Aguilar Oscar A, Hadj-Moussa Hanane, Storey Kenneth B
Institute of Biochemistry and Department of Biology, Carleton University, 1125 Colonel By Drive, Ottawa, Ontario, Canada K1S 5B6.
Institute of Biochemistry and Department of Biology, Carleton University, 1125 Colonel By Drive, Ottawa, Ontario, Canada K1S 5B6.
J Therm Biol. 2017 Jul;67:1-8. doi: 10.1016/j.jtherbio.2017.04.007. Epub 2017 Apr 19.
The wood frog survives frigid North American winters by retreating into a state of suspended animation characterized by the freezing of up to 65% of total body water as extracellular ice and displaying no heartbeat, breathing, brain activity, or movement. Physiological and biochemical adaptations are in place to facilitate global metabolic depression and protect against the consequences of whole body freezing. This study examined the myocyte enhancer factor 2 (MEF2) transcription factor family, proteins responsible for coordinating selective gene expression of a myriad of cellular functions from muscle development and remodelling to various stress responses. Immunoblotting, subcellular localization, and RT-PCR were used to analyze the regulation of MEF2A and MEF2C transcription factors and selected downstream targets under their control at transcriptional, translational, and post-translational levels in skeletal and cardiac muscles from control, frozen and thawed frogs. Both MEF2A/C proteins were freeze-responsive in skeletal muscle, displaying increases of 1.7-2 fold for phosphorylated MEF2A and MEF2C during freezing with an enrichment of nuclear phosphorylated MEF2 proteins (by 1.7-2.1 fold) observed as early as 4h post-freezing. Despite the reduced response of total and phosphorylated MEF2A/C protein levels observed in cardiac muscle, the MEF2 downstream gene targets (glucose transporter-4, calreticulin, and creatine kinase brain and muscle isozymes) displayed similar increases in transcript levels (1.7-4.8 fold) after 24h freezing in both muscle types. This study describes a novel freeze-responsive function for MEF2 transcription factors and further elaborates our understanding of the molecular mechanisms underlying natural freeze tolerance. This novel freeze-responsive regulation suggests a role for MEF2s and downstream genes in cryoprotectant glucose distribution, calcium homeostasis, and maintenance of energy reserves vital for successful freeze tolerance.
林蛙通过进入假死状态来度过北美寒冷的冬天，这种状态的特征是高达65%的全身水分以细胞外冰的形式冻结，且没有心跳、呼吸、脑活动或运动。生理和生化适应机制有助于全面降低代谢，并防止全身冻结带来的后果。本研究考察了肌细胞增强因子2（MEF2）转录因子家族，这些蛋白质负责协调众多细胞功能的选择性基因表达，从肌肉发育和重塑到各种应激反应。免疫印迹、亚细胞定位和逆转录聚合酶链反应（RT-PCR）被用于分析在对照、冷冻和解冻青蛙的骨骼肌和心肌中，MEF2A和MEF2C转录因子及其控制下的选定下游靶点在转录、翻译和翻译后水平的调控情况。MEF2A/C蛋白在骨骼肌中对冷冻有反应，在冷冻过程中磷酸化的MEF2A和MEF2C增加了1.7至2倍，早在冷冻后4小时就观察到核磷酸化MEF2蛋白富集（增加1.7至2.1倍）。尽管在心肌中观察到总MEF2A/C蛋白水平和磷酸化MEF2A/C蛋白水平的反应有所降低，但在两种肌肉类型中，冷冻24小时后，MEF2下游基因靶点（葡萄糖转运蛋白4、钙网蛋白以及脑型和肌型肌酸激酶同工酶）的转录水平都有类似的增加（1.7至4.8倍）。本研究描述了MEF2转录因子一种新的冷冻反应功能，并进一步阐述了我们对自然抗冻能力潜在分子机制的理解。这种新的冷冻反应调控表明MEF2及其下游基因在冷冻保护剂葡萄糖分布、钙稳态以及维持对成功抗冻至关重要的能量储备方面发挥作用。
