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，... More
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.
