Cytotoxicity induced in myotubes by a Lys49 phospholipase A 2 homologue from the venom of the snake Bothrops asper: Evidence of rapid plasma membrane damage and a dual role for extracellular calcium Juan Carlos Villalobos a , Rodrigo Mora a,b , Bruno Lomonte a , Jose ´ Marı ´a Gutie ´rrez a , Yamileth Angulo a,c, * a Instituto Clodomiro Picado, Facultad de Microbiologı ´a, Universidad de Costa Rica, San Jose ´, Costa Rica b Departamento de Microbiologı ´a e Inmunologı ´a, Facultad de Microbiologı ´a, Universidad de Costa Rica, San Jose ´, Costa Rica c Departamento de Bioquı ´mica, Escuela de Medicina, Universidad de Costa Rica, San Jose ´, Costa Rica Received 13 March 2007; accepted 4 April 2007 Available online 29 April 2007 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 phos- pholipase A 2 homologue. Mt-II induced a dose- and time-dependent cytotoxic effect associated with plasma membrane disruption, evi- denced 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. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Myotoxic PLA 2 ; Snake venom; Ca 2+ homeostasis; Cytotoxicity; Mitochondria 1. Introduction Muscle tissue degeneration occurs in many pathologic phenomena, including dystrophies, mechanical and cold- induced damage, various infections, and intoxications by synthetic or natural agents (Carpenter and Karpati, 2001). Envenomations by snakes often concur with prominent local and systemic muscle degeneration, owing to either the direct action of myotoxins or to the ischemia that occurs in muscle tissue as a consequence of venom-induced micro- vascular alterations (Gutie ´rrez and Rucavado, 2000; Gutie ´rrez and Ownby, 2003). The most abundant myo- toxic components in snake venoms correspond to 0887-2333/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2007.04.010 * Corresponding author. Address: Instituto Clodomiro Picado, Facultad de Microbiologı ´a, Universidad de Costa Rica, San Jose ´, Costa Rica. Tel.: +506 2293135; fax: +506 2920485. E-mail address: yangulo@icp.ucr.ac.cr (Y. Angulo). www.elsevier.com/locate/toxinvit Available online at www.sciencedirect.com Toxicology in Vitro 21 (2007) 1382–1389