Regulación β-adrenérgica sobre las cinéticas de inactivación del canal de calcio tipo L durante el potencial de acción cardiaco
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2017
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es
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Universidad Andrés Bello
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Resumen
La activación de los receptores β-adrenérgicos provoca un aumento en el ritmo cardiaco, la contractibilidad y la frecuencia de la relajación del corazón. En las células contráctiles ventriculares, los cardiomiocitos, la activación de estos receptores induce cambios en el potencial de acción (AP) como consecuencia de la regulación de diversos canales involucrados en este proceso. Entre ellos, el canal de calcio (Ca2+) Cav1.2, lo que genera un aumento en la corriente de Ca2+ tipo L (ICaL). Los canales de Ca2+ dependientes de potencial tipo L (L-VDCC) son la principal vía de entrada de Ca2+ en el corazón; éstos son activados por la despolarización de la membrana durante el AP, y posteriormente inactivados a través de mecanismos dependientes de potencial, y de Ca2+. Ha sido descrito que, bajo estimulación β-adrenérgica, los AP de rata adulta disminuyen su duración, sin embargo, el cómo ocurre este acortamiento no ha sido completamente dilucidado. En particular, cómo los mecanismos de inactivación del canal Cav1.2 influyen en la morfología es desconocido, por lo que la hipótesis de este trabajo es que el estímulo con Isoproterenol sobre los cardiomiocitos de rata neonata modifica el potencial de acción mediante la modulación de las cinéticas de inactivación del canal Cav1.2. Es por ello, que en esta tesis fueron estudiadas las cinéticas de inactivación del canal Cav1.2 durante el AP de cardiomiocitos neonatos bajo estimulación con Isoproterenol en base al uso de técnicas de electrofisiología. Los resultados de este trabajo demuestran que el aumento de la densidad de corrientes de Ca2+, efecto que fue logrado aumentando la concentración de Ca2+ extracelular o induciendo la facilitación del canal mediante el aumento de la frecuencia del estímulo, produce principalmente cambios en la cinética de los AP y no es responsable del acortamiento de los AP observado en cardiomiocitos de ratas neonatas estimuladas con Isoproterenol; por otro lado, la inhibición de la inactivación dependiente de potencial (VDI) o la inactivación dependiente de Ca2+ (CDI) demostraron ser capaces de modificar la duración del AP. Para observar directamente las corrientes de Ca2+ durante el AP fue utilizada la técnica de AP-clamp dinámico. Estos experimentos demuestran que la corriente de Ca2+ tipo L en cardiomiocitos estimulados con Isoproterenol es inactivada más rápido, sin existir diferencia en la cantidad de Ca2+ que ingresa en cada AP. La manipulación de cada tipo de inactivación demostró que la inhibición de cualquiera de ellas produce, paradójicamente, un aumento en la velocidad de decaimiento
de la corriente durante el AP, produciendo que la corriente de Ca2+ terminase incluso antes del término del AP. En su conjunto los resultados de esta tesis demuestran que el estímulo β-adrenérgico, sobre cardiomiocitos de ratas neonatas, modula al canal de Ca2+ tipo L durante el AP, aumentando las velocidades de la VDI y de la CDI, siendo el efecto de esta última el que tiene un mayor aporte durante el AP.
Under β-adrenergic stimulation, action potential (AP) duration in adult rat shortens as a consequence of the regulation of several ion channels involved at this process, among them, the L-type calcium channels (LTCC). These channels are the main entrances of Ca2+ in the cardiac cells and are activated by membrane depolarization during the AP, and inactivated later through potential and Ca2+ dependent mechanisms. Although it is well established that L-type current is involved in the AP plateau duration, how the inactivation processes of these channels participate in this process has not been elucidated. Thus, the hypothesis of this study was that the AP from Isoproterenol-stimulate newborn rat cardiomyocytes are modified as a consequence of the modulation of LTCC inactivation. In order to answer this hypothesis, we used different electrophysiolical techniques with the purpose of study the LTCC-inactivation kinetic changes from Isoproterenol-stimulated newborn rat cardiomyocytes. The results of this study shows that an increase in the Ca2+ current density achieved, either by an increase in the extracellular Ca2+ concentration or by the calcium-dependent facilitation induction through a higher stimulus frequency, mainly produces changes in the AP kinetic. In contrast, voltage dependent inactivation (VDI) or Ca2+ dependent inactivation (CDI) inhibition modify AP duration, suggesting that changes in the inactivation processes, and not the increase in the current, are responsible for the AP shortening observed in Isoproterenol-stimulated newborn rat cardiomyocytes. In order to directly observe the L-type calcium current during the AP, the dynamic AP-clamp technique was used. These experiments show that the L-type Ca2+ current from Isoproterenol-stimulated cardiomyocytes inactivate faster, with no difference in the total Ca2+ influx in each AP, while the inhibition of either type of inactivation increase the speed of current decay during the AP, resulting in the termination of the Ca2+ current before than the AP. Finally, AP from Isoproterenol-stimulated cardiomyocytes with the VDI inhibited has a reduced duration, suggesting that β-adrenergic stimulus mainly modulates CDI. In summary, in this work is demonstrated that the main effect of β-adrenergic stimulation over cardiomyocyte AP is the modulation of L-type calcium channel inactivation, most likely CDI, and not the increase of the current magnitude. Moreover, the experiments shown here provide strong evidence that the augmented contraction observed after β-adrenergic stimulation is independent of L-type calcium channels.
Under β-adrenergic stimulation, action potential (AP) duration in adult rat shortens as a consequence of the regulation of several ion channels involved at this process, among them, the L-type calcium channels (LTCC). These channels are the main entrances of Ca2+ in the cardiac cells and are activated by membrane depolarization during the AP, and inactivated later through potential and Ca2+ dependent mechanisms. Although it is well established that L-type current is involved in the AP plateau duration, how the inactivation processes of these channels participate in this process has not been elucidated. Thus, the hypothesis of this study was that the AP from Isoproterenol-stimulate newborn rat cardiomyocytes are modified as a consequence of the modulation of LTCC inactivation. In order to answer this hypothesis, we used different electrophysiolical techniques with the purpose of study the LTCC-inactivation kinetic changes from Isoproterenol-stimulated newborn rat cardiomyocytes. The results of this study shows that an increase in the Ca2+ current density achieved, either by an increase in the extracellular Ca2+ concentration or by the calcium-dependent facilitation induction through a higher stimulus frequency, mainly produces changes in the AP kinetic. In contrast, voltage dependent inactivation (VDI) or Ca2+ dependent inactivation (CDI) inhibition modify AP duration, suggesting that changes in the inactivation processes, and not the increase in the current, are responsible for the AP shortening observed in Isoproterenol-stimulated newborn rat cardiomyocytes. In order to directly observe the L-type calcium current during the AP, the dynamic AP-clamp technique was used. These experiments show that the L-type Ca2+ current from Isoproterenol-stimulated cardiomyocytes inactivate faster, with no difference in the total Ca2+ influx in each AP, while the inhibition of either type of inactivation increase the speed of current decay during the AP, resulting in the termination of the Ca2+ current before than the AP. Finally, AP from Isoproterenol-stimulated cardiomyocytes with the VDI inhibited has a reduced duration, suggesting that β-adrenergic stimulus mainly modulates CDI. In summary, in this work is demonstrated that the main effect of β-adrenergic stimulation over cardiomyocyte AP is the modulation of L-type calcium channel inactivation, most likely CDI, and not the increase of the current magnitude. Moreover, the experiments shown here provide strong evidence that the augmented contraction observed after β-adrenergic stimulation is independent of L-type calcium channels.
Notas
Tesis (Magíster en Biotecnología)
Palabras clave
Cardiopatías, Tratamiento, Receptores Adrenérgicos Beta, Canales de Calcio