Envenomation by the Venezuelan bushmaster snake (Lachesis muta muta) (Serpentes: Viperidae) is characterized by local and cardiac alterations. This study investigates the in vivo cardiac dysfunction, tissue destruction, and cellular processes triggered by Lachesis muta muta snake crude venom and a C-type lectin (CTL)-like toxin named Mutacytin-1 (MC-1). The 28 kDa MC-1 was obtained by molecular exclusion, ion exchange, and C-18 (checking pureness) reverse-phase chromatographies. N-terminal sequencing of the first eight amino acids (NNCPQ LLM) revealed 100% identity with Mutina (CTL-like) isolated from Lachesis stenophrys, which is a Ca2+-dependent-type galactoside-binding lectin from Bothrops jararaca and CTL BpLec from Bothrops pauloensis. The cardiotoxicity in zebrafish of MC-1 was evaluated by means of specific phenotypic expressions and larvae behavior at 5, 15, 30, 40 and 60 min post-treatment. The L. muta muta venom and MC-1 also produced heart rate/rhythm alterations, circulation modifications, and the presence of thrombus and apoptotic phenomenon with pericardial damages. Acridine orange (100 μg/mL) was used to visualize apoptosis cellular process in control and treated whole embryos. The cardiotoxic alterations happened in more than 90% of all larvae under the action of L. muta muta venom and MC-1. The findings have demonstrated the potential cardiotoxicity by L. muta muta venom, suggesting the possibility of cardiovascular damages to patients after bushmaster envenoming.
Get full access to this article
View all access options for this article.
References
1.
ClemetsonKJ. Snaclecs (snake C-type lectins) that inhibit or activate platelets by binding to receptors. Toxicon, 2010; 56:1236–1246.
2.
ToyamaMH, CarneiroE, MarangoniS, AmaralMEC, VellosoLA, BoscheroAC. Isolation and characterization of a convulxin-like protein from Crotalus durissus collilineatus venom. J Protein, 2001; 20:585–591.
3.
KilpatrickDC. Animal lectins: a historical introduction and overview. Biochim Biophys Acta, 2002; 1572:187–197.
DrickamerK. Demonstration of carbohydrate-recognition activity in diverse proteins which share a common primary structure motif. Biochem Soc Trans, 1989; 17:13–15.
6.
DrickamerK. Evolution of Ca(2+)-dependent animal lectins. Prog Nucleic Acid Res Mol Biol, 1993; 45:207–232.
7.
FoxJW, SerranoSM. Structural considerations of the snake venom metalloproteinases, key members of the M12 reprolysin family of metalloproteinases. Toxicon, 2005; 45:969–985.
8.
PifanoF. Investigacion y docencia en medicina tropical. Arch Venez Med Trop Parasitol Med, 1961; 4:1–203.
9.
StoscheckCM. Quantitation of protein. Methods Enzymol, 1990; 182:50–68.
10.
LaemmliUK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 1970; 227:680–685.
11.
SchäggerH, von JagowG. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem, 1987; 166:368–379.
12.
FoulgerJH. The User of the Molisch (α-Naphthol) reactions in the study of sugars in biological fluids. J Biol Chem, 1931; 92:345–353.
13.
WesterfieldM: The Zebrafish Book, 5th Edition; A Guide for the Laboratory Use of Zebrafish (Danio rerio). University of Oregon Press, Eugene OR, 2007.
14.
D'amicoLS, SengWl, YangY, SuterW: Assessment of drug-induced cardiotoxicity in zebrafish. In: Zebrafish: Methods for Assessing Drug Safety and Toxicity. McGrathP (eds), pp. 45–54, John Wiley & Sons, Inc., New Jersey, 2012.
15.
Aragon-OrtizR, MenteleA, AuerswaldEA. Amino acid sequence of a lectin-like protein from Lachesis muta stenophyrs venom. Toxicon, 1996; 34:763–769.
16.
OzekiY, MatsuiT, HamakoJ, SuzukiM, FujimuraY, YoshidaE, et al.C-type galactoside-binding lectin from Bothrops jararaca venom: comparison of its structure and function with those of botrocetin. Arch Biochem Biophys, 1994; 308:306–310.
17.
CastanheiraLE, NunesDC, CardosoT, Santos PdeS, GoulartLR, RodriguesRS, et al.Biochemical and functional characterization of a C-type lectin (BpLec) from Bothrops pauloensis snake Venom. Int J Biol Macromol, 2013; 54:57–64.
18.
Rodriguez-RauseoC, BlancoM, GonzalezL, GirónME, AguilarI, TorresJR, et al.A case of ophidic envenomation by cuaima (Lachesis muta muta) en Venezuela. Bol Malariol Saneam Amb, 1995; 35:30–33.
19.
KangY, KimJ, AndersonJP, WuJ, GleimSR, KunduRK, et al.Apelin-APJ signaling is a critical regulator of endothelial MEF2 activation in cardiovascular development. Circ Res, 2013; 113:22–31.
20.
ShielsHA, CalaghanSC, WhiteE. The cellular basis for enhanced volume-modulated cardiac output in fish hearts. J Gen Physiol, 2006; 128:37–44.
21.
MilanDJ, JonesIL, EllinorPT, MacRaeCA. In vivo recording of adult zebrafish electrocardiogram and assessment of drug-induced QT prolongation. Am J Physiol Heart Circ Physiol, 2006; 291:269–273.
22.
PieperhoffS, WilsonKS, BailyJ, de MoraK, MaqsoodS, VassS, et al.Heart on a plate: histological and functional assessment of isolated adult zebrafish hearts maintained in culture. PLoS ONE, 2014; 9:e96771.
23.
MoritaT. Structures and functions of snake venom CLPs (C-type lectin-like proteins) with anticoagulant-, procoagulant-, and platelet-modulating activities. Toxicon, 2005; 45:1099–1114.
24.
KiniRM. Are C-type lectin-related proteins derived by proteolysis of metalloproteinase/disintegrin precursor proteins?. Toxicon, 1996; 34:1287–1294.
25.
KumarTKS, JayaramanG, LeeCS, ArunkumarAI, SivaramanT, SamuelD, et al.Snake venom cardiotoxins -structure, dynamics, function and folding. J Biomol Struct Dyn, 1997; 15:431–463.
26.
KonshinaAG, DubovskiiPV, EfremovRG. Structure and dynamics of cardiotoxins. Curr Protein Pept Sci, 2012; 13:570–584.
27.
KonshinaAG, KrylovNA, EfremovRG. Cardiotoxins: functional role of local conformational changes. J Chem Inf Model, 2017; 57:2799–2810.
28.
MurakamiMT, ZelaSP, GavaLM, Michelan-DuarteS, CintraAC, ArniRK. Crystal structure of the platelet activator convulxin, a disulfide-linked α4β4 cyclic tetramer from the venom of Crotalus durissus terrificus. Biochem Biophys Res Commun, 2003; 310:478–482.
29.
PolgárJ, ClemetsonJM, KehrelBE, WiedemannM, MagnenatEM, WellsTN, et al.Platelet activation and signal transduction by convulxin, a C-type lectin from Crotalus durissus terrificus (tropical rattlesnake) venom via the p62/GPVI collagen receptor. J Biol Chem, 1997; 272:13576–13583.
30.
ParkerT, LibourelPA, HetheridgeMJ, CummingRI, SutcliffeTP, GoonesingheAC, et al.A multi-endpoint in vivo larval zebrafish (Danio rerio) model for the assessment of integrated cardiovascular function. J Pharm Toxicol Meth, 2014; 69:30–38.
31.
SchwerteT, FritscheR. Understanding cardiovascular physiology in zebrafish and Xenopus larvae: the use of microtechniques. Comp Biochem Physiol A Mol Integr Physiol, 2003; 135:131–145.
32.
DenvirMA, TuckerCS, MullinsJJ. Systolic and diastolic ventricular function in zebrafish embryos: influence of norepenephrine, MS-222 and temperature. BMC Biotechnol, 2008; 8:21.
RanaN, MoondM, MarthiA, BapatlaS, SarvepalliT, ChattiK, et al.Caffeine-induced effects on heart rate in zebrafish embryos and possible mechanisms of action: an effective system for experiments in chemical biology. Zebrafish, 2010; 7:69–81.
35.
RochaLL, PessoaCM, CorrêaTD, PereiraAJ, de AssunçãoMS, SilvaE. Current concepts on hemodynamic support and therapy in septic shock. Braz J Anesthesiol, 2015; 65:395–402.
36.
OrfanidouT, PapaefthimiouC, AntonopoulouE, LeonardosI, TheophilidisG. The force of the spontaneously contracting zebrafish heart, in the assessment of cardiovascular toxicity: application on adriamycin. Toxicol In Vitro, 2013; 27:1440–1444.
37.
GibbinsJM. Platelet adhesion signalling and the regulation of thrombus formation. J Cell Sci, 2004; 117:3415–3425.
38.
ColeL, RossL. Apoptosis in the developing zebrafish embryo. Dev Biol, 2001; 240:123–142.
39.
DanialNN, KorsmeyerSJ. Cell death: critical control points. Cell, 2004; 116:205–219.
40.
BurattiniS, FalcieriE. Analysis of cell death by electron microscopy. Methods Mol Biol, 2013; 1004:77–89.
41.
YamazakiY, MoritaT. Snake venom components affecting blood coagulation and the vascular system: structural similarities and marked diversity. Curr Pharm Des, 2007; 13:2872–2886.
42.
Al-AsmariAK, RiyasdeenA, Al-ShahraniMH. Snake venom causes apoptosis by increasing the reactive oxygen species in colorectal and breast cancer cell lines. Onco Targets Ther, 2016; 9:6485–6498.
43.
AlvarezM, Villanueva Á, AcedoP, CañeteM, StockertJC. Cell death causes relocalization of photosensitizing fluorescent probes. Acta Histochem, 2011; 113:363–368.
