Interação do antiarrítmico amiodarona e dronedarona com o canal de sódio Nav1.5 de humano depende do ph extracelular: novas perspectivas para o tratamento de doenças arrítmicas
Data
2024-04-08
Autores
Conceição, Michael Ramon de Lima 
Orientadores
Compos, Danilo Roman
Tipo
Tese de doutorado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
Introdução: A amiodarona (AMD, pKa 6.56) e dronedarona (DRN, pKa 9.40) são drogas antiarrítmicas que atuam em diversos canais iônicos, incluindo o canal para sódio Nav1.5, expresso majoritariamente no coração. Ambas as drogas apresentam grupos ionizáveis, porém em faixas de pHs distintos. Dessa forma, alterações no pH podem alterar a forma de interação dessas drogas com os canais iônicos. Objetivo: Investigar o papel do pH extracelular (pHe) sobre as propriedades farmacológicas da AMD e DRN sobre o Nav1.5. Métodos: Foram utilizadas células embrionárias de rim humano (HEK293) para expressar de forma heteróloga o Nav1.5 de humano. Coração e átrio de rato isolado foram utilizados para registro eletrocardiográfico e aferição da força e cronotropismo em função do pHe. Foi utilizada a neurotoxina do tipo 2, ATX, para aumentar o componente tardio da corrente de sódio (INaLate), o qual é capaz de gerar arritmias. Para o estudo da eletrofisiologia celular foi empregada a técnica de patch-clamp, por meio da configuração de célula inteira na modalidade “voltage-clamp” e “current-clamp”, utilizando diversos protolos. Resultados: A potência da AMD sobre o pico da INa foi ~25x maior em pHe 7.0 quando comparada a pHe 7.4. A DNR apresentou a mesma potência sobre o pico da INa para ambos os pHes. A dependência da voltagem para ativação não diferiu entre todos os grupos, tanto para AMD como para DNR. A AMD mudou a curva de inativação em estado estacionário para potenciais mais hiperpolarizados, com magnitudes semelhantes para ambos os pHes. Já a DNR não alterou esse parâmetro. A recuperação da inativação da INa foi atrasada na presença de AMD com perfil semelhante em ambos os pHes. A DNR também atrasou o recobro da inativação, porém em menor magnitude e independente do pHe. As propriedades do uso dependente de frequência da AMD foram distintas em pHe 7.0 e 7.4. Além disso, a AMD foi capaz de alterar o perfil do ECG ex vivo, porém em pHe 7.0+AMD causou maior aumento na duração de RR e QRS e no intervalo QT quando comparado ao pHe 7.4+AMD. A alteração do pHe não modificou as propriedades farmacológicas da AMD sobre o átrio esquerdo (AE) e direito (AD) isolados. O pHe 7.0 atenuou a velocidade máxima de despolarização e amplitude do potencial de ação (PA) de cardiomiócito atrial isolado que foi potencializada na presença de AMD. A AMD não teve efeito na repolarização do PA, independente do pHe. Utilizando transfecção transitória da subunidade alfa humana de Nav1.5, descobriu-se que a 10 M de AMD foi capaz de bloquear a INalate induzida por ATX apenas em pHe 7.0. Adicionalmente, 5 nM de ATX foi capaz de gerar arritmias em AD e AE isolados, além de prolongar a duração da PA e aumentar a dispersão da repolarização em cardiomiócitos atriais isolados em pHe 7.4 e pHe 7.0. A pré-incubação de AD e AE e cardiomiócitos atriais isolados com AMD foi capaz de prevenir arritmias induzidas por ATX apenas em pHe 7.0. Conclusões: A AMD em pHe ácido altera algumas propriedades biofísicas do canal para sódio de maneira diferente da DRN, além de reverter a INalate e arritmias em cardiomiócitos atriais e átrio isolado gerado por ATX apenas em pHe 7.0.
Introduction: Amiodarone (AMD, pKa 6.56) and dronedarone (DRN, pKa 9.40) are antiarrhythmic drugs that act on several ion channels, including the sound channel Nav1.5, expressed mainly in the heart. Both drugs have ionizable groups, but in different pH ranges. Therefore, changes in pH can alter the way these drugs interact with ion channels. Objective: To investigate the role of extracellular pH (pHe) on the pharmacological properties of AMD and DRN on Nav1.5. Methods: Human embryonic kidney cells (HEK293) were used to heterologously express human Nav1.5. Isolated rat heart and atrium were used for electrocardiographic recording and measurement of strength and chronotropism as a function of pHe. A type 2 neurotoxin, ATX, was used to increase the late component of the sodium current (INaLate), which is capable of generating arrhythmias. To study cellular electrophysiology, the patchclamp technique was used, through the configuration of whole cells in “voltageclamp” and "currentclamp” modes, using different protocols. Results: The potency of AMD over the INa peak was ~25x greater at pHe 7.0 when compared to pHe 7.4. DNR showed the same power over the INa peak for both pHes. The voltage dependence for activation did not differ between all groups for both AMD and DNR. AMD shifted the steadystate inactivation curve to more hyperpolarized potentials, with similar magnitudes for both pHes. The DNR did not change this parameter. Recovery from INa inactivation was delayed in the presence of AMD with a similar profile at both pHes. DNR also delayed recovery from inactivation, but to a lesser extent and independent of pHe. The frequencydependent usage properties of AMD were distinct at pHe 7.0 and 7.4. Furthermore, AMD was able to change the ex vivo ECG profile, however at pHe 7.0+AMD it caused a greater increase in RR and QRS duration and QT interval when compared to pHe 7.4+AMD. The change in pHe did not modify the pharmacological properties of AMD on the isolated left (LA) and right (RA) atrium. pHe 7.0 attenuated the maximum depolarization velocity and action potential (AP) amplitude of isolated atrial cardiomyocytes that were potentiated in the presence of AMD. AMD had no effect on PA repolarization, regardless of pHe. Using transient transfection of the human alpha subunit of Nav1.5, it was discovered that 10 μM AMD was able to block INaLate caused by ATX only at pHe 7.0. Furthermore, 5 nM of ATX was able to generate arrhythmias in isolated AD and LA, in addition to prolonging the duration of AP and increasing the dispersion of repolarization in isolated atrial cardiomyocytes at pHe 7.4 and pHe 7.0. Preincubation of AD and AE and isolated atrial cardiomyocytes with AMD was able to prevent arrhythmias caused by ATX only at pHe 7.0. Conclusions: AMD in acidic pHe alters some biophysical properties of the sodium channel differently than DRN, in addition to reversing INaLate and arrhythmias in atrial cardiomyocytes and isolated atrium generated by ATX only at pHe 7.0.
Introduction: Amiodarone (AMD, pKa 6.56) and dronedarone (DRN, pKa 9.40) are antiarrhythmic drugs that act on several ion channels, including the sound channel Nav1.5, expressed mainly in the heart. Both drugs have ionizable groups, but in different pH ranges. Therefore, changes in pH can alter the way these drugs interact with ion channels. Objective: To investigate the role of extracellular pH (pHe) on the pharmacological properties of AMD and DRN on Nav1.5. Methods: Human embryonic kidney cells (HEK293) were used to heterologously express human Nav1.5. Isolated rat heart and atrium were used for electrocardiographic recording and measurement of strength and chronotropism as a function of pHe. A type 2 neurotoxin, ATX, was used to increase the late component of the sodium current (INaLate), which is capable of generating arrhythmias. To study cellular electrophysiology, the patchclamp technique was used, through the configuration of whole cells in “voltageclamp” and "currentclamp” modes, using different protocols. Results: The potency of AMD over the INa peak was ~25x greater at pHe 7.0 when compared to pHe 7.4. DNR showed the same power over the INa peak for both pHes. The voltage dependence for activation did not differ between all groups for both AMD and DNR. AMD shifted the steadystate inactivation curve to more hyperpolarized potentials, with similar magnitudes for both pHes. The DNR did not change this parameter. Recovery from INa inactivation was delayed in the presence of AMD with a similar profile at both pHes. DNR also delayed recovery from inactivation, but to a lesser extent and independent of pHe. The frequencydependent usage properties of AMD were distinct at pHe 7.0 and 7.4. Furthermore, AMD was able to change the ex vivo ECG profile, however at pHe 7.0+AMD it caused a greater increase in RR and QRS duration and QT interval when compared to pHe 7.4+AMD. The change in pHe did not modify the pharmacological properties of AMD on the isolated left (LA) and right (RA) atrium. pHe 7.0 attenuated the maximum depolarization velocity and action potential (AP) amplitude of isolated atrial cardiomyocytes that were potentiated in the presence of AMD. AMD had no effect on PA repolarization, regardless of pHe. Using transient transfection of the human alpha subunit of Nav1.5, it was discovered that 10 μM AMD was able to block INaLate caused by ATX only at pHe 7.0. Furthermore, 5 nM of ATX was able to generate arrhythmias in isolated AD and LA, in addition to prolonging the duration of AP and increasing the dispersion of repolarization in isolated atrial cardiomyocytes at pHe 7.4 and pHe 7.0. Preincubation of AD and AE and isolated atrial cardiomyocytes with AMD was able to prevent arrhythmias caused by ATX only at pHe 7.0. Conclusions: AMD in acidic pHe alters some biophysical properties of the sodium channel differently than DRN, in addition to reversing INaLate and arrhythmias in atrial cardiomyocytes and isolated atrium generated by ATX only at pHe 7.0.
Descrição
Citação
Conceição. MRL
Michael Ramon de Lima Conceição. Interação do antiarrítmico amiodarona e dronedarona com o canal de sódio NaV 1.5 de humano dependente de pH extracelular: novas perspectivas para o tratamento de doenças arrítmicas. 2024. 126 f. Tese (Doutorado em Farmacologia) - Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, 2024.
Michael Ramon de Lima Conceição. Interação do antiarrítmico amiodarona e dronedarona com o canal de sódio NaV 1.5 de humano dependente de pH extracelular: novas perspectivas para o tratamento de doenças arrítmicas. 2024. 126 f. Tese (Doutorado em Farmacologia) - Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, 2024.