ABSTRACT
Beta-cardiotoxin (ß-CTX), the three-finger toxin isolated from king cobra (Ophiophagus hannah) venom, possesses ß-blocker activity as indicated by its negative chronotropy and its binding property to both ß-1 and ß-2 adrenergic receptors and has been proposed as a novel ß-blocker candidate. Previously, ß-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: ß-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 ß-CTX concentration. Results: Purified ß-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. ß-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 ß-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 ß-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 ß-CTX.(AU)
Subject(s)
Animals , Charybdotoxin/isolation & purification , Myocytes, Cardiac , Cobra Cardiotoxin Proteins , Elapid Venoms , Cardiotoxins , Ophiophagus hannah , Suppression , Cytotoxicity, ImmunologicABSTRACT
Background: Russell’s viper venom-factor X activator (RVV-X) is a major procoagulant in Russell’s viper venom, and is composed of a heavy chain (RVV-XH) and two light chains (RVV-XL). It directly activates factor X in the final common coagulation pathway, which leads to rapid formation of blood clots. Objective: Produce rabbit anti-recombinant protein antibodies and identify their cross-reactivity with two viperine snake venoms. Methods: cDNA clones encoding RVV-XH and one of the light chains (RVV-XL; LC1) were recombinantly expressed in E. coli BL21 and used as antigens for rabbit immunization. The cross-reactivity of these anti-recombinant protein antibodies with two viperine snake venoms was determined using Western blot analysis. Results: rRVV-XH was more immunogenic than rRVV-XL. Rabbit anti-rRVV-XH and rRVV-XL IgG antibodies bind specifically to RVV-X, but they do not neutralize purified RVV-X. In addition, rabbit anti-rRVV-XH IgG antibody also bind to an 18-kDa protein in C. rhodostoma venom, and many proteins in C. albolabris venom. Rabbit antirRVV- XL IgG antibody recognized protein bands of crude venoms of C. rhodostoma and C. albolabris at about 25-kDa and 23-kDa, respectively. Conclusion: Rabbit anti-rRVV-XH and rRVV-XL IgG antibodies cross-reacted with molecules in other viperine venoms, which could have molecules with similar antigenic determinants. These antibodies could be useful to purify snake venom molecules by affinity chromatography as the first step in purification of factor X activator and other cross reacting molecules.