Cytotoxicity induced in myotubes by a Lys49 phospholipase A2 homologue from the venom of the snake Bothrops asper: Evidence of rapid plasma membrane damage and a dual role for extracellular calcium

Acute muscle tissue damage, myonecrosis, is a typical consequence of envenomations by snakes of the family Viperidae. Catalytically-inactive Lys49 phospholipase A₂ homologues are abundant myotoxic components in viperid venoms, causing plasma membrane damage by a mechanism independent of phospholipid hydrolysis. However, the precise mode of action of these myotoxins remains unsolved. In this work, a cell culture model of C2C12 myotubes was used to assess the action of Bothrops asper myotoxin II (Mt-II), a Lys49 phospholipase A₂ homologue. Mt-II induced a dose- and time-dependent cytotoxic effect associated with plasma membrane disruption, evidenced by the release of the cytosolic enzyme lactate dehydrogenase and the penetration of propidium iodide. A rapid increment in cytosolic Ca²⁺ occurred after addition of Mt-II. Such elevation was associated with hypercontraction of myotubes and blebbing of plasma membrane. An increment in the Ca²⁺ signal was observed in myotube nuclei. Elimination of extracellular Ca²⁺ resulted in increased cytotoxicity upon incubation with Mt-II, suggesting a membrane-protective role for extracellular Ca²⁺. Chelation of cytosolic Ca²⁺ with BAPTA-AM did not modify the cytotoxic effect, probably due to the large increment induced by Mt-II in cytosolic Ca²⁺ which overrides the chelating capacity of BAPTA-AM. It is concluded that Mt-II induces rapid and drastic plasma membrane lesion and a prominent Ca²⁺ influx in myotubes. Extracellular Ca²⁺ plays a dual role in this model: it protects the membrane from the cytolytic action of the toxin; at the same time, the Ca²⁺ influx that occurs after membrane disruption is likely to play a key role in the intracellular degenerative events associated with Mt-II-induced myotube damage.