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Suppression of cardiomyocyte functions by β-CTX isolated from the Thai king cobra (Ophiophagus hannah) venom via an alternative method

BACKGROUND: Beta-cardiotoxin (β-CTX), the three-finger toxin isolated from king cobra (Ophiophagus hannah) venom, possesses β-blocker activity as indicated by its negative chronotropy and its binding property to both β-1 and β-2 adrenergic receptors and has been proposed as a novel β-blocker candida...

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Autores principales: Lertwanakarn, Tuchakorn, Suntravat, Montamas, Sanchez, Elda E., Boonhoh, Worakan, Solaro, R. John, Wolska, Beata M., Martin, Jody L., de Tombe, Pieter P., Tachampa, Kittipong
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Centro de Estudos de Venenos e Animais Peçonhentos 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7375408/
https://www.ncbi.nlm.nih.gov/pubmed/32742278
http://dx.doi.org/10.1590/1678-9199-JVATITD-2020-0005
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author Lertwanakarn, Tuchakorn
Suntravat, Montamas
Sanchez, Elda E.
Boonhoh, Worakan
Solaro, R. John
Wolska, Beata M.
Martin, Jody L.
de Tombe, Pieter P.
Tachampa, Kittipong
author_facet Lertwanakarn, Tuchakorn
Suntravat, Montamas
Sanchez, Elda E.
Boonhoh, Worakan
Solaro, R. John
Wolska, Beata M.
Martin, Jody L.
de Tombe, Pieter P.
Tachampa, Kittipong
author_sort Lertwanakarn, Tuchakorn
collection PubMed
description BACKGROUND: Beta-cardiotoxin (β-CTX), the three-finger toxin isolated from king cobra (Ophiophagus hannah) venom, possesses β-blocker activity as indicated by its negative chronotropy and its binding property to both β-1 and β-2 adrenergic receptors and has been proposed as a novel β-blocker candidate. Previously, β-CTX was isolated and purified by FPLC. Here, we present an alternative method to purify this toxin. In addition, we tested its cytotoxicity against different mammalian muscle cell types and determined the impact on cardiac function in isolated cardiac myocyte so as to provide insights into the pharmacological action of this protein. METHODS: β-CTX was isolated from the crude venom of the Thai king cobra using reverse-phased and cation exchange HPLC. In vitro cellular viability MTT assays were performed on mouse myoblast (C2C12), rat smooth muscle (A7r5), and rat cardiac myoblast (H9c2) cells. Cell shortening and calcium transient dynamics were recorded on isolated rat cardiac myocytes over a range of β-CTX concentration. RESULTS: Purified β-CTX was recovered from crude venom (0.53% w/w). MTT assays revealed 50% cytotoxicity on A7r5 cells at 9.41 ± 1.14 µM (n = 3), but no cytotoxicity on C2C12 and H9c2 cells up to 114.09 µM. β-CTX suppressed the extend of rat cardiac cell shortening in a dose-dependent manner; the half-maximal inhibition concentration was 95.97 ± 50.10 nM (n = 3). In addition, the rates of cell shortening and re-lengthening were decreased in β-CTX treated myocytes concomitant with a prolongation of the intracellular calcium transient decay, indicating depression of cardiac contractility secondary to altered cardiac calcium homeostasis. CONCLUSION: We present an alternative purification method for β-CTX from king cobra venom. We reveal cytotoxicity towards smooth muscle and depression of cardiac contractility by this protein. These data are useful to aid future development of pharmacological agents derived from β-CTX.
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spelling pubmed-73754082020-07-31 Suppression of cardiomyocyte functions by β-CTX isolated from the Thai king cobra (Ophiophagus hannah) venom via an alternative method Lertwanakarn, Tuchakorn Suntravat, Montamas Sanchez, Elda E. Boonhoh, Worakan Solaro, R. John Wolska, Beata M. Martin, Jody L. de Tombe, Pieter P. Tachampa, Kittipong J Venom Anim Toxins Incl Trop Dis Research BACKGROUND: Beta-cardiotoxin (β-CTX), the three-finger toxin isolated from king cobra (Ophiophagus hannah) venom, possesses β-blocker activity as indicated by its negative chronotropy and its binding property to both β-1 and β-2 adrenergic receptors and has been proposed as a novel β-blocker candidate. Previously, β-CTX was isolated and purified by FPLC. Here, we present an alternative method to purify this toxin. In addition, we tested its cytotoxicity against different mammalian muscle cell types and determined the impact on cardiac function in isolated cardiac myocyte so as to provide insights into the pharmacological action of this protein. METHODS: β-CTX was isolated from the crude venom of the Thai king cobra using reverse-phased and cation exchange HPLC. In vitro cellular viability MTT assays were performed on mouse myoblast (C2C12), rat smooth muscle (A7r5), and rat cardiac myoblast (H9c2) cells. Cell shortening and calcium transient dynamics were recorded on isolated rat cardiac myocytes over a range of β-CTX concentration. RESULTS: Purified β-CTX was recovered from crude venom (0.53% w/w). MTT assays revealed 50% cytotoxicity on A7r5 cells at 9.41 ± 1.14 µM (n = 3), but no cytotoxicity on C2C12 and H9c2 cells up to 114.09 µM. β-CTX suppressed the extend of rat cardiac cell shortening in a dose-dependent manner; the half-maximal inhibition concentration was 95.97 ± 50.10 nM (n = 3). In addition, the rates of cell shortening and re-lengthening were decreased in β-CTX treated myocytes concomitant with a prolongation of the intracellular calcium transient decay, indicating depression of cardiac contractility secondary to altered cardiac calcium homeostasis. CONCLUSION: We present an alternative purification method for β-CTX from king cobra venom. We reveal cytotoxicity towards smooth muscle and depression of cardiac contractility by this protein. These data are useful to aid future development of pharmacological agents derived from β-CTX. Centro de Estudos de Venenos e Animais Peçonhentos 2020-07-17 /pmc/articles/PMC7375408/ /pubmed/32742278 http://dx.doi.org/10.1590/1678-9199-JVATITD-2020-0005 Text en This article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Research
Lertwanakarn, Tuchakorn
Suntravat, Montamas
Sanchez, Elda E.
Boonhoh, Worakan
Solaro, R. John
Wolska, Beata M.
Martin, Jody L.
de Tombe, Pieter P.
Tachampa, Kittipong
Suppression of cardiomyocyte functions by β-CTX isolated from the Thai king cobra (Ophiophagus hannah) venom via an alternative method
title Suppression of cardiomyocyte functions by β-CTX isolated from the Thai king cobra (Ophiophagus hannah) venom via an alternative method
title_full Suppression of cardiomyocyte functions by β-CTX isolated from the Thai king cobra (Ophiophagus hannah) venom via an alternative method
title_fullStr Suppression of cardiomyocyte functions by β-CTX isolated from the Thai king cobra (Ophiophagus hannah) venom via an alternative method
title_full_unstemmed Suppression of cardiomyocyte functions by β-CTX isolated from the Thai king cobra (Ophiophagus hannah) venom via an alternative method
title_short Suppression of cardiomyocyte functions by β-CTX isolated from the Thai king cobra (Ophiophagus hannah) venom via an alternative method
title_sort suppression of cardiomyocyte functions by β-ctx isolated from the thai king cobra (ophiophagus hannah) venom via an alternative method
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7375408/
https://www.ncbi.nlm.nih.gov/pubmed/32742278
http://dx.doi.org/10.1590/1678-9199-JVATITD-2020-0005
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