Hoyeck Myriam P, Hadj-Moussa Hanane, Storey Kenneth B
Institute of Biochemistry, Departments of Biology and Chemistry, Carleton University, Ottawa, Canada.
PeerJ. 2017 Nov 9;5:e4014. doi: 10.7717/peerj.4014. eCollection 2017.
The wood frog () can endure freezing of up to 65% of total body water during winter. When frozen, wood frogs enter a dormant state characterized by a cessation of vital functions (i.e., no heartbeat, blood circulation, breathing, brain activity, or movement). Wood frogs utilize various behavioural and biochemical adaptations to survive extreme freezing and component anoxia and dehydration stresses, including a global suppression of metabolic functions and gene expression. The stress-responsive myocyte enhancer factor-2 (MEF2) transcription factor family regulates the selective expression of genes involved in glucose transport, protein quality control, and phosphagen homeostasis. This study examined the role of MEF2A and MEF2C proteins as well as select downstream targets (glucose transporter-4, calreticulin, and muscle and brain creatine kinase isozymes) in 40% dehydration and 24 h anoxia exposure at the transcriptional, translational, and post-translational levels using qRT-PCR, immunoblotting, and subcellular localization. transcript levels remained constant during dehydration and anoxia. Total, cytoplasmic, and nuclear MEF2A/C and phospho-MEF2A/C protein levels remained constant during dehydration, whereas a decrease in total MEF2C levels was observed during rehydration. Total and phospho-MEF2A levels remained constant during anoxia, whereas total MEF2C levels decreased during 24 h anoxia and P-MEF2C levels increased during 4 h anoxia. In contrast, cytoplasmic MEF2A levels and nuclear phospho-MEF2A/C levels were upregulated during anoxia. MEF2 downstream targets remained constant during dehydration and anoxia, with the exception of which was upregulated during anoxia. These results suggest that the upregulated MEF2 response reported in wood frogs during freezing may in part stem from their cellular responses to surviving prolonged anoxia, rather than dehydration, leading to an increase in GLUT4 expression which may have an important role during anoxia survival.
林蛙在冬季能够忍受高达全身65%的水分被冻结。当被冻结时，林蛙进入一种休眠状态，其特征是生命功能停止（即没有心跳、血液循环、呼吸、大脑活动或运动）。林蛙利用各种行为和生化适应机制来在极端冻结以及伴随的缺氧和脱水应激中存活，包括对代谢功能和基因表达的全面抑制。应激反应性的肌细胞增强因子2（MEF2）转录因子家族调节参与葡萄糖转运、蛋白质质量控制和磷酸肌酸稳态的基因的选择性表达。本研究使用qRT-PCR、免疫印迹和亚细胞定位，在转录、翻译和翻译后水平上研究了MEF2A和MEF2C蛋白以及选定的下游靶点（葡萄糖转运蛋白4、钙网蛋白以及肌肉和脑肌酸激酶同工酶）在40%脱水和24小时缺氧暴露中的作用。转录水平在脱水和缺氧期间保持恒定。在脱水期间，总MEF2A/C、细胞质和细胞核中的MEF2A/C以及磷酸化MEF2A/C蛋白水平保持恒定，而在复水期间观察到总MEF2C水平下降。在缺氧期间，总MEF2A和磷酸化MEF2A水平保持恒定，而在24小时缺氧期间总MEF/C水平下降，在4小时缺氧期间磷酸化MEF2C水平升高。相反，在缺氧期间细胞质MEF2A水平和细胞核磷酸化MEF2A/C水平上调。除了在缺氧期间上调的 外，MEF2下游靶点在脱水和缺氧期间保持恒定。这些结果表明，林蛙在冻结期间报告的上调的MEF2反应可能部分源于它们对在长期缺氧而非脱水状态下存活的细胞反应，导致葡萄糖转运蛋白4（GLUT4）表达增加，这在缺氧存活期间可能具有重要作用。
