Torpor-responsive microRNAs in the heart of the Monito del monte, Dromiciops gliroides.

The marsupial Monito del monte (Dromiciops gliroides) utilizes both daily and seasonal bouts of torpor to preserve energy and prolong survival during periods of cold and unpredictable food availability. Torpor involves changes in cellular metabolism, including specific changes to gene expression that is coordinated in part, by the posttranscriptional gene silencing activity of microRNAs (miRNA). Previously, differential miRNA expression has been identified in D. gliroides liver and skeletal muscle; however, miRNAs in the heart of Monito del monte remained unstudied. In this study, the expression of 82 miRNAs was assessed in the hearts of active and torpid D. gliroides, finding that 14 were significantly differentially expressed during torpor. These 14 miRNAs were then used in bioinformatic analyses to identify Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways that were predicted to be most affected by these differentially expressed miRNAs. Overexpressed miRNAs were predicted to primarily regulate glycosaminoglycan biosynthesis, along with various signaling pathways such as Phosphoinositide-3-kinase/protein kinase B and transforming growth factor-ß. Similarly, signaling pathways including phosphatidylinositol and Hippo were predicted to be regulated by the underexpression of miRNAs during torpor. Together, these results suggest potential molecular adaptations that protect against irreversible tissue damage and enable continued cardiac and vascular function despite hypothermia and limited organ perfusion during torpor.

Cardiac microRNA expression profile in response to estivation.

Couch's spadefoot toad (Scaphiopus couchii) spends most of the year underground in a hypometabolic state known as estivation. During this time, they overcome significant dehydration and lack of food through many mechanisms including employing metabolic rate depression (MRD), increasing urea concentration, switching to lipid oxidation as the primary energy source, and decreasing their breathing and heart rate. MicroRNA (miRNA) are known to regulate translation by targeting messenger RNA (mRNA) for degradation or temporary storage, with several studies having reported that miRNA is differentially expressed during MRD, including estivation. Thus, we hypothesized that miRNA would be involved in gene regulation during estivation in S. couchii heart. Next-generation sequencing and bioinformatic analyses were used to assess changes in miRNA expression in response to two-month estivation and to predict the downstream effects of this expression. KEGG and GO analyses indicated that ribosome and cardiac muscle contraction are among the pathways predicted to be upregulated, whereas cell signaling and fatty acid metabolism were predicted to be downregulated. Together these results suggest that miRNAs contribute to the regulation of gene expression related to cardiac muscle physiology and energy metabolism during estivation.

Sub-zero microRNA expression in the liver of the frozen hatchling painted turtle, Chrysemys picta marginata.

The Midland painted turtle (Chrysemys picta marginata) are the highest known vertebrate species to experience and survive freezing and sub-zero temperatures. Painted turtles typically hatch from their eggs in the fall and remain underground in their nests until the following spring. While in these nests over the winter, hatchling turtles withstand over 50 % of their total extracellular body water freezing. Herein, the expression of microRNAs (miRNAs) was investigated in response to freezing stress in the hatchling painted turtle liver. A total of 204 known miRNAs were identified to be expressed in turtles, with 17 being upregulated and 13 being downregulated during freezing. KEGG and GO analyses suggested that upregulated miRNAs inhibit genes of cell cycle and Focal adhesion and Adherens junction, suggesting their role in downregulation of central metabolic processes necessary for metabolic rate depression (MRD) and maintaining the tissue homeostasis. Only 9 of the 36 enriched KEGG pathways were less targeted by miRNAs during freezing, including linoleic acid metabolism and multiple signaling pathways. These predicted upregulated pathways likely promote homeoviscous adaptation and expression of pro-survival/protective proteins for metabolic adaptations necessary for defence of liver during MRD. Overall, miRNA-seq analysis of liver revealed a strong role of miRNA in the adaptive strategy that not only enables hatchlings to substantially suppress their nonessential energy needs but also makes them flexible enough to restore and protect their basal organ functions by activating pro-survival processes.

Activation of p53 in anoxic freshwater crayfish, Faxonius virilis.

Tumor suppressing transcription factor p53 regulates multiple pathways including DNA repair, cell survival, apoptosis and autophagy. Here, we studied the stress-induced activation of p53 in anoxic crayfish (Faxonius virilis). Relative levels of target proteins and mRNAs involved in the DNA damage response were measured in normoxic control and anoxic hepatopancreas and tail muscle. Phosphorylation levels of p53 were assessed using immunoblotting at sites known to be phosphorylated (serine 15 and 37) in response to DNA damage or reduced oxygen signaling. The capacity for DNA binding by phosphorylated p53 (p-p53) was also measured, followed by transcript analysis of a potentially pro-apoptotic downstream target, the etoposide induced (ei24) gene. Following this, both inhibitor (MDM2) and activator (p19-ARF) protein levels in response to low-oxygen stress were studied. The results showed an increase in p-p53 levels during anoxia in both hepatopancreas and tail muscle. Increased transcript levels of ei24 support the activation of p53 under anoxic stress. Cytoplasmic accumulation of Ser15 phosphorylated p53 was observed during anoxia when proteins from cytoplasmic and nuclear fractions were measured. Increased cytoplasmic concentration is known to initiate an apoptotic response, which can be assumed as a preparatory step to prevent autophagy. The results suggest that p53 might play a protective role in crayfish defense against low-oxygen stress. Understanding how anoxia-tolerant organisms are able to protect themselves against DNA damage could provide important clues towards survival under metabolic rate depression and preparation for recovery to minimize damage.

Regulation of Apoptosis and Autophagy During Anoxia in the Freshwater Crayfish, Faxonius virilis.

The ability of an animal to survive prolonged periods of oxygen deprivation is a critical area of study, both in terms of its importance to better understanding the physiology of these incredible animals and to its potential applicability to medical fields. The freshwater crayfish, Faxonius virilis, is one such animal capable of resisting anoxia, but it remains understudied and much of the metabolic mechanisms underlying this anoxia tolerance remain largely unprofiled. This study examines the activity and regulation of apoptosis and autophagy in F. virilis in response to 20-h anoxia. Apoptosis signaling was assessed through pro- and anti-apoptosis targets, whereas autophagy was assessed via expression response of multiple autophagy proteins. An anoxia-triggered, tissue-specific result arose, potentially based on the importance of individual organ integrity through hypometabolism. Tail muscle, which showed increased expression profiles of all three target groups, contrasted with hepatopancreas, which appeared to not be susceptible to either apoptotic or autophagic signaling during anoxia. This is likely due to the importance of the hepatopancreas, given that apoptosis or autophagy of this organ at any significant level could be fatal to the organism. The data provides a comprehensive overview of the responses and integration of multiple stress-responsive signaling pathways in F. virilis that provide a novel contribution to our understanding of pro-survival mechanisms supporting invertebrate anoxia resistance.

Nrf2 activates antioxidant enzymes in the anoxia-tolerant red-eared slider turtle, Trachemys scripta elegans.

The freshwater red-eared slider turtle, Trachemys scripta elegans, experiences weeks to months of anoxia at the bottom of ice-locked bodies of water in the winter. While this introduces anoxia-reoxygenation cycles similar to the ischemia-reperfusion events that mammals experience, T. s. elegans does not suffer any apparent tissue damage. To survive prolonged anoxia and prevent cellular damage associated with reactive oxygen species, these turtles have developed numerous adaptions, including highly effective antioxidant defenses. Herein, we examined the subcellular localization and protein expression of nuclear factor erythroid-2-related factor 2 (Nrf2), a central transcription factor responsible for modulating cellular antioxidant responses, that was found to be upregulated and localized to the nucleus in anoxic turtles. Additionally, we examined protein levels of glutathione S-transferases (GSTs) and manganese superoxide dismutase (MnSOD) antioxidant enzymes in anoxic liver, kidney, heart, and skeletal muscle tissues. MnSOD levels were significantly higher in heart and muscle during anoxia, and the four GST isozymes (GSTK1, GSTT1, GSTP1, and GSTM3) were elevated in a tissue-specific manner during anoxia and/or aerobic recovery. Together, these results indicate that Nrf2 is likely involved in activating downstream antioxidant genes in response to anoxic stress. These results provide a possible Nrf2-mediated transcriptional mechanism that supports existing findings of enhanced antioxidant defenses that allow T. s. elegans to cope with anoxia-reoxygenation cycles, and subsequent oxidative stress.

Modulating Nrf2 transcription factor activity: Revealing the regulatory mechanisms of antioxidant defenses during hibernation in 13-lined ground squirrels.

Mammalian hibernators undergo major behavioural, physiological and biochemical changes to survive hypothermia, ischaemia-reperfusion and finite fuel reserves during days or weeks of continuous torpor. During hibernation, the 13-lined ground squirrel (Ictidomys tridecemlineatus) undergoes a global suppression of energetically expensive processes such as transcription and translation, while selectively upregulating certain genes/proteins to mitigate torpor-related damage. Antioxidant defenses are critical for preventing damage caused by reactive oxygen species (ROS) during torpor and arousal, and Nrf2 is a critical regulator of these antioxidant genes. This study analysed the relative protein expression levels of Nrf2, KEAP1, small Mafs (MafF, MafK and MafG) and catalase and the regulation of Nrf2 transcription factors by post-translational modifications (PTMs) and protein-protein interactions with a negative regulator (KEAP1) during hibernation. It was found that a significant increase in MafK during late torpor predicated an increase in relative Nrf2 and catalase levels seen in arousal. Additionally, Nrf2-KEAP1 protein-protein interactions and Nrf2 PTMs, including serine phosphorylation and lysine acetylation, were responsive to cycles of torpor-arousal with peak responses occurring during arousal. These peaks seen during arousal correspond to a surge in oxygen consumption, which causes increased ROS production. Thus, these regulatory mechanisms could be important during hibernation because they provide mechanisms for mitigating the deleterious effects of oxidative stress by modifying Nrf2 expression and function in an energetically inexpensive manner.