Cytotoxicity of snake venom Lys49 PLA2-like myotoxin on rat cardiomyocytes ex vivo does not involve a direct action on the contractile apparatus

Alfredo Jesús López-Dávila,Natalie Weber,Faramarz Matinmehr,Julián Fernández,Mariela Arias-Hidalgo,Theresia Kraft,Bruno Lomonte,José María Gutiérrez

doi:10.1038/s41598-021-98594-5

Alfredo Jesús López-Dávila, Natalie Weber + Show 6 more

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https://doi.org/10.1038/s41598-021-98594-5

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Abstract

Viperid snake venoms contain a unique family of cytotoxic proteins, the Lys49 PLA2 homologs, which are devoid of enzymatic activity but disrupt the integrity of cell membranes. They are known to induce skeletal muscle damage and are therefore named ‘myotoxins’. Single intact and skinned (devoid of membranes and cytoplasm but with intact sarcomeric proteins) rat cardiomyocytes were used to analyze the cytotoxic action of a myotoxin, from the venom of Bothrops asper. The toxin induced rapid hypercontraction of intact cardiomyocytes, associated with an increase in the cytosolic concentration of calcium and with cell membrane disruption. Hypercontraction of intact cardiomyocytes was abrogated by the myosin inhibitor para-aminoblebbistatin (AmBleb). No toxin-induced changes of key parameters of force development were observed in skinned cardiomyocytes. Thus, although myosin is a key effector of the observed hypercontraction, a direct effect of the toxin on the sarcomeric proteins -including the actomyosin complex- is not part of the mechanism of cytotoxicity. Owing to the sensitivity of intact cardiomyocytes to the cytotoxic action of myotoxin, this ex vivo model is a valuable tool to explore in further detail the mechanism of action of this group of snake venom toxins.

Highlights

IntroductionAbbreviations AmBleb Para-aminoblebbistatin PLA2 Phospholipase A2 Lys49 PLA2 PLA2 homologs characterized by having a Lys residue in substitution for the canonical Asp at position 49 Mt-II Lys49 PLA2 homolog Myotoxin II kTR The rate constant of isometric force redevelopment after a short period of isotonic shortening pCa − LOG10[Ca2+] (see Hill equation in the methods section) nH Hill coefficient (see Hill equation in the methods section)
Abbreviations AmBleb Para-aminoblebbistatin PLA2 Phospholipase A2 Lys49 PLA2 PLA2 homologs characterized by having a Lys residue in substitution for the canonical Asp at position 49 Mt-II Lys49 PLA2 homolog Myotoxin II kTR The rate constant of isometric force redevelopment after a short period of isotonic shortening pCa − LOG10[Ca2+] nH Hill coefficient
Our findings demonstrate the cytotoxic action of a Lys[49] P LA2 homolog on rat cardiomyocytes and provide evidence for a rapid disruption of plasma membrane integrity, associated with a prominent increment in cytosolic calcium and hypercontraction

Summary

Introduction

Abbreviations AmBleb Para-aminoblebbistatin PLA2 Phospholipase A2 Lys49 PLA2 PLA2 homologs characterized by having a Lys residue in substitution for the canonical Asp at position 49 Mt-II Lys49 PLA2 homolog Myotoxin II kTR The rate constant of isometric force redevelopment after a short period of isotonic shortening pCa − LOG10[Ca2+] (see Hill equation in the methods section) nH Hill coefficient (see Hill equation in the methods section). Studies on the mechanism of action of Lys49 PLA2 homologs have revealed their ability to interact with and disrupt natural and artificial m embranes[13,14,15,16,17,18]. Such plasma membrane disruption leads to a prominent calcium influx, following an electrochemical gradient across the plasma m embrane[17,19]. It is of interest to analyze the action of Lys[49] toxins on cardiomyocytes, in order to identify whether they are susceptible to their actions, and to understand the underlying mechanisms of toxicity

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