Frozen but alive: Molecular responses to autophagy, angiogenesis and energy metabolism in the stress-tolerant wood frog, Rana sylvatica

The freeze-tolerant wood frogs (Rana sylvatica) are incredible creatures that can tolerate the freezing of up to ~70% of their total body water during winter. Once frozen, these frogs are considered clinically dead, exhibiting no signs of breathing, heartbeat, muscle movement and nerve conductance; yet, they come back to life, unharmed, after a few hours of thawing. Freezing is associated with ischemia due to the freezing of the blood, with hyperglycemia due to the production of large quantities of glucose for cryoprotection, and with dehydration as water moves from inside the cell to the extracellular space to prevent intracellular freezing. Interestingly, wood frogs can tolerate all these stresses independently of freezing, thereby creating a multifactorial model for studying vertebrate freeze-tolerance. Oxygen availability is very low to non-existing during freezing, anoxia, and dehydration; therefore, wood frogs are hypothesized to reduce their overall metabolic rates to balance energy production with energy expenditure in a process called metabolic rate depression (MRD). Animals that undergo MRD reduce energy expensive or detrimental processes and allocate the limited energy available only to pro-survival responses. This thesis examined the effects of freezing and its associated stress on responses to autophagy, angiogenesis, select group of antioxidant enzymes, and energy metabolism. Molecular responses to autophagy demonstrate a significant reduction in autophagosome formation and lysosomal biogenesis in response to anoxia/reoxygenation and to a lesser degree in response to dehydration/rehydration in liver, whereas these two processes were significantly reduced under all conditions in skeletal muscle. Current results also indicate that angiogenesis is regulated in a temporal and stress-dependent manner, where wood frogs increase the expression of certain pro- and anti-angiogenic factors in anticipation of potential damage to capillaries or injury to tissues. Investigation into the role of ETS1 as a transcriptional activator and repressor demonstrated its potential involvement in promoting the expression of select antioxidant enzymes, while repressing the expression of certain nuclear-encoded mitochondrial proteins. Overall, findings in this thesis demonstrate the complexity of the mechanisms involved in controlling metabolic rate depression in adaptive responses in wood frogs.