Also, cardiac and pulmonary morphometric, and the presence of cardiomyocytes hypertrophy and pulmonary media hypertrophy allowed the validation of the PAH model. In vitro functional studies were performed in spontaneously beating atria and RV strips electrically stimulated to evaluate the role of adenosine A1 and A2 receptors on cardiac function. The results obtained with R-PIA (0,001-1 μM), a selective A1 receptor agonist, and NECA (0,01-100 μM), a non-selective agonist, demonstrated that A1 receptors decrease auricular frequency (negative chronotropic effect) and force of contraction (negative inotropic effect) in both groups. The activation of A2A receptors with CGS 21680 (0,003-1 μM), and A2AB receptors with BAY 60-6583 (0,01-10 μM) did not change the auricular function. At the ventricular level, the activation of A1 receptors with R-PIA and NECA did not change ventricular contractility, in spite of the presence of these receptors in RV myocardium as demonstrated by confocal microscopy. Likewise, the activation of A2A receptors with CGS 21680 had no effect on ventricular contractility. On the other hand, in MCT animals, A2B receptor blockade with PSB 603 (100 nM) resulted in a positive inotropic effect promoted by NECA, suggesting that these receptors could be involved in a negative inotropic effect. Immunolocalization studies confirmed the presence of small amounts of A2B receptors staining in the RV myocardium of both CTRL and MCT animals. However, we only observed in PAH rats a marked expression of this receptor in cells present in the interstitial spaces of RV. In conclusion, adenosine seems to modulate cardiac function in PAH through the activation of A1 and A2B receptors. The functional significance of A2B positive interstitial cells should be evaluated in the near future.
Pulmonary arterial hypertension (PAH) is a progressive disease characterized by increases in pulmonary arterial pressure and pulmonary vascular remodelling, leading to right ventricle (RV) hypertrophy, heart failure and death. Despite RV dysfunction is closely tied to survival in PAH, little is known about RV adaptation and failure within the context of PAH. Adenosine is an important modulator of cardiovascular function, via the activation of A1, A2A, A2B and A3 receptors. Recent studies have implicated adenosine in PAH, due to its role on the regulation of pulmonary vascular tonus. Nevertheless, the role of this nucleoside in RV dysfunction secondary to this pathology is still unknown. In this context, our main goal was to study the role of adenosine in ventricular dysfunction associated to PAH. To carry out the study, an animal model of monocrotaline-induced PAH was used. Male Wistar rats randomly received monocrotaline (MCT group) or vehicle (CTRL group). Hemodynamic, myographic and sample collection for morphometric, histological, immunofluorescence, and molecular studies were performed 21 to 25 days after monocrotaline administration. Hemodynamic evaluation confirmed the presence of PAH in MCT animals, as demonstrated by the elevation of systolic pressure of RV. Also, cardiac and pulmonary morphometric, and the presence of cardiomyocytes hypertrophy and pulmonary media hypertrophy allowed the validation of the PAH model. In vitro functional studies were performed in spontaneously beating atria and RV strips electrically stimulated to evaluate the role of adenosine A1 and A2 receptors on cardiac function. The results obtained with R-PIA (0,001-1 μM), a selective A1 receptor agonist, and NECA (0,01-100 μM), a non-selective agonist, demonstrated that A1 receptors decrease auricular frequency (negative chronotropic effect) and force of contraction (negative inotropic effect) in both groups. The activation of A2A receptors with CGS 21680 (0,003-1 μM), and A2AB receptors with BAY 60-6583 (0,01-10 μM) did not change the auricular function. At the ventricular level, the activation of A1 receptors with R-PIA and NECA did not change ventricular contractility, in spite of the presence of these receptors in RV myocardium as demonstrated by confocal microscopy. Likewise, the activation of A2A receptors with CGS 21680 had no effect on ventricular contractility. On the other hand, in MCT animals, A2B receptor blockade with PSB 603 (100 nM) resulted in a positive inotropic effect promoted by NECA, suggesting that these receptors could be involved in a negative inotropic effect. Immunolocalization studies confirmed the presence of small amounts of A2B receptors staining in the RV myocardium of both CTRL and MCT animals. However, we only observed in PAH rats a marked expression of this receptor in cells present in the interstitial spaces of RV. In conclusion, adenosine seems to modulate cardiac function in PAH through the activation of A1 and A2B receptors. The functional significance of A2B positive interstitial cells should be evaluated in the near future.
Descrição
Dissertação de Mestrado em Biotecnologia para as Ciências da Saúde
Palavras-chave
Hipertensão (arterial pulmonar) , Monocrotalina , Adenosina