Remodelling of Skeletal Muscle Myosin Metabolic States in Hibernating Mammals.

Hibernation is a period of metabolic suppression utilized by many small and large mammal species to survive during winter periods. As the underlying cellular and molecular mechanisms remain incompletely understood, our study aimed to determine whether skeletal muscle myosin and its metabolic efficiency undergo alterations during hibernation to optimize energy utilization. We isolated muscle fibers from small hibernators, Ictidomys tridecemlineatus and Eliomys quercinus and larger hibernators, Ursus arctos and Ursus americanus. We then conducted loaded Mant-ATP chase experiments alongside X-ray diffraction to measure resting myosin dynamics and its ATP demand. In parallel, we performed multiple proteomics analyses. Our results showed a preservation of myosin structure in U. arctos and U. americanus during hibernation, whilst in I. tridecemlineatus and E. quercinus, changes in myosin metabolic states during torpor unexpectedly led to higher levels in energy expenditure of type II, fast-twitch muscle fibers at ambient lab temperatures (20°C). Upon repeating loaded Mant-ATP chase experiments at 8°C (near the body temperature of torpid animals), we found that myosin ATP consumption in type II muscle fibers was reduced by 77-107% during torpor compared to active periods. Additionally, we observed Myh2 hyper-phosphorylation during torpor in I. tridecemilineatus, which was predicted to stabilize the myosin molecule. This may act as a potential molecular mechanism mitigating myosin-associated increases in skeletal muscle energy expenditure during periods of torpor in response to cold exposure. Altogether, we demonstrate that resting myosin is altered in hibernating mammals, contributing to significant changes to the ATP consumption of skeletal muscle. Additionally, we observe that it is further altered in response to cold exposure and highlight myosin as a potentially contributor to skeletal muscle non-shivering thermogenesis.

Climate change is altering the physiology and phenology of an arctic hibernator.

Climate warming is rapid in the Arctic, yet impacts to biological systems are unclear because few long-term studies linking biophysiological processes with environmental conditions exist for this data-poor region. In our study spanning 25 years in the Alaskan Arctic, we demonstrate that climate change is affecting the timing of freeze-thaw cycles in the active layer of permafrost soils and altering the physiology of arctic ground squirrels (Urocitellus parryii). Soil freeze has been delayed and, in response, arctic ground squirrels have delayed when they up-regulate heat production during torpor to prevent freezing. Further, the termination of hibernation in spring has advanced 4 days per decade in females but not males. Continued warming and phenological shifts will alter hibernation energetics, change the seasonal availability of this important prey species, and potentially disrupt intraspecific interactions.

A Repeat Pattern of Founder Events for SARS-CoV-2 Variants in Alaska.

Alaska is a unique US state because of its large size, geographically disparate population density, and physical distance from the contiguous United States. Here, we describe a pattern of SARS-CoV-2 variant emergence across Alaska reflective of these differences. Using genomic data, we found that in Alaska, the Omicron sublineage BA.2.3 overtook BA.1.1 by the week of 27 February 2022, reaching 48.5% of sequenced cases. On the contrary, in the contiguous United States, BA.1.1 dominated cases for longer, eventually being displaced by BA.2 sublineages other than BA.2.3. BA.2.3 only reached a prevalence of 10.9% in the contiguous United States. Using phylogenetics, we found evidence of potential origins of the two major clades of BA.2.3 in Alaska and with logistic regression estimated how it emerged and spread throughout the state. The combined evidence is suggestive of founder events in Alaska and is reflective of how Alaska's unique dynamics influence the emergence of SARS-CoV-2 variants.

Pattern of SARS-CoV-2 variant B.1.1.519 emergence in Alaska.

Alaska has the lowest population density in the United States (US) with a mix of urban centers and isolated rural communities. Alaska's distinct population dynamics compared to the contiguous US may have contributed to unique patterns of SARS-CoV-2 variants observed in early 2021. Here we examined 2323 SARS-CoV-2 genomes from Alaska and 278,635 from the contiguous US collected from December 2020 through June 2021 because of the notable emergence and spread of lineage B.1.1.519 in Alaska. We found that B.1.1.519 was consistently detected from late January through June of 2021 in Alaska with a peak prevalence in April of 77.9% unlike the rest of the US at 4.6%. The earlier emergence of B.1.1.519 coincided with a later peak of Alpha (B.1.1.7) compared to the contiguous US. We also observed differences in variant composition over time between the two most populated regions of Alaska and a modest increase in COVID-19 cases during the peak incidence of B.1.1.519. However, it is difficult to disentangle how social dynamics conflated changes in COVID-19 during this time. We suggest that the viral characteristics, such as amino acid substitutions in the spike protein, likely contributed to the unique spread of B.1.1.519 in Alaska.

Effects of Spring Warming on Seasonal Neuroendocrinology and Activation of the Reproductive Axis in Hibernating Arctic Ground Squirrels.

Many animals adjust the timing of seasonal events, such as reproduction, molt, migration, and hibernation, in response to interannual variation and directional climate-driven changes in temperature. However, the mechanisms by which temperature influences seasonal timing are relatively under-explored. Seasonal timing involves retrograde signaling in which thyrotropin (TSH) in the pars tuberalis (PT) alters expression of thyroid hormone (TH) deiodinases (Dio2/Dio3) in tanycyte cells lining the third ventricle of the hypothalamus. This, in turn, affects the availability of triiodothyronine (T3) within the mediobasal hypothalamus-increased hypothalamic T3 restores a summer phenotype and activates the reproductive axis in long-day breeders. Recently, we showed that retrograde TH signaling is activated during late hibernation in arctic ground squirrels (Urocitellus parryii) held in constant darkness and constant ambient temperature. Sensitivity of seasonal pathways to nonphotic cues, such as temperature, is likely particularly important to hibernating species that are sequestered in hibernacula during spring. To address this issue, we exposed captive arctic ground squirrels of both sexes to an ecologically relevant increase in ambient temperature (from -6 to -1°C) late in hibernation and examined the effects of warming on the seasonal retrograde TSH/Dio/T3 signaling pathway, as well as downstream elements of the reproductive axis. We found that warmed males tended to have higher PT TSHß expression and significantly heavier testis mass whereas the TSH/Dio/T3 signaling pathway was unaffected by warming in females, although warmed females exhibited a slight decrease in ovarian mass. Our findings suggest that temperature could have different effects on gonadal growth in male and female arctic ground squirrels, which could lead to mismatched timing in response to rapid climate change.

A repeat pattern of founder events for SARS-CoV-2 variants in Alaska.

Alaska is a unique US state because of its large size, geographically disparate population density, and physical distance from the contiguous United States. Here, we describe a pattern of SARS-CoV-2 variant emergence across Alaska reflective of these differences. Using genomic data, we found that in Alaska the Omicron sublineage BA.2.3 overtook BA.1.1 by the week of 2022-02-27, reaching 48.5% of sequenced cases. On the contrary in the contiguous United States, BA.1.1 dominated cases for longer, eventually being displaced by BA.2 sublineages other than BA.2.3. BA.2.3 only reached a prevalence of 10.9% in the contiguous United States. Using phylogenetics, we found evidence of potential origins of the two major clades of BA.2.3 in Alaska and with logistic regression estimated how it emerged and spread throughout the state. The combined evidence is suggestive of founder events in Alaska and is reflective of how Alaska’s unique dynamics influence the emergence of SARS-CoV-2 variants.

Hypothalamic remodeling of thyroid hormone signaling during hibernation in the arctic ground squirrel.

Hibernation involves prolonged intervals of profound metabolic suppression periodically interrupted by brief arousals to euthermy, the function of which is unknown. Annual cycles in mammals are timed by a photoperiodically-regulated thyroid-hormone-dependent mechanism in hypothalamic tanycytes, driven by thyrotropin (TSH) in the pars tuberalis (PT), which regulates local TH-converting deiodinases and triggers remodeling of neuroendocrine pathways. We demonstrate that over the course of hibernation in continuous darkness, arctic ground squirrels (Urocitellus parryii) up-regulate the retrograde TSH/Deiodinase/TH pathway, remodel hypothalamic tanycytes, and activate the reproductive axis. Forcing the premature termination of hibernation by warming animals induced hypothalamic deiodinase expression and the accumulation of secretory granules in PT thyrotrophs and pituitary gonadotrophs, but did not further activate the reproductive axis. We suggest that periodic arousals may allow for the transient activation of hypothalamic thyroid hormone signaling, cellular remodeling, and re-programming of brain circuits in preparation for the short Arctic summer.

Transcriptional changes and preservation of bone mass in hibernating black bears.

Physical inactivity leads to losses of bone mass and strength in most mammalian species. In contrast, hibernating bears show no bone loss over the prolonged periods (4-6 months) of immobility during winter, which suggests that they have adaptive mechanisms to preserve bone mass. To identify transcriptional changes that underlie molecular mechanisms preventing disuse osteoporosis, we conducted a large-scale gene expression screening in the trabecular bone and bone marrow, comparing hibernating and summer active bears through sequencing of the transcriptome. Gene set enrichment analysis showed a coordinated down-regulation of genes involved in bone resorption, osteoclast differentiation and signaling, and apoptosis during hibernation. These findings are consistent with previous histological findings and likely contribute to the preservation of bone during the immobility of hibernation. In contrast, no significant enrichment indicating directional changes in gene expression was detected in the gene sets of bone formation and osteoblast signaling in hibernating bears. Additionally, we revealed significant and coordinated transcriptional induction of gene sets involved in aerobic energy production including fatty acid beta oxidation, tricarboxylic acid cycle, oxidative phosphorylation, and mitochondrial metabolism. Mitochondrial oxidation was likely up-regulated by transcriptionally induced AMPK/PGC1α pathway, an upstream stimulator of mitochondrial function.

Transcriptional changes in muscle of hibernating arctic ground squirrels (Urocitellus parryii): implications for attenuation of disuse muscle atrophy.

Physical inactivity generates muscle atrophy in most mammalian species. In contrast, hibernating mammals demonstrate limited muscle loss over the prolonged intervals of immobility during winter, which suggests that they have adaptive mechanisms to reduce disuse muscle atrophy. To identify transcriptional programs that underlie molecular mechanisms attenuating muscle loss, we conducted a large-scale gene expression profiling in quadriceps muscle of arctic ground squirrels, comparing hibernating (late in a torpor and during torpor re-entry after arousal) and summer active animals using next generation sequencing of the transcriptome. Gene set enrichment analysis showed a coordinated up-regulation of genes involved in all stages of protein biosynthesis and ribosome biogenesis during both stages of hibernation that suggests induction of translation during interbout arousals. Elevated proportion of down-regulated genes involved in apoptosis, NFKB signaling as well as significant under expression of atrogenes, upstream regulators (FOXO1, FOXO3, NFKB1A), key components of the ubiquitin proteasome pathway (FBXO32, TRIM63, CBLB), and overexpression of PPARGC1B inhibiting proteolysis imply suppression of protein degradation in muscle during arousals. The induction of protein biosynthesis and decrease in protein catabolism likely contribute to the attenuation of disuse muscle atrophy through prolonged periods of immobility of hibernation.

Tissue-specific telomere dynamics in hibernating arctic ground squirrels (Urocitellus parryii).

Hibernation is used by a variety of mammals to survive seasonal periods of resource scarcity. Reactive oxygen species (ROS) released during periodic rewarming throughout hibernation, however, may induce oxidative damage in some tissues. Telomeres, which are the terminal sequences of linear chromosomes, may shorten in the presence of ROS, and thus the telomere length of an individual reflects the degree of accrued oxidative damage. This study quantified telomere length dynamics throughout hibernation in arctic ground squirrels (Urocitellus parryii). We hypothesized that telomere dynamics are tissue specific and predicted that telomere shortening would be most pronounced in brown adipose tissue (BAT), the organ that directly supports non-shivering thermogenesis during arousals. We used qPCR to determine relative telomere length (RTL) in DNA extracted from liver, heart, skeletal muscle (SM) and BAT of 45 juvenile and adult animals sampled either at mid- or late hibernation. Age did not have a significant effect on RTL in any tissue. At mid-hibernation, RTL of juvenile females was longer in BAT and SM than in liver and heart. In juvenile females, RTL in BAT and SM, but not in liver and heart, was shorter at late hibernation than at mid-hibernation. At late hibernation, juvenile males had longer RTL in BAT than did juvenile females, perhaps due to the naturally shorter hibernation duration of male arctic ground squirrels. Finally, BAT RTL at late hibernation negatively correlated with arousal frequency. Overall, our results suggest that, in a hibernating mammal, telomere shortening is tissue specific and that metabolically active tissues might incur higher levels of molecular damage.

Coping with differences in snow cover: the impact on the condition, physiology and fitness of an arctic hibernator.

The Earth's climate is changing at an unprecedented rate and, as ecologists, we are challenged with the difficult task of predicting how individuals and populations will respond to climate-induced changes to local and global ecosystems. Although we are beginning to understand some of the responses to changing seasonality, the physiological mechanisms that may drive these responses remain unknown. Using long-term data comparing two nearby populations (<20 km apart) of free-living arctic ground squirrels in northern Alaska, we have previously shown that the timing of spring snowmelt greatly influences their phenology of hibernation and reproduction in a population and site-specific manner. Here, we integrate these site-specific phenologies with body condition, stress physiology, reproductive success and juvenile recruitment to understand phenotypic selection in the two populations. We found that at the site with relatively late spring snowmelt and early autumn snow cover: (i) adult females were larger and in better body condition but had significantly higher stress hormone levels; (ii) females had similar numbers of comparably sized offspring, but offspring had higher stress hormone levels; and (iii) offspring density was lower just prior to hibernation. Thus, adult females at the two sites appear to use different coping strategies that allow them to maintain reproductive fitness; however, marked shortening of the active season because of later snowmelt in spring and earlier snow cover in autumn may compromise juvenile recruitment. We discuss the significance of these findings within the broader context of changing animal-environment relationships.

Sex-Dependent Phenological Plasticity in an Arctic Hibernator.

Hibernation provides a means of escaping the metabolic challenges associated with seasonality, yet the ability of mammals to prolong or reenter seasonal dormancy in response to extreme weather events is unclear. Here, we show that Arctic ground squirrels in northern Alaska exhibited sex-dependent plasticity in the physiology and phenology of hibernation in response to a series of late spring snowstorms in 2013 that resulted in the latest snowmelt on record. Females and nonreproductive males responded to the >1-month delay in snowmelt by extending heterothermy or reentering hibernation after several days of euthermy, leading to a >2-week delay in reproduction compared to surrounding years. In contrast, reproductive males neither extended nor reentered hibernation, likely because seasonal gonadal growth and development and subsequent testosterone release prevents a return to torpor. Our findings reveal intriguing differences in responses of males and females to climatic stressors, which can generate a phenological mismatch between the sexes.

Seasonal reproductive tactics: annual timing and the capital-to-income breeder continuum.

Tactics of resource use for reproduction are an important feature of life-history strategies. A distinction is made between 'capital' breeders, which finance reproduction using stored energy, and 'income' breeders, which pay for reproduction using concurrent energy intake. In reality, vertebrates use a continuum of capital-to-income tactics, and, for many species, the allocation of capital towards reproduction is a plastic trait. Here, we review how trophic interactions and the timing of life-history events are influenced by tactics of resource use in birds and mammals. We first examine how plasticity in the allocation of capital towards reproduction is linked to phenological flexibility via interactions between endocrine/neuroendocrine control systems and the sensory circuits that detect changes in endogenous state, and environmental cues. We then describe the ecological drivers of reproductive timing in species that vary in the degree to which they finance reproduction using capital. Capital can be used either as a mechanism to facilitate temporal synchrony between energy supply and demand or as a means of lessening the need for synchrony. Within many species, an individual's ability to cope with environmental change may be more tightly linked to plasticity in resource allocation than to absolute position on the capital-to-income breeder continuum.This article is part of the themed issue 'Wild clocks: integrating chronobiology and ecology to understand timekeeping in free-living animals'.

Entraining to the polar day: circadian rhythms in arctic ground squirrels.

Circadian systems are principally entrained to 24 h light-dark cycles, but this cue is seasonally absent in polar environments. Although some resident polar vertebrates have weak circadian clocks and are seasonally arrhythmic, the arctic ground squirrel (AGS) maintains daily rhythms of physiology and behavior throughout the summer, which includes 6 weeks of constant daylight. Here, we show that persistent daily rhythms in AGS are maintained through a circadian system that readily entrains to the polar day yet remains insensitive to entrainment by rapid light-dark transitions, which AGS generate naturally as a consequence of their semi-fossorial behavior. Additionally, AGS do not show 'jet lag', the slow realignment of circadian rhythms induced by the inertia of an intrinsically stable master circadian clock in the suprachiasmatic nucleus (SCN). We suggest this is due to the low expression of arginine vasopressin in the SCN of AGS, as vasopressin is associated with inter-neuronal coupling and robust rhythmicity.

Clock Gene Expression in the Suprachiasmatic Nucleus of Hibernating Arctic Ground Squirrels.

Most organisms have a circadian system, entrained to daily light-dark cycles, that regulates 24-h rhythms of physiology and behavior. It is unclear, however, how circadian systems function in animals that exhibit seasonal metabolic suppression, particularly when this coincides with the long-term absence of a day-night cycle. The arctic ground squirrel, Urocytellus parryii, is a medium-sized, semi-fossorial rodent that appears above-ground daily during its short active season in spring and summer before re-entering a constantly dark burrow for 6 to 9 months of hibernation. This hibernation consists of multiple week-long torpor bouts interrupted by short (< 20 h) arousal intervals when metabolism and body temperature (Tb) return to normal levels. Here, we used immunohistochemistry to measure the expression of daily or circadian rhythms of the protein products of 3 circadian clock genes, PER1, PER2, BMAL1, and the neural activity marker c-FOS in the suprachiasmatic nucleus (SCN) of arctic ground squirrels before, during, and after the first torpor bout of hibernation. Before torpor, while under 12:12-h light:dark conditions, animals showed significant daily rhythms in their Tb, as well as in protein expression levels of PER1 and PER2, but not BMAL1. Upon entering first torpor (Tb < 30°C), animals were moved into constant darkness. When sampled at 6-h intervals-beginning 24 h after the last light out, with Tb 3°C to 4°C-there were no circadian oscillations in PER1, PER2, or c-FOS expression. Sampling across 24 h during the first spontaneous arousal interval, c-FOS expression was elevated only when Tb reached 20°C and PER1 and PER2 expression did not show any Tb- or time-dependent changes. These results suggest that the central circadian clock might have stopped functioning during hibernation in this species, and the timing of arousal from torpor in arctic ground squirrels is unlikely to be controlled by the circadian clock within the SCN.

Lipid emulsion enhances cardiac performance after ischemia-reperfusion in isolated hearts from summer-active arctic ground squirrels.

Hibernating mammals, like the arctic ground squirrel (AGS), exhibit robust resistance to myocardial ischemia/reperfusion (IR) injury. Regulated preference for lipid over glucose to fuel metabolism may play an important role. We tested whether providing lipid in an emulsion protects hearts from summer-active AGS better than hearts from Brown Norway (BN) rats against normothermic IR injury. Langendorff-prepared AGS and BN rat hearts were perfused with Krebs solution containing 7.5 mM glucose with or without 1% Intralipid™. After stabilization and cardioplegia, hearts underwent 45-min global ischemia and 60-min reperfusion. Coronary flow, isovolumetric left ventricular pressure, and mitochondrial redox state were measured continuously; infarct size was measured at the end of the experiment. Glucose-only AGS hearts functioned significantly better on reperfusion than BN rat hearts. Intralipid™ administration resulted in additional functional improvement in AGS compared to glucose-only and BN rat hearts. Infarct size was not different among groups. Even under non-hibernating conditions, AGS hearts performed better after IR than the best-protected rat strain. This, however, appears to strongly depend on metabolic fuel: Intralipid™ led to a significant improvement in return of function in AGS, but not in BN rat hearts, suggesting that year-round endogenous mechanisms are involved in myocardial lipid utilization that contributes to improved cardiac performance, independent of the metabolic rate decrease during hibernation. Comparative lipid analysis revealed four candidates as possible cardioprotective lipid groups. The improved function in Intralipid™-perfused AGS hearts also challenges the current paradigm that increased glucose and decreased lipid metabolism are favorable during myocardial IR.

Stable isotope analysis of CO2 in breath indicates metabolic fuel shifts in torpid arctic ground squirrels.

Stable carbon isotope ratios (δ13C) in breath show promise as an indicator of immediate metabolic fuel utilization in animals because tissue lipids have a lower δ13C value than carbohydrates and proteins. Metabolic fuel consumption is often estimated using the respiratory exchange ratio (RER), which has lipid and carbohydrate boundaries, but does not differentiate between protein and mixed fuel catabolism at intermediate values. Because lipids have relatively low δ13C values, measurements of stable carbon isotopes in breath may help distinguish between catabolism of protein and mixed fuel that includes lipid. We measured breath δ13C and RER concurrently in arctic ground squirrels (Urocitellus parryii) during steady-state torpor at ambient temperatures from -2 to -26°C. As predicted, we found a correlation between RER and breath δ13C values; however, the range of RER in this study did not reach intermediate levels to allow further resolution of metabolic substrate use with the addition of breath δ13C measurements. These data suggest that breath δ13C values are 1.1‰ lower than lipid tissue during pure lipid metabolism. From RER, we determined that arctic ground squirrels rely on nonlipid fuel sources for a significant portion of energy during torpor (up to 37%). The shift toward nonlipid fuel sources may be influenced by adiposity of the animals in addition to thermal challenge.

Seasonal loss and resumption of circadian rhythms in hibernating arctic ground squirrels.

Circadian clocks are near universal among organisms and play a key role in coordinating physiological and metabolic functions to anticipate or coincide with predictable daily changes in the physical and social environment. However, whether circadian rhythms persist and are functionally important during hibernation in all mammals is currently unclear. We examined whether circadian rhythms of body temperature (T b) persist during multi-day, steady-state torpor and investigated the association between timing of animal emergence, exposure to light, and resumption of activity and T b rhythms in free-living and captive male arctic ground squirrels. High-resolution (0.02 °C) temperature loggers revealed that circadian rhythms of T b were not present during deep torpor in free-living arctic ground squirrels. Significant circadian rhythms of T b resumed, however, following the resumption of euthermia, but prior to emergence, though rhythms became much more robust coincident with aboveground emergence. Additionally, squirrels maintained in captivity under conditions of constant darkness spontaneously developed significant circadian rhythms of T b and activity soon after ending torpor. Exposing animals to a 5-s pulse of light within a week when they ended torpor increased the strength of rhythms. Our results are consistent with the hypothesis that circadian clock function is inhibited during hibernation in arctic ground squirrels, and we postulate that exposure to external stimuli, such as light in free-living animals, and meals or acute disturbance for captive squirrels, may enhance T b rhythmicity by synchronizing loosely coupled circadian oscillators within the suprachiasmatic nucleus.

The secret life of ground squirrels: accelerometry reveals sex-dependent plasticity in above-ground activity.

The sexes differ in how and when they allocate energy towards reproduction, but how this influences phenotypic plasticity in daily activity patterns is unclear. Here, we use collar-mounted light loggers and triaxial accelerometers to examine factors that affect time spent above ground and overall dynamic body acceleration (ODBA), an index of activity-specific energy expenditure, across the active season of free-living, semi-fossorial arctic ground squirrels (Urocitellus parryii). We found high day-to-day variability in time spent above ground and ODBA with most of the variance explained by environmental conditions known to affect thermal exchange. In both years, females spent more time below ground compared with males during parturition and early lactation; however, this difference was fourfold larger in the second year, possibly, because females were in better body condition. Daily ODBA positively correlated with time spent above ground in both sexes, but females were more active per unit time above ground. Consequently, daily ODBA did not differ between the sexes when females were early in lactation, even though females were above ground three to six fewer hours each day. Further, on top of having the additional burden of milk production, ODBA data indicate females also had fragmented rest patterns and were more active during late lactation. Our results indicate that sex differences in reproductive requirements can have a substantial influence on activity patterns, but the size of this effect may be dependent on capital resources accrued during gestation.