Lertwanakarn Tuchakorn, Suntravat Montamas, Sanchez Elda E, Boonhoh Worakan, Solaro R John, Wolska Beata M, Martin Jody L, de Tombe Pieter P, Tachampa Kittipong
Department of Physiology, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand.
National Natural Toxins Research Center, Texas A&M University-Kingsville, Kingsville, TX, USA.
J Venom Anim Toxins Incl Trop Dis. 2020 Jul 17;26:e20200005. doi: 10.1590/1678-9199-JVATITD-2020-0005. eCollection 2020.
Beta-cardiotoxin (β-CTX), the three-finger toxin isolated from king cobra () 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.
β-CTX was isolated from the crude venom of the Thai king cobra using reverse-phased and cation exchange HPLC. 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.
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.
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.
β-心脏毒素（β-CTX）是从眼镜王蛇毒液中分离出的三指毒素，具有负性变时作用以及与β-1和β-2肾上腺素能受体的结合特性，表明其具有β受体阻滞剂活性，并已被提议作为一种新型β受体阻滞剂候选物。此前，β-CTX是通过快速蛋白质液相色谱法（FPLC）分离纯化的。在此，我们提出了一种纯化该毒素的替代方法。此外，我们测试了其对不同哺乳动物肌肉细胞类型的细胞毒性，并确定了其对分离的心肌细胞心脏功能的影响，以便深入了解该蛋白质的药理作用。
使用反相和阳离子交换高效液相色谱法（HPLC）从泰国眼镜王蛇的粗毒液中分离β-CTX。对小鼠成肌细胞（C2C12）、大鼠平滑肌细胞（A7r5）和大鼠心肌成肌细胞（H9c2）进行细胞活力MTT测定。在一系列β-CTX浓度下，记录分离的大鼠心肌细胞的细胞缩短和钙瞬变动力学。
从粗毒液中回收了纯化的β-CTX（0.53% w/w）。MTT测定显示，在浓度为9.41±1.14 μM时，β-CTX对A7r5细胞具有50%的细胞毒性（n = 3），但在高达114.09 μM时，对C2C12和H9c2细胞无细胞毒性。β-CTX以剂量依赖性方式抑制大鼠心肌细胞缩短的程度；半数最大抑制浓度为95.97±50.10 nM（n = 3）。此外，在β-CTX处理的心肌细胞中，细胞缩短和重新延长的速率降低，同时细胞内钙瞬变衰减延长，表明由于心脏钙稳态改变导致心脏收缩力下降。
我们提出了一种从眼镜王蛇毒液中纯化β-CTX的替代方法。我们揭示了该蛋白质对平滑肌的细胞毒性和对心脏收缩力的抑制作用。这些数据有助于促进未来源自β-CTX的药物制剂的开发。
