Calcium imaging of muscle cells treated with snake myotoxins reveals toxin synergism and presence of acceptors

Abstract

Snake myotoxins have a great impact on human health worldwide. Most of them adopt a phospholipase A2 fold and occur in two forms which often co-exist in the same venom: the Asp49 toxins hydrolyse phospholipids, whilst Lys49 toxins are enzymatically inactive. To gain insights into their mechanism of action, muscle cells were exposed to Bothrops myotoxins, and cytosolic Ca(2+) and cytotoxicity were measured. In both myoblasts and myotubes, the myotoxins induced a rapid and transient rise in cytosolic [Ca(2+)], derived from intracellular stores, followed, only in myotubes, by a large Ca(2+) influx and extensive cell death. Myoblast viability was unaffected. Notably, in myotubes Asp49 and Lys49 myotoxins acted synergistically to increase the plasma membrane Ca(2+) permeability, inducing cell death. Therefore, these myotoxins may bind to acceptor(s) coupled to intracellular Ca(2+) mobilization in both myoblasts and myotubes. However, in myotubes only, the toxins alter plasma membrane permeability, leading to death.

Bothrops snake myotoxins induce a large efflux of ATP and potassium with spreading of cell damage and pain

“…This latter increase could originate from the myotoxin diffusing out from the site of injection and from ATP released by the myotubes directly hit by the myotoxin, or from both effects. Myoblasts close to the injection site remained blue, in agreement with previous findings that they are Mt-II insensitive (15,16); however, their cytosolic Ca 2+ did increase at later times ( Fig. 2 A and D, traces a-c).…”

“…We showed previously that the snake myotoxins, including the Mt-II myotoxin, caused cell death of myotubes in cultured C2C12 cells (15), and this was evident also in the present study, as shown in These results unravel a previously uncharacterized aspect of great significance in the action of B. asper Lys-49 Mt-II myotoxins, as the results show that this myotoxin may damage muscle cells at a distance from the injection site via an indirect action mediated by the ATP released from cells directly hit by the venom, i.e., those close to the tip of the snake fangs. This event can rapidly expand the anatomical dimensions of the direct myotoxin effect and, consequently, the ability of the snake to capture prey.…”

“…Both types of myotoxins cause a large influx of Ca 2+ in muscle cells, which triggers a cascade of events such as loss of mitochondrial function, widespread proteolysis, myofibrillar hypercontraction, and additional degenerative events that still await a detailed description (6,14). We recently reported that a Lys49 Bothrops myotoxin is more direct and rapid in its cytotoxic action than its Asp49 counterpart, although they both eventually cause cell death (15).…”

“…The action of Lys49 myotoxins has been extensively studied in various animal models, in isolated muscle preparations, and in cells in culture. The key role of the Ca 2+ entry from the extracellular medium into the cell, following a steep concentration gradient, is one of the best-characterized consequence of the rapid plasma membrane perturbation induced by these toxins (6,(15)(16)(17). However, no attention has been paid so far to the possible role of the efflux of cytosolic molecules that may act as alarm signals or amplifiers of the damage.…”

Antibothropic Action of Camellia sinensis Extract Against the Neuromuscular Blockade by Bothrops jararacussu Snake Venom and Its Main Toxin, Bothropstoxin-I

“…3D), showing a parallelism between neurotoxic and myotoxic effects of the myotoxin. Cintra-Francischinelli et al (2009) excluded the possibility that the inactive Lys49 toxins act by binding to a membrane channel, thus increasing its permeability to Ca2+. The authors have shown that the action of myotoxins from snake venoms on muscle cells begins with the activation of membrane acceptors coupled to intracellular Ca 2+ stores, which is rapidly followed by the toxin dependent alteration of membrane permeability to ions (and other molecules).…”