Complex Yet Coordinated: Regulation of Transcriptional Factors and Cell Signaling Pathways to Endure Anoxia in Rana sylvatica

Abstract

Wood frogs (Rana sylvatica) are a well-studied vertebrate model of natural freeze tolerance, surviving several months of winter subzero temperatures with 65-70% of total body water frozen as extracellular ice. Freezing halts blood circulation, heartbeat and breathing, restricting oxygen availability throughout the body and requiring a switch to anaerobic glycolysis for energy production, with its much lower ATP yield. To survive, wood frogs suppress their metabolic rate by about 90% to match ATP availability from glycolysis alone. Multiple cellular processes are regulated and suppressed, sustaining only pro-survival pathways until thawing occurs. Episodes of anoxia/reoxygenation also elevate reactive oxygen species (ROS) production that can surpass the antioxidant capacity of cells causing oxidative stress and tissue damage. This thesis examined a network of stress-responsive transcription factors (NRF2, OCT1, OCT4, YAP/TEAD, and RBPJ) and their associated pathways to determine their response and regulation over the anoxia/reoxygenation cycle. Decreased binding of transcriptional complexes to the promoter regions of target genes indicated a global reduction in transcription/translation processes. The data show also "functional switching" of OCT1, OCT4, and MAML while selectively upregulating antioxidants in a stress/organ specific manner. The present studies also shed new light on tissue repair mechanisms by demonstrating upregulation of selected pathway proteins. An increase in AHCY levels in liver also suggests maintenance of redox control, and elevated JMJD2C, TAZ, and MAML in skeletal and cardiac muscles indicates a potential increase in the expression of MyoD for muscle regeneration. Overall, the findings of this thesis document a complex yet coordinated network of transcriptional factors that support metabolic rate depression during freezing, combat oxidative stress, and initiate tissue repair mechanisms to endure prolonged anoxia and maintain cellular homeostasis in frozen wood frogs.