S- and P-type cobra venom cardiotoxins differ in their action on isolated rat heart

Background: The cardiovascular system is one of the first systems to be affected by snake toxins; but not many toxins exert a direct effect on the heart. Cobra venom cardiotoxins are among those few toxins that attack the heart. Although the two cardiotoxin types (S and P) differ in their central-loop structure, it is not known whether they differ in their effect on the mammalian heart. We compared the effects of S- and P-type cardiotoxins, CTÐ¥-1 and CTÐ¥-2, respectively, from the cobra Naja oxiana, on the isolated rat heart. Methods: An isolated rat heart perfused according to the Langendorff technique was used in this study to investigate the activity of cardiotoxins CTX-1 and CTX-2. The following parameters were registered: the left ventricular developed pressure, calculated as the difference between systolic and diastolic pressure in the left ventricle, the end-diastolic pressure, the heart rate, time to maximal end-diastolic pressure (heart contracture), and time to depression of the heart contraction. Results: Both cardiotoxins at the concentration of 5 µg/mL initially produce a slight increase in systolic intraventricular pressure, followed by its rapid decrease with a simultaneous increase in diastolic intraventricular pressure until reaching contracture. CTX-2 blocks cardiac contractions faster than CTX-1; in its presence the maximum diastolic pressure is reached faster and the magnitude of the developed contracture is higher. Conclusion: The P-type cardiotoxin CTX-2 more strongly impairs rat heart functional activity than the S-type cardiotoxin CTX-1, as expressed in its faster blockage of cardiac contractions as well as in more rapid development and greater magnitude of contracture in its presence.(AU)

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

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)

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

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)

Biochemical and toxinological characterization of Naja sumatrana (Equatorial spitting cobra) venom

The lethal and enzymatic activities of venom from Naja sumatrana (Equatorial spitting cobra) were determined and compared to venoms from three other Southeast Asian cobras (Naja sputatrix, Naja siamensis and Naja kaouthia). All four venoms exhibited the common characteristic enzymatic activities of Asiatic cobra venoms: low protease, phosphodiesterase, alkaline phosphomonoesterase and L-amino acid oxidase activities, moderately high acetylcholinesterase and hyaluronidase activities and high phospholipase A2. Fractionation of N. sumatrana venom by Resource® S cation exchange chromatography (GE Healthcare, USA) yielded nine major protein peaks, with all except the acidic protein peak being lethal to mice. Most of the protein peaks exhibit enzymatic activities, and L-amino acid oxidase, alkaline phosphomonoesterase, acetylcholinesterase, 5'-nucleotidase and hyaluronidase exist in multiple forms. Comparison of the Resource® S chromatograms of the four cobra venoms clearly indicates that the protein composition of N. sumatrana venom is distinct from venoms of the other two spitting cobras, N. sputatrix (Javan spitting cobra) and N. siamensis (Indochinese spitting cobra). The results support the revised systematics of the Asiatic cobra based on multivariate analysis of morphological characters. The three spitting cobra venoms exhibit two common features: the presence of basic, potentially pharmacologically active phospholipases A2 and a high content of polypeptide cardiotoxin, suggesting that the pathophysiological actions of the three spitting cobra venoms may be similar.(AU)

Biochemical and toxinological characterization of Naja sumatrana (Equatorial spitting cobra) venom

The lethal and enzymatic activities of venom from Naja sumatrana (Equatorial spitting cobra) were determined and compared to venoms from three other Southeast Asian cobras (Naja sputatrix, Naja siamensis and Naja kaouthia). All four venoms exhibited the common characteristic enzymatic activities of Asiatic cobra venoms: low protease, phosphodiesterase, alkaline phosphomonoesterase and L-amino acid oxidase activities, moderately high acetylcholinesterase and hyaluronidase activities and high phospholipase A2. Fractionation of N. sumatrana venom by Resource® S cation exchange chromatography (GE Healthcare, USA) yielded nine major protein peaks, with all except the acidic protein peak being lethal to mice. Most of the protein peaks exhibit enzymatic activities, and L-amino acid oxidase, alkaline phosphomonoesterase, acetylcholinesterase, 5'-nucleotidase and hyaluronidase exist in multiple forms. Comparison of the Resource® S chromatograms of the four cobra venoms clearly indicates that the protein composition of N. sumatrana venom is distinct from venoms of the other two spitting cobras, N. sputatrix (Javan spitting cobra) and N. siamensis (Indochinese spitting cobra). The results support the revised systematics of the Asiatic cobra based on multivariate analysis of morphological characters. The three spitting cobra venoms exhibit two common features: the presence of basic, potentially pharmacologically active phospholipases A2 and a high content of polypeptide cardiotoxin, suggesting that the pathophysiological actions of the three spitting cobra venoms may be similar.(AU)