Remodeling 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.

Many animals use hibernation as a tactic to survive harsh winters. During this dormant, inactive state, animals reduce or limit body processes, such as heart rate and body temperature, to minimise their energy use. To conserve energy during hibernation, animals can use different approaches. For example, garden dormice undergo periodic states of extremely low core temperatures (down to 4­8^oC); whereas Eurasian brown bears see milder temperature drops (down to 23­25^oC). An important organ that changes during hibernation is skeletal muscle. Skeletal muscle typically uses large amounts of energy, making up around 50% of body mass. To survive, hibernating animals must change how their skeletal muscle uses energy. Traditionally, active myosin ­ a protein found in muscles that helps muscles to contract ­ was thought to be responsible for most of the energy use by skeletal muscle. But, more recently, resting myosin has also been found to use energy when muscles are relaxed. Lewis et al. studied myosin and skeletal muscle energy use changes during hibernation and whether they could impact the metabolism of hibernating animals. Lewis et al. assessed myosin changes in muscle samples from squirrels, dormice and bears during hibernation and during activity. Experiments showed changes in resting myosin in squirrels and dormice (whose temperature drops to 4­8^oC during hibernation) but not in bears. Further analysis revealed that cooling samples from non-hibernating muscle to 4­8^oC increased energy use in resting myosin, thereby generating heat. However, no increase in energy use was found after cooling hibernating muscle samples to 4­8^oC. This suggest that resting myosin generates heat at cool temperatures ­ a mechanism that is switched off in hibernating animals to allow them to cool their body temperature. These findings reveal key insights into how animals conserve energy during hibernation. In addition, the results show that myosin regulates energy use in skeletal muscles, which indicates myosin may be a potential drug target in metabolic diseases, such as obesity.

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.

The relationship between hypoxia exposure and circulating cortisol levels in social and solitary African mole-rats: An initial report.

Hypoxemia from exposure to intermittent and/or acute environmental hypoxia (lower oxygen concentration) is a severe stressor for many animal species. The response to hypoxia of the hypothalamic-pituitary-adrenal axis (HPA-axis), which culminates in the release of glucocorticoids, has been well-studied in hypoxia-intolerant surface-dwelling mammals. Several group-living (social) subterranean species, including most African mole-rats, are hypoxia-tolerant, likely due to regular exposure to intermittent hypoxia in their underground burrows. Conversely, solitary mole-rat species, lack many adaptive mechanisms, making them less hypoxia-tolerant than the social genera. To date, the release of glucocorticoids in response to hypoxia has not been measured in hypoxia-tolerant mammalian species. Consequently, this study exposed three social African mole-rat species and two solitary mole-rat species to normoxia, or acute hypoxia and then measured their respective plasma glucocorticoid (cortisol) concentrations. Social mole-rats had lower plasma cortisol concentrations under normoxia than the solitary genera. Furthermore, individuals of all three of the social mole-rat species exhibited significantly increased plasma cortisol concentrations after hypoxia, similar to those of hypoxia-intolerant surface-dwelling species. By contrast, individuals of the two solitary species had a reduced plasma cortisol response to acute hypoxia, possibly due to increased plasma cortisol under normoxia. If placed in perspective with other closely related surface-dwelling species, the regular exposure of the social African mole-rats to hypoxia may have reduced the basal levels of the components for the adaptive mechanisms associated with hypoxia exposure, including circulating cortisol levels. Similarly, the influence of body mass on plasma cortisol levels cannot be ignored. This study demonstrates that both hypoxia-tolerant rodents and hypoxia-intolerant terrestrial laboratory-bred rodents may possess similar HPA-axis responses from exposure to hypoxia. Further research is required to confirm the results from this pilot study and to further confirm how the cortisol concentrations may influence responses to hypoxia in African mole-rats.

Ventilatory Sensitivity to Ambient CO2 at Different Hibernation Temperatures in Thirteen-Lined Ground Squirrels (Ictidomys tridecemlineatus).

AbstractMammals entering hibernation undergo drastic reductions in metabolic rate and body temperature (Tb; to as low as ∼2% of euthermic metabolic rate and 1°C to -2°C). Although ventilation (V˙E) is also greatly reduced in hibernating ground squirrels, their relative ventilatory response (%ΔV˙E) to increases in inspired CO2 (∼400% increase to 7% CO2) dwarfs that of euthermic squirrels (∼60% increase). On the basis of data from earlier studies on hypothermic animals, we hypothesized that this switch in apparent ventilatory sensitivity was the result of the change in state (from euthermic to hibernating) and not due to the change in core Tb. Thus, we used whole-body plethysmography to assess the hypercapnic ventilatory response (HCVR) in thirteen-lined ground squirrels in steady-state hibernation at 20°C, 15°C, 10°C, 7°C, and 5°C. With the transition into hibernation as Tb fell, the breathing pattern became irregular and then episodic. Total V˙E and the oxygen consumption rate (V˙O2) decreased progressively as Tb fell. Hibernating squirrels with a core Tb of 20°C increased V˙E by 150% from normocapnic levels when given 7% CO2 to breathe, while squirrels with a Tb of 7°C increased V˙E by 650% when exposed to the same inspired CO2. When Tb was cooled from 7°C to 5°C, however, the increase in the HCVR fell to 450% and was associated with a rise in V˙O2 and total V˙E. These results reveal progressive changes in breathing pattern and the HCVR with decreasing Tb and suggest that the effects of hibernation state may be Tb dependent. V˙E did not fall in proportion to metabolic rate, and the HCVR increased progressively in both absolute terms and relative terms until a Tb of 7°C, both of which potentially constrain the extent of the metabolic suppression.

Changes in CO2 sensitivity during entrance into, and arousal from hibernation in Ictidomys tridecemlineatus.

At the onset of entrance into hibernation in many mammals, there is a reduction in the respiratory exchange ratio (RER) thought to result in a retention of CO2 that contributes to the ensuing metabolic suppression. In steady-state hibernation, the relative hypercapnic ventilatory response (HCVR; the % change in ventilation to CO2 exposure) is elevated. These two observations, paradoxically, suggest a transient decrease in CO2 sensitivity at the onset of entrance into hibernation, allowing the retention of CO2, then a subsequent increase in CO2 sensitivity giving rise to the elevated HCVR in steady-state hibernation. We examined the time course of the changes in ventilation, O2 consumption rates ([Formula: see text]o2), CO2 excretion rates, body temperature, and hence the RER and ACR (air convection ratio, ventilation/[Formula: see text]o2) and the HCVR throughout entrance and arousal into and out of hibernation in 13-lined ground squirrels to confirm this. We observed a significant drop (entrance) and rise (arousal) in the RER produced by hypo- and hyperventilation, respectively. CO2 chemo-sensitivity while the RER was reduced on entrance was blunted and rose late in entrance. On arousal, CO2 chemo-sensitivity was elevated when the RER was elevated and fell immediately after RER returned to normal values. At any given Tb, the HCVR was lower during entrance compared to arousal producing a significant hysteresis. The HCVR, however, was the same at any given [Formula: see text]o2 during entrance and arousal. These data suggest that both the changes in [Formula: see text]o2 and in the HCVR are associated with changes in central regulation of the effector limbs establishing steady-state hibernation.

Respiratory development in burrowing rodents: Effect of perinatal hypercapnia.

Burrowing rodents have a blunted hypercapnic ventilatory response compared to non-burrowing rodents, but semi-fossorial ground squirrels and hamsters are not born with this blunted response when raised in room conditions. This study examined the hypercapnic ventilatory response of rats, hamsters, and ground squirrels raised in burrow-like hypercapnia (∼3 % CO2) through development (embryonic day 16-18 to postnatal day 30) to determine if chronic hypercapnia exerts any effect on the developing and adult semi-fossorial response. Chronic hypercapnia attenuated the ventilatory response to 5 % CO2 by 60 % (rats), 150 % (hamsters), and 70 % (squirrels) in newborns when compared to newborns raised in normal conditions. When raised in burrow conditions, squirrels and hamsters reached the blunted adult response ∼8-12 days sooner in development than their room air counterparts, while burrow-reared rats maintained a consistently blunted response until removal from chronic hypercapnia. Our study revealed no lasting effect of chronic hypercarbia on the ventilatory responses to CO2 in burrowing rodents, but rather a change in the developmental profile such that the blunted adult response was reached earlier in development.

The hypoxia tolerance of eight related African mole-rat species rivals that of naked mole-rats, despite divergent ventilatory and metabolic strategies in severe hypoxia.

AIMS: Burrowing mammals tend to be more hypoxia tolerant than non-burrowing mammals and rely less on increases in ventilation and more on decreases in metabolic rate to tolerate hypoxia. Naked mole-rats (Heterocephalus glaber, NMRs), eusocial mammals that live in large colonies, are among the most hypoxia-tolerant mammals, and rely almost solely on decreases in metabolism with little change in ventilation during hypoxia. We hypothesized that the remarkable hypoxia tolerance of NMRs is an evolutionarily conserved trait derived from repeated exposure to severe hypoxia owing to their burrow environment and eusocial colony organization. METHODS: We used whole-body plethysmography and indirect calorimetry to measure the hypoxic ventilatory and metabolic responses of eight mole-rat species closely related to the NMR. RESULTS: We found that all eight species examined had a strong tolerance to hypoxia, with most species tolerating 3 kPa O2 , Heliophobius emini tolerating 2 kPa O2 and Bathyergus suillus tolerating 5 kPa O2 . All species examined employed a combination of increases in ventilation and decreases in metabolism in hypoxia, a response midway between that of the NMR and that of other fossorial species (larger ventilatory responses, lesser reductions in metabolism). We found that eusociality is not fundamental to the physiological response to hypoxia of NMRs as Fukomys damarensis, another eusocial species, was among this group. CONCLUSIONS: Our data suggest that, while the NMR is unique in the pattern of their physiological response to hypoxia, eight closely related mole-rat species share the ability to tolerate hypoxia like the current "hypoxia-tolerant champion," the NMR.

Postnatal changes in O2 and CO2 sensitivity in rodents.

In rodents, the ventilatory responses to hypoxia (low O2) and hypercarbia (high CO2) change significantly over postnatal development. In hypoxia, most adult rodents increase ventilation and decrease metabolism to some degree. Hypercarbia, however, leads to an increase in ventilation with little, to no change in metabolism. Neonates, on the other hand, respond to hypoxia with a profound metabolic depression, and a severely attenuated ventilatory response. In hypercarbia, they exhibit a strong ventilatory response early in development that blunts, reaches a nadir, and then rises back to the adult-like response, thus, stabilizing postnatally. In this review we discuss how the O2 and CO2 ventilatory responses develop in rodents, the possible mechanisms that drive these postnatal changes, and how being raised in a burrow, an environment putatively low in O2 and high in CO2, may affect the development of O2 and CO2 sensitivity in rodents.

Comparison of the CO2 ventilatory response through development in three rodent species: Effect of fossoriality.

Burrowing rodents have a blunted ventilatory response to CO2 in comparison to non-burrowing rodents. Non-burrowing rats display a period during development where ventilatory responses to hypercarbia become transiently blunted. This study examined the ventilatory responses to CO2 of rats, hamsters and ground squirrels through neonatal development to determine whether the blunted adult response of burrowing species is a retention of the blunting period seen in rats or present from birth. All three species increased ventilation in response to hypercarbia on the day of birth (70-170% in response to 5% CO2; 100-250% in response to 7% CO2). Rats in our study exhibited the triphasic ventilatory response (when expressed as %Δ) to CO2 previously described. In golden-Syrian hamsters, the ventilatory response slowly and progressively waned to a blunted adult response while in the 13-lined ground squirrels, the early ventilatory response to CO2 decreased within days and remained attenuated through development. Our study shows three distinct developmental patterns in the hypercarbic ventilatory response.

Developing a Model of Vitamin A Deficiency in a Hibernating Mammal, the 13-Lined Ground Squirrel (Ictidomys tridecemlineatus).

Retinoic acid, a bioactive metabolite of vitamin A, plays key roles in immune function and vision and adipose tissue development. Our goal was to study the effect of vitamin A deficiency in physiologic changes seen in hibernating 13-lined ground squirrels (Ictidomys tridecemlineatus). In this study, we first developed a model of vitamin A deficiency that was based on published mouse models; we then examined the role of RA in the circannual cycle of and adipose accumulation in this hibernating species. Gravid female ground squirrels began consuming a deficient diet during the last 2 wk of their 4-wk gestation; pups received the diet until they were 8 wk old, when severe symptoms of hypovitaminosis were observed, requiring the animals' removal from the protocol. Body size and adipose mass were significantly lower in vitamin-deficient pups than controls. To avoid these complications, we developed a second model, in which pups started on the deficient diet after weaning. The revised model produced few symptoms of deficiency, and squirrels were able to remain on the diet through spring emergence. Liver retinol analysis showed that deficient squirrels essentially had no vitamin A stores. Our data suggest that 13-lined ground squirrels maintain higher concentrations of stored retinol than other rodent species, such that their dietary needs may differ from those of traditional laboratory rodent models. Our results indicate that ground squirrels are especially susceptible to vitamin A deficiency, and ground squirrels should not be fed a deficient diet until after weaning, to avoid severe symptoms. Interestingly, vitamin A deficiency does not seem to affect this species' ability to hibernate successfully.

Hibernation alters the diversity and composition of mucosa-associated bacteria while enhancing antimicrobial defence in the gut of 13-lined ground squirrels.

The gut microbiota plays important roles in animal nutrition and health. This relationship is particularly dynamic in hibernating mammals where fasting drives the gut community to rely on host-derived nutrients instead of exogenous substrates. We used 16S rRNA pyrosequencing and caecal tissue protein analysis to investigate the effects of hibernation on the mucosa-associated bacterial microbiota and host responses in 13-lined ground squirrels. The mucosal microbiota was less diverse in winter hibernators than in actively feeding spring and summer squirrels. UniFrac analysis revealed distinct summer and late winter microbiota clusters, while spring and early winter clusters overlapped slightly, consistent with their transitional structures. Communities in all seasons were dominated by Firmicutes and Bacteroidetes, with lesser contributions from Proteobacteria, Verrucomicrobia, Tenericutes and Actinobacteria. Hibernators had lower relative abundances of Firmicutes, which include genera that prefer plant polysaccharides, and higher abundances of Bacteroidetes and Verrucomicrobia, some of which can survive solely on host-derived mucins. A core mucosal assemblage of nine operational taxonomic units shared among all individuals was identified with an average total sequence abundance of 60.2%. This core community, together with moderate shifts in specific taxa, indicates that the mucosal microbiota remains relatively stable over the annual cycle yet responds to substrate changes while potentially serving as a pool for 'seeding' the microbiota once exogenous substrates return in spring. Relative to summer, hibernation reduced caecal crypt length and increased MUC2 expression in early winter and spring. Hibernation also decreased caecal TLR4 and increased TLR5 expression, suggesting a protective response that minimizes inflammation.