Suppression of snake-venom cardiotoxin-induced cardiomyocyte degeneration by blockage of Ca2+ influx or inhibition of non-lysosomal proteinases

Abstract

The incubation of 10(5) single neonatal rat cardiomyocytes with 1 microM-cardiotoxin in a bath medium, Tyrode solution in the presence of 1 mM-Ca2+, at 37 degrees C evoked the following chain of events. Firstly, there appeared a latent period of about 10 min during which the cells behaved normally. Neither lactate dehydrogenase nor ATP leaked from the cells. Cytosolic free Ca2+ increased considerably, as measured by the fluorescence intensity of fura-2-Ca2+ complex. At the same time a large portion of endogenous ATP was depleted. Secondly, after the latent period, the cell beating became irregular and eventually stopped. Thirdly, blebs appeared on the cell surface, leading to cell degeneration. If, before the appearance of blebs, the cells were washed with the bath medium exhaustively or incubated in the presence of the toxin antibody, cytosolic free Ca2+ and endogenous ATP returned to normal levels and cells resumed regular beating. Preincubation of the cells with 3.75 microM-flunarizine or 3.75 microM-diltiazem (both are Ca2+ antagonists), or 1.5 microM-fura-2 acetoxymethyl ester (a chelate for Ca2+), or 200 microM-leupeptin or 50 microM-antipain (both are proteinase inhibitors) considerably suppressed the toxin's ability to degenerate the cells. On the other hand, lysosomal proteinase inhibitor, autophage inhibitor, serine proteinase inhibitor, phospholipase inhibitor and calmodulin antagonist did not inhibit the toxin's activity. The results suggest that the toxin may act on the extracellular surface of intact cardiomyocytes to increase cytosolic free Ca2+. The subsequent cell degeneration may result from the activation of a Ca2+-dependent non-lysosomal proteolytic system.

Amphiphilic β‐sheet cobra cardiotoxin targets mitochondria and disrupts its network

“…Although it has long been established that amphiphilic polypeptides of CTX A3 induces muscle contraction and systolic heart arrest, due to irreversible cell membrane depolarization, the long term effect of CTX action on muscle to cause tissue necrosis and perturbed wound healing process is less understood [21–23]. In fact, as shown in Fig.…”

“…The establishment of cobra α‐neurotoxin as a specific ligand to target acetylcholine receptor provides clear evidence to explain the neurotoxicity of cobra venom action. Recent demonstration of CTX as a pore forming polypeptide also justifies its role in causing irreversible membrane depolarization, cytosolic calcium homeostasis, muscle contraction and general cytotoxicity [3,5,10], although it is likely that many extracellular proteins and carbohydrate components may be also involved to impart the specificity observed in CTX actions on cardiomyocytes [21,29].…”

Cell Death Mechanisms and the Immune System

“…Secondly, calcium may activate calcium-dependent proteases acting on the cytoskeleton (Meilgren 1987) and on other essential cell structures. In line with this possibility, inhibitors of calcium-dependent proteases suppress or retard death due to chemical toxins in a number of cell types (Nicotera et al 1986b, Tzeng & Chen 1988, Mirabelli et al 1989. And thirdly, calcium may stimulate phospholipase Aj, which cleaves phospholipids, generating arachidonic acid and lysophospholipids (Chien et al 1977, Shier & DuBourdieu 1982, Glende & Pushpendran 1986, Nicotera et al 1986a.…”

Mechanisms of cell death

“…An increase in the cytosolic calcium concentration activates the calpains, which can degrade both cytoskeletal elements and integral membrane proteins implicated in plasma membrane blebbing (Kosower et al 1983;Mirabelli et al 1989). The toxicity of some compounds to the liver, myocardium and platelets can be prevented or delayed by inhibition of these calciumactivated proteases (Nicotera et al 1986a,b;Tzeng and Chen 1988;Mirabelli et al 1989). Although cytoskeletal proteins are a target for the calcium-activated proteases (Mirabelli et al 1989) their involvement in cell injury is still unclear.…”