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

Abstract

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.

Negative inotropic mechanisms of β-cardiotoxin in cardiomyocytes by depression of myofilament ATPase activity without activation of the classical β-adrenergic pathway

“…Interestingly, our findings revealed that β-CTX attenuated the stimulatory effect of ISO in most parameters. The deterioration effects of the myocyte functions, inotropy, and lusitropy by β-CTX were consistent with those found in the basal condition as in previous study 3 . However, the blunting effect of the peak CaT was found only in the ISO-stimulating condition.…”

“…Lyophilized Thai king cobra venom (TKCV) was purchased from Queen Saovabha Memorial Institute (QSMI), Bangkok, Thailand. β-CTX was isolated and purified from the TKCV by the sequential use of reverse phase following with cation exchange chromatography as previously described 3 . The presence of the compound was confirmed using SDS-PAGE and automated Edman’s degradation N-terminal sequencer (PPSQ™-33B, Shimadzu®).…”

“…Interestingly, our recent study demonstrated the calcium-independent negative inotropic effects of β-CTX in the isolated cardiomyocyte 3 (i.e., β-CTX reduced myocyte contractility without alteration in calcium transient at the basal state), indicating the possibility of involvement of a non-classical-β-adrenergic pathway by β-CTX. As a novel BBs candidate, the need of understanding molecular mechanisms is required to provide basic knowledge for the drug developmental process.…”

“…Comparing the homology, β-CTX shows a structural similarity of 35–58% with other cobra cardiotoxins (CTXs) 2 . Unlike CTXs, β-CTX possesses no cytotoxic effect in myoblast and cardiomyoblast due to its unique structural properties 3 . Interestingly, when injected β-CTX intraperitoneally into rodents, a negative chronotropic effect is exhibited without changing the cardiac contractility index 1 .…”

Cardiovascular Effects of Snake Toxins: Cardiotoxicity and Cardioprotection

“…This leads to a decrease in the heart rate in vivo and in vitro without noticeable cytotoxicity, which may be associated with the inability of β-cardiotoxin to directly interact with the membrane, due to some of its structural features [ 73 ]. Later, a cytotoxic effect on cultured smooth muscle cells and no effect on skeletal cells and cardiac myocytes were shown in [ 74 ]. Interestingly, the study revealed direct negative inotropic and lusitropic effects, with the intracellular calcium concentration in systole remaining unchanged.…”