Efecto de cortisol sobre genes asociados a contracción muscular esquelética de trucha arcoíris (Oncorhynchus mykiss)
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2023
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es
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Universidad Andrés Bello
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Actualmente Chile se encuentra entre los diez primeros países que generan peces para exportación,
y hasta el año 2018 era el principal exportador de trucha arcoíris (Oncorhynchus mykiss) en América del
Sur. Bajo condiciones de estrés los teleósteos pueden desencadenar problemas que afectan el crecimiento,
reproducción, u otros cambios en el desarrollo del pez. Para combatir el estrés, los teleósteos desarrollaron
como respuesta primaria secretar la hormona glucocorticoide (cortisol) al torrente sanguíneo. La acción de
cortisol es llevada a cabo por la formación de un complejo con receptores intracelulares de glucocorticoides
(GR) o mineralocorticoides (MR). La mayoría de los teleósteos presentan dos GR (gr1 y gr2) y un MR (mr)
Una vez formado este complejo se translocará al núcleo, reconociendo sitios específicos en las regiones
promotoras llamados sitios de reconocimientos de glucocorticoides (GRE), donde una vez unido el complejo
este puede actuar como regulador de la transcripción. El músculo esquelético en teleósteos constituye más
del 50% de la musculatura del pez, y este tejido está asociado al proceso de contracción muscular, el cual
es de suma importancia para la sobrevivencia del pez. La contracción del músculo es llevada a cabo por
diferentes proteínas motoras donde se destacan, Troponina C, encargada de realizar el cambio estructural y
es el sitio de unión de Ca2+, Troponina I, que es la encargada de inhibir la interacción entre miosina y actina,
y finalmente miosina, donde se puede encontrar Miosina 7 que está relacionada con la contracción muscular
interactuando con los filamentos de actina. El rol de cortisol frente a distintos tipos de estrés en trucha
arcoíris ha sido ampliamente estudiado, pero cómo el cortisol afecta la regulación de proteínas motoras
asociadas a la contracción muscular todavía presenta un gran foco de estudio. Es por esto que en este trabajo
se evaluó el efecto de cortisol sobre la regulación de los genes de troponina I (tni), troponina C (tnc) y
miosina 7 (myh7), y si su regulación es llevada a cabo mediante las vías de GR o MR. Para esto se realizaron
ensayos in vivo con truchas arcoíris juveniles, inyectando vía intraperitoneal cortisol en dosis fisiológicas,
e inhibidores farmacológicos de GR (Mifepristona) y MR (Eplerenona). Además, se realizaron ensayos en
cultivo miotubos de músculo esquelético, y se evaluó la expresión de GR y MR. Adicionalmente, se
identificaron sitios GRE en las regiones promotoras de cada gen para determinar si pudiesen ser blancos
directos de cortisol. Los resultados obtenidos sugieren que la expresión de tnc posiblemente sea regulado
directamente por cortisol a través de la vía de GR bajo un estrés agudo. No así para los genes de myh7 y tni,
donde, si bien se cree que cortisol podría regularlos debido a resultados in silico de los sitios GRE, sus
regulaciones podrían estar asociadas a una regulación indirecta por cortisol. Para los genes gr1 y mr se
obtuvo que su expresión fue regulada negativamente con la administración de cortisol, pero luego estos
vuelven a sus niveles. Este trabajo contribuye al conocimiento sobre el rol de cortisol en la regulación de
genes asociados a contracción muscular y su potencial uso en la acuicultura como potenciales marcadores
de estrés y crecimiento entre salmónidos.
Chile is currently among the top ten countries generating fish for export, and until 2018 was the leading exporter of rainbow trout (Oncorhynchus mykiss) in South America. Under stress conditions teleosts can trigger issues that affect growth, reproduction, or other developmental changes in the fish. To combat stress, teleosts developed as primary response secreting the glucocorticoid hormone (cortisol) into the bloodstream. The action of cortisol is carried out by the formation of a complex with intracellular glucocorticoid receptors (GR) or mineralocorticoid receptors (MR). Most teleosts have two GR (gr1 and gr2) and one MR (mr). Once formed this complex will translocate to the nucleus, recognizing specific sites in the promoter regions called glucocorticoid response elements (GRE), where once bound, the complex can act as a transcriptional regulator. Skeletal muscle in teleosts constitutes more than 50% of the fish musculature, and this tissue is associated with the process of muscle contraction, which is of utmost importance for the survival of the fish. Muscle contraction is carried out by different motor proteins such as Troponin C, which is in charge of the structural change and is the Ca2+ binding site, Troponin I, which is in charge of inhibiting the interaction between myosin and actin, and finally myosin, where we can find Myosin 7, which is related to muscle contraction by interacting with actin filaments The role of cortisol against different types of stress in rainbow trout has been extensively studied, but how cortisol affects the regulation of motor proteins associated with muscle contraction still presents a great focus of study. This is why in this work we evaluated the effect of cortisol on the regulation of troponin I (tni), troponin C (tnc) and myosin 7 (myh7) genes, and whether their regulation is carried out through the GR or MR pathways. For this purpose, in vivo assays were performed with juvenile rainbow trout, injecting intraperitoneally cortisol at physiological doses, and pharmacological inhibitors of GR (Mifepristone) and MR (Epleronone). In addition, to validate these results, skeletal muscle myotubes were cultured and GR and MR expression was evaluated. Additionally, GRE sites were identified in the promoter regions of each gene to determine if they could be direct targets of cortisol. The results obtained suggest that tnc expression is likely to be directly regulated by cortisol through the GR pathway under acute stress. Not so for the myh7 and tni genes, where, although it is believed that cortisol could regulate them due to in silico results from GRE sites, their regulations could be associated with an indirect regulation by cortisol. For the gr1 and mr genes, it was obtained that their expression was negatively regulated by cortisol administration, but then they return to their levels. This work contributes to the knowledge about the role of cortisol in the regulation of genes associated with muscle contraction and its potential use in aquaculture as potential markers of stress and growth among salmonids.
Chile is currently among the top ten countries generating fish for export, and until 2018 was the leading exporter of rainbow trout (Oncorhynchus mykiss) in South America. Under stress conditions teleosts can trigger issues that affect growth, reproduction, or other developmental changes in the fish. To combat stress, teleosts developed as primary response secreting the glucocorticoid hormone (cortisol) into the bloodstream. The action of cortisol is carried out by the formation of a complex with intracellular glucocorticoid receptors (GR) or mineralocorticoid receptors (MR). Most teleosts have two GR (gr1 and gr2) and one MR (mr). Once formed this complex will translocate to the nucleus, recognizing specific sites in the promoter regions called glucocorticoid response elements (GRE), where once bound, the complex can act as a transcriptional regulator. Skeletal muscle in teleosts constitutes more than 50% of the fish musculature, and this tissue is associated with the process of muscle contraction, which is of utmost importance for the survival of the fish. Muscle contraction is carried out by different motor proteins such as Troponin C, which is in charge of the structural change and is the Ca2+ binding site, Troponin I, which is in charge of inhibiting the interaction between myosin and actin, and finally myosin, where we can find Myosin 7, which is related to muscle contraction by interacting with actin filaments The role of cortisol against different types of stress in rainbow trout has been extensively studied, but how cortisol affects the regulation of motor proteins associated with muscle contraction still presents a great focus of study. This is why in this work we evaluated the effect of cortisol on the regulation of troponin I (tni), troponin C (tnc) and myosin 7 (myh7) genes, and whether their regulation is carried out through the GR or MR pathways. For this purpose, in vivo assays were performed with juvenile rainbow trout, injecting intraperitoneally cortisol at physiological doses, and pharmacological inhibitors of GR (Mifepristone) and MR (Epleronone). In addition, to validate these results, skeletal muscle myotubes were cultured and GR and MR expression was evaluated. Additionally, GRE sites were identified in the promoter regions of each gene to determine if they could be direct targets of cortisol. The results obtained suggest that tnc expression is likely to be directly regulated by cortisol through the GR pathway under acute stress. Not so for the myh7 and tni genes, where, although it is believed that cortisol could regulate them due to in silico results from GRE sites, their regulations could be associated with an indirect regulation by cortisol. For the gr1 and mr genes, it was obtained that their expression was negatively regulated by cortisol administration, but then they return to their levels. This work contributes to the knowledge about the role of cortisol in the regulation of genes associated with muscle contraction and its potential use in aquaculture as potential markers of stress and growth among salmonids.
Notas
Memoria de título (Ingeniero en Biotecnología)
Esta memoria de título fue llevada a cabo en el Laboratorio de Biotecnología Molecular de la Facultad de Ciencias de la Vida en Universidad Andrés Bello, bajo la dirección del Dr. Juan Antonio Valdés y fue financiada a través de los proyectos FONDAP INCAR/15110027 and FONDECYT/1201498.
Esta memoria de título fue llevada a cabo en el Laboratorio de Biotecnología Molecular de la Facultad de Ciencias de la Vida en Universidad Andrés Bello, bajo la dirección del Dr. Juan Antonio Valdés y fue financiada a través de los proyectos FONDAP INCAR/15110027 and FONDECYT/1201498.
Palabras clave
Trucha Arcoiris, Genética, Cortisol