Pharmacological characterization of crotamine effects on mice hind limb paralysis employing both ex vivo and in vivo assays: Insights into the involvement of voltage-gated ion channels in the crotamine action on skeletal muscles

Abstract

The high medical importance of Crotalus snakes is unquestionable, as this genus is the second in frequency of ophidian accidents in many countries, including Brazil. With a relative less complex composition compared to other genera venoms, as those from the Bothrops genus, the Crotalus genus venom from South America is composed basically by the neurotoxin crotoxin (a phospholipase A2), the thrombin-like gyroxin (a serinoprotease), a very potent aggregating protein convulxin, and a myotoxic polypeptide named crotamine. Interestingly not all Crotalus snakes express crotamine, which was first described in early 50s due to its ability to immobilize animal hind limbs, contributing therefore to the physical immobilization of preys and representing an important advantage for the envenoming efficacy, and consequently, for the feeding and survival of these snakes in nature. Representing about 10–25% of the dry weight of the crude venom of crotamine-positive rattlesnakes, the polypeptide crotamine is also suggested to be of importance for antivenom therapy, although the contribution of this toxin to the main symptoms of envenoming process remains far unknown until now. Herein, we concomitantly performed in vitro and in vivo assays to show for the first time the dose-dependent response of crotamine-triggered hind limbs paralysis syndrome, up to now believed to be observable only at high (sub-lethal) concentrations of crotamine. In addition, ex vivo assay performed with isolated skeletal muscles allowed us to suggest here that compounds active on voltage-sensitive sodium and/or potassium ion channels could both affect the positive inotropic effect elicited by crotamine in isolated diaphragm, besides also affecting the hind limbs paralysis syndrome imposed by crotamine in vivo. By identifying the potential molecular targets of this toxin, our data may contribute to open new roads for translational studies aiming to improve the snakebite envenoming treatment in human. Interestingly, we also demonstrate that the intraplantal or intraperitoneal (ip) injections of crotamine in mice do not promote pain. Therefore, this work may also suggest the profitable utility of non-toxic analogs of crotamine as a potential tool for targeting voltage-gated ion channels in skeletal muscles, aiming its potential use in the therapy of neuromuscular dysfunctions and envenoming therapy.

Effects of the Natural Peptide Crotamine from a South American Rattlesnake on Candida auris, an Emergent Multidrug Antifungal Resistant Human Pathogen

“…Defensins are members of a class of multifaceted AMPs that are efficient in killing most microbes, and for which the development of resistance is rare, mainly due to their characteristic mechanism(s) of action based on the interaction of defensins with the lipid bilayer of cell membranes [16,17,18], as also demonstrated by us for crotamine [19]. Interestingly, recent reports have identified human beta defensins as a new type of potassium ion channel inhibitors [20,21], as it was also proposed for crotamine [11,22,23], confirming the striking degree of structural and phylogenetic congruence with functional reciprocity between these antimicrobial polypeptides. The perspective of using host defense AMPs for treating infections caused by bacteria, viruses, or fungi is seriously considered lately by many, mainly due to the emergence of drug-resistance mechanisms, which threaten the efficacy of all current antimicrobial agents [24].…”

“…The native polypeptide crotamine from the venom of the South American rattlesnake Crotalus durissus terrificus was originally isolated in the late 1950s [9,10]. However, its real contribution to the envenoming process remains largely unknown, in contrast to the well-exploited molecular pathways underlying its assigned roles in skeletal muscles [11] and as a theranostic agent against cancer [12,13]. The peculiar (three-dimensional) structural similarity of crotamine with the human antimicrobial peptide (AMP) beta defensins [14] stimulated us to study and demonstrate the candicidal effect of crotamine against non-resistant reference strains and against the treatment-resistant clinical isolates, and more importantly, with no hemolytic activity [15].…”

Crotalus Durissus Ruruima: Current Knowledge on Natural History, Medical Importance, and Clinical Toxinology

“…Although C. d. ruruima venom can present crotamine in their venom cocktail, its abundance is very low (1.5%) as shown in preliminary results ( 5 ) when compared to C. d. collilineatus venom (20.8%) ( 44 ). Crotamine has an evident myotoxic action during envenomings, which was demonstrated through increased levels of creatine phosphokinase (CK) in vivo ( 53 , 58 , 95 ), as well as neuromuscular blocking effects ( 96 ) and hemostasis modulation ( 79 , 97 ).…”

“…Hypocalcemia may occur in patients with intense rhabdomyolysis after C. d. terrificus bites ( 116 ). Both crotoxin and crotamine are the major myotoxic components within C. durissus venoms ( 96 , 127 ). Moreover, crotoxin exhibits a capacity to spread to distant muscles and induce rhabdomyolysis, due to its systemic distribution ability mediated by the heterodimeric complex formation of the toxin to bind to specific cell targets ( 128 ).…”

“…Neurological impairment observed in C. durissus envenomings is associated with the action of neurotoxins, which are found to act in both peripheral and central nervous systems ( 96 , 144 , 145 ). In the clinical description from the Extra-Amazonian region, neuroparalytic signs, especially the neurotoxic facies/palpebral ptosis, are the most frequent signs of Crotalus envenomings.…”