Dynamic temperature-sensitive A-to-I RNA editing in the brain of a heterothermic mammal during hibernation.

RNA editing diversifies genomically encoded information to expand the complexity of the transcriptome. In ectothermic organisms, including Drosophila and Cephalopoda, where body temperature mirrors ambient temperature, decreases in environmental temperature lead to increases in A-to-I RNA editing and cause amino acid recoding events that are thought to be adaptive responses to temperature fluctuations. In contrast, endothermic mammals, including humans and mice, typically maintain a constant body temperature despite environmental changes. Here, A-to-I editing primarily targets repeat elements, rarely results in the recoding of amino acids, and plays a critical role in innate immune tolerance. Hibernating ground squirrels provide a unique opportunity to examine RNA editing in a heterothermic mammal whose body temperature varies over 30°C and can be maintained at 5°C for many days during torpor. We profiled the transcriptome in three brain regions at six physiological states to quantify RNA editing and determine whether cold-induced RNA editing modifies the transcriptome as a potential mechanism for neuroprotection at low temperature during hibernation. We identified 5165 A-to-I editing sites in 1205 genes with dynamically increased editing after prolonged cold exposure. The majority (99.6%) of the cold-increased editing sites are outside of previously annotated coding regions, 82.7% lie in SINE-derived repeats, and 12 sites are predicted to recode amino acids. Additionally, A-to-I editing frequencies increase with increasing cold-exposure, demonstrating that ADAR remains active during torpor. Our findings suggest that dynamic A-to-I editing at low body temperature may provide a neuroprotective mechanism to limit aberrant dsRNA accumulation during torpor in the mammalian hibernator.

Precise Control of Target Temperature Using N6-Cyclohexyladenosine and Real-Time Control of Surface Temperature.

Targeted temperature management is standard of care for cardiac arrest and is in clinical trials for stroke. N6-cyclohexyladenosine (CHA), an A1 adenosine receptor (A1AR) agonist, inhibits thermogenesis and induces onset of hibernation in hibernating species. Despite promising thermolytic efficacy of CHA, prior work has failed to achieve and maintain a prescribed target core body temperature (Tb) between 32°C and 34°C for 24 hours. We instrumented Sprague-Dawley rats (n = 19) with indwelling arterial and venous cannulae and a transmitter for monitoring Tb and ECG, then administered CHA via continuous IV infusion or intraperitoneal (IP) injection. In the first experiment (n = 11), we modulated ambient temperature and increased the dose of CHA in an attempt to manage Tb. In the second experiment (n = 8), we administered CHA (0.25 mg/[kg·h]) via continuous IV infusion and modulated cage surface temperature to control Tb. We rewarmed animals by increasing surface temperature at 1°C h-1 and discontinued CHA after Tb reached 36.5°C. Tb, brain temperature (Tbrain), heart rate, blood gas, and electrolytes were also monitored. Results show that titrating dose to adjust for individual variation in response to CHA led to tolerance and failed to manage a prescribed Tb. Starting with a dose (0.25 mg/[kg·h]) and modulating surface temperature to prevent overcooling proved to be an effective means to achieve and maintain Tb between 32°C and 34°C for 24 hours. Increasing surface temperature to 37°C during CHA administration brought Tb back to normothermic levels. All animals treated in this way rewarmed without incident. During the initiation of cooling, we observed bradycardia within 30 minutes of the start of IV infusion, transient hyperglycemia, and a mild hypercapnia; the latter normalized via metabolic compensation. In conclusion, we describe an intravenous delivery protocol for CHA at 0.25 mg/(kg·h) that, when coupled with conductive cooling, achieves and maintains a prescribed and consistent target Tb between 32°C and 34°C for 24 hours.

Optimization of Thermolytic Response to A1 Adenosine Receptor Agonists in Rats.

Cardiac arrest is a leading cause of death in the United States, and, currently, therapeutic hypothermia, now called targeted temperature management (TTM), is the only recent treatment modality proven to increase survival rates and reduce morbidity for this condition. Shivering and subsequent metabolic stress, however, limit application and benefit of TTM. Stimulating central nervous system A1 adenosine receptors (A1AR) inhibits shivering and nonshivering thermogenesis in rats and induces a hibernation-like response in hibernating species. In this study, we investigated the pharmacodynamics of two A1AR agonists in development as antishivering agents. To optimize body temperature (Tb) control, we evaluated the influence of every-other-day feeding, dose, drug, and ambient temperature (Ta) on the Tb-lowering effects of N6-cyclohexyladenosine (CHA) and the partial A1AR agonist capadenoson in rats. The highest dose of CHA (1.0 mg/kg, i.p.) caused all ad libitum-fed animals tested to reach our target Tb of 32°C, but responses varied and some rats overcooled to a Tb as low as 21°C at 17.0°C Ta Dietary restriction normalized the response to CHA. The partial agonist capadenoson (1.0 or 2.0 mg/kg, i.p.) produced a more consistent response, but the highest dose decreased Tb by only 1.6°C. To prevent overcooling after CHA, we studied continuous i.v. administration in combination with dynamic surface temperature control. Results show that after CHA administration control of surface temperature maintains desired target Tb better than dose or ambient temperature.

Comparative tissue transcriptomics highlights dynamic differences among tissues but conserved metabolic transcript prioritization in preparation for arousal from torpor.

During the hibernation season, 13-lined ground squirrels spend days to weeks in torpor with body temperatures near freezing then spontaneously rewarm. The molecular drivers of the drastic physiological changes that orchestrate and permit torpor are not well understood. Although transcription effectively ceases at the low body temperatures of torpor, previous work has demonstrated that some transcripts are protected from bulk degradation in brown adipose tissue (BAT), consistent with the importance of their protein products for metabolic heat generation during arousal from torpor. We examined the transcriptome of skeletal muscle, heart, and liver to determine the patterns of differentially expressed genes in these tissues, and whether, like BAT, a subset of these were relatively increased during torpor. EDGE-tags were quantified from five distinct physiological states representing the seasonal and torpor-arousal cycles of 13-lined ground squirrels. Supervised clustering on relative transcript abundances with Random Forest separated the two states bracketing prolonged torpor, entrance into and aroused from torpor, in all three tissues. Independent analyses identified 3347, 6784, and 2433 differentially expressed transcripts among all sampling points in heart, skeletal muscle, and liver, respectively. There were few differentially expressed genes in common across all three tissues; these were enriched in mitochondrial and apoptotic pathway components. Divisive clustering of these data revealed unique cohorts of transcripts that increased across the torpor bout in each tissue with patterns reflecting various combinations of cycling within and between seasons as well as between torpor and arousal. Transcripts that increased across the torpor bout were likewise tissue specific. These data shed new light on the biochemical pathways that alter in concert with hibernation phenotype and provide a rich resource for further hypothesis-based studies.

Comfortably Numb and Back: Plasma Metabolomics Reveals Biochemical Adaptations in the Hibernating 13-Lined Ground Squirrel.

Hibernation is an evolutionary adaptation that affords some mammals the ability to exploit the cold to achieve extreme metabolic depression (torpor) while avoiding ischemia/reperfusion or hemorrhagic shock injuries. Hibernators cycle periodically out of torpor, restoring high metabolic activity. If understood at the molecular level, the adaptations underlying torpor-arousal cycles may be leveraged for translational applications in critical fields such as intensive care medicine. Here, we monitored 266 metabolites to investigate the metabolic adaptations to hibernation in plasma from 13-lined ground squirrels (57 animals, 9 time points). Results indicate that the periodic arousals foster the removal of potentially toxic oxidative stress-related metabolites, which accumulate in plasma during torpor while replenishing reservoirs of circulating catabolic substrates (free fatty acids and amino acids). Specifically, we identified metabolic fluctuations of basic amino acids lysine and arginine, one-carbon metabolism intermediates, and sulfur-containing metabolites methionine, cysteine, and cystathionine. Conversely, reperfusion injury markers such as succinate/fumarate remained relatively stable across cycles. Considering the cycles of these metabolites with the hibernator's cycling metabolic activity together with their well-established role as substrates for the production of hydrogen sulfide (H2S), we hypothesize that these metabolic fluctuations function as a biological clock regulating torpor to arousal transitions and resistance to reperfusion during arousal.

The effects of hibernation and forced disuse (neurectomy) on bone properties in arctic ground squirrels.

Bone loss is a well-known medical consequence of disuse such as in long-term space flight. Immobilization in many animals mimics the effects of space flight on bone mineral density. Decreases in metabolism are also thought to contribute to a loss of skeletal mass. Hibernating mammals provide a natural model of disuse and metabolic suppression. Hibernating ground squirrels have been shown to maintain bone strength despite long periods of disuse and decreased metabolism during torpor. This study examined if the lack of bone loss during torpor was a result of the decrease in metabolic rate during torpor or an evolutionary change in these animals affording protection against disuse. We delineated changes in bone density during natural disuse (torpor) and forced disuse (sciatic neurectomy) in the hind limbs of the arctic ground squirrel (AGS) over an entire year. We hypothesized that the animals would be resistant to bone loss due to immobilization and disuse during the winter hibernation season when metabolism is depressed but not the summer active season. This hypothesis was not supported. The animals maintained bone density (dual-energy X-ray absorptiometry) and most bone structural and mechanical properties in both seasons. This was observed in both natural and forced disuse, regardless of the known metabolic rate increase during the summer. However, trabecular bone volume fraction (microcomputed tomography) in the distal femur was lower in neurectomized AGS at the study endpoint. These results demonstrate a need to better understand the relationship between skeletal load (use) and bone density that may lead to therapeutics or strategies to maintain bone density in disuse conditions.

1H-NMR metabolomic biomarkers of poor outcome after hemorrhagic shock are absent in hibernators.

BACKGROUND: Hemorrhagic shock (HS) following trauma is a leading cause of death among persons under the age of 40. During HS the body undergoes systemic warm ischemia followed by reperfusion during medical intervention. Ischemia/reperfusion (I/R) results in a disruption of cellular metabolic processes that ultimately lead to tissue and organ dysfunction or failure. Resistance to I/R injury is a characteristic of hibernating mammals. The present study sought to identify circulating metabolites in the rat as biomarkers for metabolic alterations associated with poor outcome after HS. Arctic ground squirrels (AGS), a hibernating species that resists I/R injury independent of decreased body temperature (warm I/R), was used as a negative control. METHODOLOGY/PRINCIPAL FINDINGS: Male Sprague-Dawley rats and AGS were subject to HS by withdrawing blood to a mean arterial pressure (MAP) of 35 mmHg and maintaining the low MAP for 20 min before reperfusing with Ringers. The animals' temperature was maintained at 37 ± 0.5 °C for the duration of the experiment. Plasma samples were taken immediately before hemorrhage and three hours after reperfusion. Hydrophilic and lipid metabolites from plasma were then analyzed via 1H-NMR from unprocessed plasma and lipid extracts, respectively. Rats, susceptible to I/R injury, had a qualitative shift in their hydrophilic metabolic fingerprint including differential activation of glucose and anaerobic metabolism and had alterations in several metabolites during I/R indicative of metabolic adjustments and organ damage. In contrast, I/R injury resistant AGS, regardless of season or body temperature, maintained a stable metabolic homeostasis revealed by a qualitative 1H-NMR metabolic profile with few changes in quantified metabolites during HS-induced global I/R. CONCLUSIONS/SIGNIFICANCE: An increase in circulating metabolites indicative of anaerobic metabolism and activation of glycolytic pathways is associated with poor prognosis after HS in rats. These same biomarkers are absent in AGS after HS with warm I/R.

Resistance to systemic inflammation and multi organ damage after global ischemia/reperfusion in the arctic ground squirrel.

INTRODUCTION: Cardiac arrest (CA) and hemorrhagic shock (HS) are two clinically relevant situations where the body undergoes global ischemia as blood pressure drops below the threshold necessary for adequate organ perfusion. Resistance to ischemia/reperfusion (I/R) injury is a characteristic of hibernating mammals. The present study sought to determine if arctic ground squirrels (AGS) are protected from systemic inflammation and multi organ damage after CA- or HS-induced global I/R and if, for HS, this protection is dependent upon their hibernation season. METHODS: For CA, rats and summer euthermic AGS (AGS-EU) were asphyxiated for 8 min, inducing CA. For HS, rats, AGS-EU, and winter interbout arousal AGS (AGS-IBA) were subject to HS by withdrawing blood to a mean arterial pressure of 35 mmHg and maintaining that pressure for 20 min before reperfusion with Ringers. For both I/R models, body temperature (Tb) was kept at 36.5-37.5°C. After reperfusion, animals were monitored for seven days (CA) or 3 hrs (HS) then tissues and blood were collected for histopathology, clinical chemistries, and cytokine level analysis (HS only). For the HS studies, additional groups of rats and AGS were monitored for three days after HS to access survival and physiological impairment. RESULTS: Rats had increased serum markers of liver damage one hour after CA while AGS did not. For HS, AGS survived 72 hours after I/R whereas rats did not survive overnight. Additionally, only rats displayed an inflammatory response after HS. AGS maintained a positive base excess, whereas the base excess in rats was negative during and after hemorrhage. CONCLUSIONS: Regardless of season, AGS are resistant to organ damage, systemic inflammation, and multi organ damage after systemic I/R and this resistance is not dependent on their ability to become decrease Tb during insult but may stem from an altered acid/base and metabolic response during I/R.

Ischemia/reperfusion injury resistance in hibernators is more than an effect of reduced body temperature or winter season.

Hibernating mammals are resistant to injury following cardiac arrest. The basis of this protection has been proposed to be due to their ability to lower body temperature or metabolic rate in a seasonally-dependent manner. However, recent studies have shown that neither reduced body temperature nor hibernation season are components this protection.

Effects of combined insulin-like growth factor 1 and macrophage colony-stimulating factor on the skeletal properties of mice.

BACKGROUND/AIM: Insulin-like growth factor-1 (IGF-1) and macrophage colony-stimulating factor (MCSF) are critical to skeletal homeostasis. We investigated the effects of combined IGF-1 plus MCSF on mice. MATERIALS AND METHODS: C57BL/6J mice, aged 7 weeks, were assigned to baseline, vehicle, IGF-1, MCSF, or combined IGF-1 plus MCSF (1 mg/kg/day each, n=12-13/group, 28-day duration). RESULTS: IGF-1 or MCSF had no effect on bone formation rate; however, IGF-1 plus MCSF produced a 169% increase in periosteal bone formation rate. Combined therapy increased femoral mechanical properties (+25% elastic force), while IGF-1, and MCSF alone did not. Combined therapy affected trabecular bone volume fraction (+40%), number (+13%), and spacing (-13%). MCSF produced similar trabecular changes, while IGF-1 had no effect. Combined therapy and MCSF alone increased bone mineral content. CONCLUSION: We have demonstrated the superior effects of combined IGF-1 and MCSF. Together, these agents may promote bone modeling to a greater extent than either therapy alone.