Early response of C2C12 myotubes to a sub-cytotoxic dose of hemorrhagic metalloproteinase HF3 from Bothrops jararaca venom

Manifestations of local tissue damage, such as hemorrhage and myonecrosis, are among the most dramatic effects of envenomation by viperid snakes. Snake venom metalloproteinases (SVMPs) of the P-III class are main players of the hemorrhagic effect due to their activities in promoting blood vessel disruption. Hemorrhagic Factor 3 (HF3), a P-III class SVMP from Bothrops jararaca, shows a minimum hemorrhagic dose of 240 fmol on rabbit skin. The aim of this study was to assess the effects of a sub-cytotoxic dose of HF3 (50 nM) on the proteomic profile of C2C12 differentiated cells (myotubes) in culture, and on the peptidomic profile of the culture supernatant. Quantitative proteomic analysis using stable-isotope dimethyl labeling showed differential abundance of various proteins including enzymes involved in oxidative stress and inflammation responses. Identification of peptides in the supernatant of HF3-treated myotubes revealed proteolysis and pointed out potential new substrates of HF3, including glyceraldehyde-3-phosphate dehydrogenase, and some damage-associated molecular patterns (DAMPs). These experiments demonstrate the subtle effects of HF3 on muscle cells and illustrate for the first time the early proteolytic events triggered by HF3 on myotubes. Moreover, they may contribute to future studies aimed at explaining the inflammation process, hemorrhage and myonecrosis caused by SVMPs.

蝰科蛇类咬伤中毒最具戏剧性的效应之一，便是局部组织损伤的表现，例如出血与肌坏死。P-III类蛇毒金属蛋白酶（snake venom metalloproteinases, SVMPs）是引发出血效应的主要致病因子，因其可促进血管壁破坏。源自贾拉拉蝰（Bothrops jararaca）的P-III类蛇毒金属蛋白酶出血因子3（Hemorrhagic Factor 3, HF3），在兔皮肤模型中的最小出血剂量为240飞摩尔（fmol）。本研究旨在评估亚细胞毒性剂量的HF3（50 nM）对体外培养的C2C12分化细胞（肌管）的蛋白质组谱，以及其细胞培养上清液的肽组谱的影响。采用稳定同位素二甲基标记法开展的定量蛋白质组学分析显示，多种蛋白的丰度出现显著差异，其中包括参与氧化应激与炎症应答的酶类。对HF3处理组肌管的培养上清液中的肽段进行鉴定后，不仅发现了蛋白水解现象，还筛选出HF3的潜在新型底物，包括甘油醛-3-磷酸脱氢酶，以及部分损伤相关分子模式（damage-associated molecular patterns, DAMPs）。本实验证实了HF3对肌细胞的细微调控效应，并首次阐明了HF3触发肌管产生的早期蛋白水解事件。此外，本研究可为后续阐明蛇毒金属蛋白酶引发的炎症过程、出血与肌坏死的相关机制研究提供参考依据。