Comparative study of the cytolytic activity of myotoxic phospholipases A(2) On mouse endothelial (tEnd) and skeletal muscle (C2C12) cells in vitro

A rapid in vitro cytolytic effect of some myotoxic phospholipases A(2) (PLA (2)s) isolated from the venoms of Viperidae snakes has been previously desc ribed. This study was undertaken to investigate if cytolytic activity is a common property of the myotoxic proteins from this group. Murine endothelia l cells (tEnd) and skeletal muscle myotubes (C2Cl2) were utilized as target s. The release of lactic dehydrogenase was quantified as a measure of cell damage, 3h after exposure of cells to the different PLA(2)s, including repr esentatives from the genera Bothrops, Agkistrodon, Trimeresurus, Crotalus ( family Viperidae), and Notechis (family Elapidae). All of the group II myot oxic PLA(2)s tested displayed rapid cytolytic activity when tested in the m icromolar range of concentrations (8-32 mu M). In contrast, the group I myo toxic PLA(2) notexin was devoid of this activity. Aspartate-49 and lysine-4 9 PLA(2) group II variants showed a comparable cytolytic effect. Skeletal m uscle myotubes, obtained after fusion and differentiation of C2Cl2 myoblast s were significantly more susceptible to the cytolytic action of myotoxins than endothelial cells, previously reported to be more susceptible than und ifferentiated myoblasts under the same assay conditions. Cytolytic activity appears to be a common characteristic of group II myotoxic PLA(2)s of the Viperidae. Bee venom PLA(2), a group III enzyme of known myotoxicity, also displayed cytotoxic activity on C2Cl2 myotubes, being devoid of activity on endothelial cells. These results suggest that bl vitro differentiated skel etal muscle myotubes may represent a suitable model target for the study of myotoxic PLA(2)s of the structural group II found in snake venoms. (C) 199 8 Elsevier Science Ltd. All rights reserved.