Anti-apoptotic response during anoxia and recovery in a freeze-tolerant wood frog (Rana sylvatica)

Abstract

The common wood frog, Rana sylvatica, utilizes freeze tolerance as a means of winter survival. Concealed beneath a layer of leaf litter and blanketed by snow, these frogs withstand subzero temperatures by allowing approximately 65–70% of total body water to freeze. Freezing is generally considered to be an ischemic event in which the blood oxygen supply is impeded and may lead to low levels of ATP production and exposure to oxidative stress. Therefore, it is as important to selectively upregulate cytoprotective mechanisms such as the heat shock protein (HSP) response and expression of antioxidants as it is to shut down majority of ATP consuming processes in the cell. The objective of this study was to investigate another probable cytoprotective mechanism, anti-apoptosis during oxygen deprivation and recovery in the anoxia tolerant wood frog. In particular, relative protein expression levels of two important apoptotic regulator proteins, Bax and p-p53 (S46), and five anti-apoptotic/pro-survival proteins, Bcl-2, p-Bcl-2 (S70), Bcl-xL, x-IAP, and c-IAP in response to normoxic, 24 Hr anoxic exposure, and 4 Hr recovery stages were assessed in the liver and skeletal muscle using western immunoblotting. The results suggest a tissue-specific regulation of the anti-apoptotic pathway in the wood frog, where both liver and skeletal muscle shows an overall decrease in apoptosis and an increase in cell survival. This type of cytoprotective mechanism could be aimed at preserving the existing cellular components during long-term anoxia and oxygen recovery phases in the wood frog.

Effects of anoxic exposure on the nuclear factor of activated T cell (NFAT) transcription factors in the stress‐tolerant wood frog

“…Previous studies have shown that superoxide dismutase, catalase, glutathione S‐transferase, glutathione reductase, and total Se‐dependent glutathione peroxidase are significantly upregulated under anoxia in different tissues of various anoxia‐tolerant organisms . Under anoxia, wood frogs also selectively downregulate proapoptotic proteins while significantly upregulating proteins that are associated with cell survival . Altogether, these data show that even during periods of extreme hypometabolism and limited oxygen availability, these organisms can selectively and strategically upregulate specific genes and signalling pathways that mediate survival.…”

“…We have previously shown that wood frogs can tolerate up to 48 hours of anoxia exposure at 5°C with no mortality . In addition, previous studies have investigated the effects of exposure to 24‐hour anoxia and 4 hours of aerobic recovery on metabolic rate depression in different wood frog tissues . As such, the current study uses the same treatment conditions to build on previous studies that focus on understanding the regulation of metabolic rate depression over the anoxia/reoxygenation cycle.…”

A potential antiapoptotic regulation: The interaction of heat shock protein 70 and apoptosis‐inducing factor mitochondrial 1 during heat stress and aestivation in sea cucumber

“…Therefore, both energy conservation and cytoprotective strategies need to be coordinated to ensure long‐term survival in sea cucumbers as they endure several months of continuous aestivation (Li et al., ). Mitigation of apoptosis has been reported as an important cytoprotective strategy to help animals preserve cellular integrity during long‐term hypometabolism like hibernating mammals and during prolonged winter freezing in freeze‐tolerant wood frogs, Rana sylvatica (Gerber et al., ; Rouble et al., ). Currently, we focused on AIFM1, a mitochondrial intermembrane protein that has been shown to translocate from the mitochondria to the cytosol and nucleus under stress conditions to stimulate apoptosis using a caspase‐independent mechanism.…”

“…Once aestivation is complete, the intestine regenerates into a functional organ in just a few days. It has been reported that there are many similarities in the mechanisms used to avoid apoptosis among vertebrates that undergo seasonal or stress‐responsive dormancy (Gerber, Wijenayake, & Storey, ; Rouble, Hefler, Mamady, Storey, & Tessier, ). Therefore, we wondered if such conserved regulatory mechanisms also exist in marine invertebrates.…”

Osmolyte regulation by TonEBP/NFAT5 during anoxia-recovery and dehydration–rehydration stresses in the freeze-tolerant wood frog (Rana sylvatica)

“…In addition, urea concentrations increase as this osmolyte is known to accumulate under dehydration stress in amphibians and it helps to slow the loss of body fluids that would otherwise lead to injury/death caused by reduced blood volume and viscosity (Costanzo & Lee, 2005; Costanzo & Lee, 2008). Adaptations to stresses including dehydration and anoxia involve profound biochemical and molecular changes at the transcriptional, post-transcriptional, translational, and post-translational levels (Roufayel, Biggar & Storey, 2011; Sullivan, Biggar & Storey, 2014; Gerber, Wijenayake & Storey, 2016). Although much has recently been discovered about how frogs adapt to stress on a molecular level, there is much that remains undiscovered, especially in the wood frog.…”