title: 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.
abstract: Acute muscle tissue damage, myonecrosis, is a typical consequence of envenomations by snakes of the family Viperidae. Catalytically-inactive Lys49 phospholipase A(2) 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 A(2) 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 Ca(2+) occurred after addition of Mt-II. Such elevation was associated with hypercontraction of myotubes and blebbing of plasma membrane. An increment in the Ca(2+) signal was observed in myotube nuclei. Elimination of extracellular Ca(2+) resulted in increased cytotoxicity upon incubation with Mt-II, suggesting a membrane-protective role for extracellular Ca(2+). Chelation of cytosolic Ca(2+) with BAPTA-AM did not modify the cytotoxic effect, probably due to the large increment induced by Mt-II in cytosolic Ca(2+) which overrides the chelating capacity of BAPTA-AM. It is concluded that Mt-II induces rapid and drastic plasma membrane lesion and a prominent Ca(2+) influx in myotubes. Extracellular Ca(2+) plays a dual role in this model: it protects the membrane from the cytolytic action of the toxin; at the same time, the Ca(2+) 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.
authors.affiliation: Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica.
authors.forename: Juan Carlos
authors.initials: JC
authors.lastname: Villalobos
authors.forename: Rodrigo
authors.initials: R
authors.lastname: Mora
authors.forename: Bruno
authors.initials: B
authors.lastname: Lomonte
authors.forename: José María
authors.initials: JM
authors.lastname: Gutiérrez
authors.forename: Yamileth
authors.initials: Y
authors.lastname: Angulo
mesh_terms: D000818:Animals; D017837:Bothrops / Q000502:physiology*; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D003435:Crotalid Venoms / Q000633:toxicity*; D003600:Cytosol / Q000378:metabolism; D054501:Group II Phospholipases A2 / Q000737:chemistry* / Q000633:toxicity*; D051379:Mice; D018485:Muscle Fibers, Skeletal / Q000187:drug effects*; D018482:Muscle, Skeletal / Q000166:cytology; D030162:Reptilian Proteins / Q000737:chemistry* / Q000633:toxicity*
chemical_list: D003435:Crotalid Venoms; D030162:Reptilian Proteins; D054501:Group II Phospholipases A2; C061056:myotoxin II, Bothrops asper; D002118:Calcium