The majority of snakebites in northeastern Argentina are caused by Bothrops alternatus (yarará grande) and Bothrops diporus (yarará chica), reptiles that belong to the Viperidae family. The specific treatment of these ophidian envenomations is serotherapy with antivenoms that ensures a rapid distribution of antibodies and controls the systemic alterations but not always the local damage in the bite site where traces of venom are capable to preclude a successful regenerative response.
In this work, we explore the characteristics of muscle tissue during the critical period after Bothrops alternatus or Bothrops diporus venom injection and their potential inhibitory effect on muscle differentiation using an in vitro study model.
Groups of CF-1 mice were injected intramuscularly in the right gastrocnemius with 50 µg of B. alternatus or B. diporus venoms. Control mice received PBS under identical conditions. Briefly, animals were sacrificed after 0, 1, 3, 24 and 168 hours, muscles were dissected out and placed in liquid nitrogen for pulverization and filtration through 0.22 µm membranes. Venom proteins present in these homogenates were quantified by the ELISA method and analyzed by Western Blotting. Myoblast cells (C2C12 cell line) were exposed for 24h to muscle homogenates and the less cytotoxic ones were used for myogenesis evaluation.
Results evidenced that the amount of both venoms in muscle homogenates decreased over time, with even traces of venom (5-13 µg/mL) being observed 168h after inoculations. No significant differences were detected between B. alternatus and B. diporus venom treatments. Identification by immunoblotting showed typical venom protein bands with molecular masses between 20 and 100 kDa for B. alternatus and 14 and 100 kDa for B. diporus whose intensities gradually decreased with time. An intense band of ~60 kDa, characteristic of metalloproteases, was mainly visualized even after 7 days of both treatments.
In addition, less cytotoxic muscle homogenates (above 85% of myoblast viability corresponding to 24 and 168 h incubation times) were used for myogenesis assay. Control showed mature myotubes formation after 72h but a complete lack of myoblast fusion occurred when myogenic cells were incubated with muscle homogenates from mice injected with bothropic venoms.
These preliminary findings suggest that a possible local treatment, complementary to serotherapy, could improve the prognosis of snakebite poisonings by accelerating muscle regeneration processes.