Mammalian hibernators experience repeated hypoxic ischaemia and reperfusion during the torpor–arousal cycle. We investigated levels of oxidative stress, antioxidant capacity, and the underlying mechanism in heart, liver, brain and kidney tissue as well as plasma during different periods of hibernation in Daurian ground squirrels ( Spermophilus dauricus ). Our data showed that the levels of hydrogen peroxide significantly increased in the heart and brain during late torpor (LT) compared with levels during the summer active (SA) state. The content of malondialdehyde (MDA) was significantly lower during interbout arousal (IBA) and early torpor (ET) than that during SA or pre-hibernation (PRE), and MDA levels in the LT brain were significantly higher than the levels in other states. Superoxide dismutase 2 protein levels increased markedly in the heart throughout the entire torpor–arousal cycle. Catalase expression remained at an elevated level in the liver during the hibernation cycle. Superoxide dismutase 1 and glutathione peroxidase 1 (GPx1) expression increased considerably in all tissues during the IBA and ET states. In addition, the activities of the various antioxidant enzymes were higher in all tissues during IBA and ET than during LT; however, GPx activity in plasma decreased significantly during the hibernation season. The expression of p-Nrf2 decreased in all tissue types during IBA, but significantly increased during LT, especially in liver tissue. Interestingly, most changed indicators recovered to SA or PRE levels in post-hibernation (POST). These results suggest that increased reactive oxygen species during LT may activate the Nrf2/Keap1 antioxidant pathway and may contribute to the decreased MDA levels found during the IBA and ET states, thereby protecting organisms from oxidative damage over the torpor-arousal cycle of hibernation. This is the first report on the remarkable controllability of oxidative stress and tissue specificity in major oxidative tissues of a hibernator.

Intracellular calcium (Ca²⁺) homeostasis plays a vital role in the preservation of skeletal muscle. In view of the well-maintained skeletal muscle found in Daurian ground squirrels (Spermophilus dauricus) during hibernation, we hypothesized that hibernators possess unique strategies of intracellular Ca²⁺ homeostasis. Here, cytoplasmic, sarcoplasmic reticulum (SR), and mitochondrial Ca²⁺ levels, as well as the potential Ca²⁺ regulatory mechanisms, were investigated in skeletal muscle fibers of Daurian ground squirrels at different stages of hibernation. The results showed that cytoplasmic Ca²⁺ levels increased in the skeletal muscle fibers during late torpor (LT) and inter-bout arousal (IBA), and partially recovered when the animals re-entered torpor (early torpor, ET). Furthermore, compared with levels in the summer active or pre-hibernation state, the activity and protein expression levels of six major Ca²⁺ channels/proteins were up-regulated during hibernation, including the store-operated Ca²⁺ entry (SOCE), ryanodine receptor 1 (RyR1), leucine zipper-EF-hand containing transmembrane protein 1 (LETM1), SR Ca²⁺ ATPase 1 (SERCA1), mitochondrial calcium uniporter complex (MCU complex), and calmodulin (CALM). Among these, the increased extracellular Ca²⁺ influx mediated by SOCE, SR Ca²⁺ release mediated by RyR1, and mitochondrial Ca²⁺ extrusion mediated by LETM1 may be triggers for the periodic elevation in cytoplasmic Ca²⁺ levels observed during hibernation. Furthermore, the increased SR Ca²⁺ uptake through SERCA1, mitochondrial Ca²⁺ uptake induced by MCU, and elevated free Ca²⁺ binding capacity mediated by CALM may be vital strategies in hibernating ground squirrels to attenuate cytoplasmic Ca²⁺ levels and restore Ca²⁺ homeostasis during hibernation. Compared with that in LT or IBA, the decreased extracellular Ca²⁺ influx mediated by SOCE and elevated mitochondrial Ca²⁺ uptake induced by MCU may be important mechanisms for the partial cytoplasmic Ca²⁺ recovery in ET. Overall, under extreme conditions, hibernating ground squirrels still possess the ability to maintain intracellular Ca²⁺ homeostasis.

Although artificial microRNA (amiRNA) technology has been used frequently in gene silencing in plants, little research has been devoted to investigating the accuracy of amiRNA precursor processing. In this work, amiRNAchs1 (amiRchs1), based on the Arabidopsis miR319a precursor, was expressed in order to suppress the expression of CHS genes in petunia. The transgenic plants showed the CHS gene-silencing phenotype. A modified 5′ RACE technique was used to map small-RNA-directed cleavage sites and to detect processing intermediates of the amiRchs1 precursor. The results showed that the target CHS mRNAs were cut at the expected sites and that the amiRchs1 precursor was processed from loop to base. The accumulation of small RNAs in amiRchs1 transgenic petunia petals was analyzed using the deep-sequencing technique. The results showed that, alongside the accumulation of the desired artificial microRNAs, additional small RNAs that originated from other regions of the amiRNA precursor were also accumulated at high frequency. Some of these had previously been found to be accumulated at low frequency in the products of ath-miR319a precursor processing and some of them were accompanied by 3′-tailing variant. Potential targets of the undesired small RNAs were discovered in petunia and other Solanaceae plants. The findings draw attention to the potential occurrence of undesired target silencing induced by such additional small RNAs when amiRNA technology is used. No appreciable production of secondary small RNAs occurred, despite the fact that amiRchs1 was designed to have perfect complementarity to its CHS-J target. This confirmed that perfect pairing between an amiRNA and its targets is not the trigger for secondary small RNA production. In conjunction with the observation that amiRNAs with perfect complementarity to their target genes show high efficiency and specificity in gene silencing, this finding has an important bearing on future applications of amiRNAs in gene silencing in plants.

Daurian ground squirrels (Spermophilus dauricus) deviate from significant increase of protein catabolism and loss of myofibrillar contents during long period of hibernation inactivity. Here we use iTRAQ based quantitative analysis to examine proteomic changes in the soleus of squirrels in pre-hibernation, hibernation and post-hibernation states. The total proteolysis rate of soleus was measured by the release of the essential amino acid tyrosine from isolated muscles. Immunofluorescent analysis was used to determine muscle fiber cross-sectional area. Western blot was used for the validation of the quantitative proteomic analysis. The proteomic responses to hibernation had a 0.4- to 0.8-fold decrease in the myofibrillar contractile protein levels of myosin-3, myosin-13 and actin, but a 2.1-fold increase in myosin-2 compared to pre-hibernation group. Regulatory proteins such as troponin C and tropomodulin-1 were 1.4-fold up-regulated and 0.7-fold down-regulated, respectively, in hibernation compared to pre-hibernation group. Moreover, 10 proteins with proteolytic function in hibernation, which was less than 14 proteins in the post-hibernation group, were up-regulated relative to the pre-hibernation group. The total proteolysis rates of soleus in hibernation and post-hibernation groups were significantly inhibited as compared with pre-hibernation group. These findings suggest that the myofibrillar remodeling and partial suppression of myofibrillar proteolysis were likely responsible for preventing skeletal muscle atrophy during prolonged disuse in hibernation. This is the first study where the myofibrillar contents and relevant synthesis and proteolytic proteins in slow soleus was discussed based on proteomic investigation performed on wild Daurian ground squirrels. Our results lay the foundation for further research in preventing disuse-induced skeletal muscle atrophy in mammals.