Mammalian hibernation is associated with wide variation in heart rate, blood flow, and oxygen delivery to tissues and is used as a model of natural ischemia/reperfusion. In non-hibernators, ischemia/reperfusion is typically associated with oxidative stress but hibernators seem to deal with potential oxidative damage by enhancing antioxidant defenses in an anticipatory manner. The present study assesses the role of the Nrf2 transcription factor in the regulation of antioxidant defenses during hibernation. Nrf2 mRNA and protein expression were enhanced in selected organs of 13-lined ground squirrels, Spermophilus tridecemlineatus during hibernation. Furthermore, Nrf2 protein in heart was elevated by 1.4-1.5 fold at multiple stages over a torpor-arousal bout including during entry, long term torpor, and early arousal. Levels returned to euthermic values when squirrels were fully aroused in interbout. Protein levels of selected downstream target genes under Nrf2 control were also measured via immunoblotting over the torpor-arousal cycle in heart. Cu/Zn superoxide dismutase and aflatoxin aldehyde reductase levels increased significantly during entry into torpor and then gradually declined falling to control levels or below in fully aroused animals. Heme oxygenase-1 also showed the same trend. This suggests a role for Nrf2 in regulating the antioxidant defenses needed for hibernation success. Heart nrf2 was amplified by PCR and sequenced. The deduced amino acid sequence showed high identity with the sequence from other mammals but with selected unique substitutions (e.g., proline residues at positions 111 and 230) that might be important for conformational stability of the protein at near 0 degrees C body temperatures in the torpid state.

Mammalian hibernation is composed of long periods of deep torpor interspersed with brief periods of arousal in which the animals, fueled by high rates of oxygen-based thermogenesis in brown adipose tissue and skeletal muscle, power themselves back to euthermic (~37 degrees C) body temperatures. Strong antioxidant defences are important both for long-term cytoprotection during torpor and for coping with high rates of reactive oxygen species generated during arousal. The present study shows that the antioxidant enzyme heme oxygenase 1 (HO1) is strongly upregulated in selected organs of thirteen-lined ground squirrels (Spermophilus tridecemlineatus) during hibernation. Compared with euthermic controls, HO1 mRNA transcript levels were 1.4- to 3.8-fold higher in 5 organs of hibernating squirrels, whereas levels of the constitutive isozyme HO2 were unchanged. Similarly, HO1 protein levels increased by 1.5- to 2.0-fold in liver, kidney, heart, and brain during torpor. Strong increases in the levels of the Nrf2 transcription factor and its heterodimeric partner protein, MafG, in several tissues indicated the mechanism of activation of hibernation-responsive HO1 gene expression. Furthermore, subcellular distribution studies with liver showed increased nuclear translocation of both Nrf2 and MafG in torpid animals. The data are consistent with the suggestion that Nrf2-mediated upregulation of HO1 expression provides enhanced antioxidant defence to counter oxidative stress in hibernating squirrels during torpor and (or) arousal.

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