Spermophilus Tridecemlineatus Research Articles

Seasonal Variation in ATP-Induced Retinal Damage in the Cone-Dominant 13-Lined Ground Squirrel.

To examine whether time of year (relative to hibernation emergence) influences the retinal degenerative effects of intravitreal injection of adenosine triphosphate (ATP) in the 13-lined ground squirrel (13-LGS). Eighteen (9 male, 9 female) 13-LGS in three experimental cohorts (early season, mid-season, late season) (n = 6 each) underwent baseline imaging using scanning light ophthalmoscopy (SLO) and optical coherence tomography (OCT). Animals then received a 10-µL intravitreal injection of 0.723 M ATP, followed by OCT and SLO imaging at 3, 10, and 21 days. Adaptive optics SLO (AOSLO) was performed in animals without retinal damage after the 21-day follow-up. Retinal thickness, choroidal thickness, and cone density measures were compared to values from wild-type controls (n = 12). Five animals (four early season, one late season) showed retinal damage post-ATP injection (Fisher's exact test, P = 0.065). Animals with retinal damage displayed areas of disrupted retinal lamination on OCT. Any changes in OCT thickness were generally present on initial follow-up and resolved at later time points. Follow-up imaging with AOSLO on animals without retinal damage showed no significant differences in the cone mosaic topography from control eyes. Axial length was increased in mid-/late-season cohorts relative to early season (P = 0.0025 and P = 0.0007). In this pilot study, the 13-LGS appears more susceptible to ATP-induced retinal damage during the early season. Future studies adjusting dose based on ocular biometry may help elucidate the impact of time of year on chemical response. Consideration of ocular biometry in this and other animal models is merited when using intravitreal methods of chemical administration.

Multi-species genome-wide CRISPR screens identify conserved suppressors of cold-induced cell death.

Cells must adapt to environmental changes to maintain homeostasis. One of the most striking environmental adaptations is entry into hibernation during which core body temperature can decrease from 37°C to as low at 4°C. How mammalian cells, which evolved to optimally function within a narrow range of temperatures, adapt to this profound decrease in temperature remains poorly understood. In this study, we conducted the first genome-scale CRISPR-Cas9 screen in cells derived from Syrian hamster, a facultative hibernator, as well as human cells to investigate the genetic basis of cold tolerance in a hibernator and a non-hibernator in an unbiased manner. Both screens independently revealed glutathione peroxidase 4 (GPX4), a selenium-containing enzyme, and associated proteins as critical for cold tolerance. We utilized genetic and pharmacological approaches to demonstrate that GPX4 is active in the cold and its catalytic activity is required for cold tolerance. Furthermore, we show that the role of GPX4 as a suppressor of cold-induced cell death extends across hibernating species, including 13-lined ground squirrels and greater horseshoe bats, highlighting the evolutionary conservation of this mechanism of cold tolerance. This study identifies GPX4 as a central modulator of mammalian cold tolerance and advances our understanding of the evolved mechanisms by which cells mitigate cold-associated damage-one of the most common challenges faced by cells and organisms in nature.

The effect of temperature and breathing pattern on the surface activity of ground squirrel pulmonary surfactant.

This study investigates how hibernation affects the surface activity of pulmonary surfactant with respect to temperature and breathing pattern. Surfactant was isolated from a hibernating species, the 13-lined ground squirrel, and a homeotherm, the rabbit, and analysed for biophysical properties on a constrained sessile drop surfactometer. The results showed that surfactant from ground squirrels reduced surface tension better at low temperatures, including when mimicking episodic breathing, as compared with rabbit surfactant. In addition, low temperature adaptation was also observed using only the hydrophobic components of surfactant from ground squirrels. Overall, the data support the conclusion that ground squirrel surfactant has adapted to maintain surface activity during low temperature episodic breathing patterns, and that temperature adaptation is maintained with the hydrophobic components of the surfactant.

Effects of the anti-inflammatory drug budesonide on the gut microbiota and cytokine production of 13-lined ground squirrels during prehibernation fattening.

The gut microbiome is essential for maintaining organismal health. Gut microbiota may be disrupted through external factors like dietary change, which can lead to gut inflammation, resulting in obesity. Hibernating mammals develop low-grade gut inflammation when they accumulate fat deposits in preparation for hibernation, making them useful models for studying the relationship between the microbiome, inflammation, and weight gain. Nonsteroidal anti-inflammatory drugs and steroids are commonly used in humans to target gut inflammation, but how these drugs affect the gut microbiome and its stability is unclear. We investigated the effect of the glucocorticoid drug budesonide on the gut microbiome and cytokine levels of an obligate hibernator, the 13-lined ground squirrel, during the fattening season. We used 16S rRNA gene sequencing to characterize bacterial communities in the lumen and mucosa of the cecum and colon and measured proinflammatory [tumor necrosis factor-α (TNF-α)/interleukin 6 (IL-6)] and anti-inflammatory (IL-10) cytokine levels. Budesonide affected the microbiome only in the cecum lumen, where bacterial diversity was higher in the control group, and communities significantly differed between treatments. Across gut sections, Marvinbryantia and Enterococcus were significantly higher in the budesonide group, whereas Sarcina was higher in the control group. TNF-α and IL-6 levels were higher in control squirrels compared with the budesonide group, but there was no difference in IL-10 levels. Overall, budesonide treatment affected the microbial community and diversity of 13-lined ground squirrels in the cecum lumen. Our study presents another step toward developing ground squirrels as a model for studying the interaction between the microbiota and host inflammation.NEW & NOTEWORTHY Disruptions of gut microbiota can lead to inflammation, resulting in weight gain. Inflammation can be treated with budesonide, but how budesonide affects gut microbiota is unclear. Thirteen-lined ground squirrels experience low-grade gut inflammation during prehibernation fattening, which compares with human inflammation-weight gain mechanisms. We showed that budesonide treatment decreased microbiome diversity and lead to a shift in community in the cecum lumen. Our study supports developing ground squirrels as a model for studying microbiome-inflammation interactions.

Abstract Mo117: Investigation of Mechanisms to Modulate Contractility in 13-Lined Ground Squirrels

Introduction: During hibernation, 13-lined ground squirrels cycle between torpor and intermittent bouts of arousal (IBA). Bouts of IBA last 12-48 hours, during which heart rates rise from 2-4 bpm in torpor to 300 bpm during IBA before returning to torpor. Thus, rates of contractility and relaxation and their mechanisms must rapidly change. Phosphorylation of cardiac troponin I (cTnI) at serine 23/24 (pSer23/24) has been shown to decrease calcium sensitivity and increase the rate of myocardial relaxation, enabling faster heart rates. Heat shock protein 27 (HSP27) has been shown to protect contractility by stabilizing troponin. Hypothesis: We hypothesized that cTnI Ser23/24 phosphorylation would be higher in summer and IBA and that HSP27 would be found in higher quantities during torpor and IBA. Methods and Results: Left ventricular tissue (summer, n=8; torpor, n=8; IBA, n=8) was solubilized, separated by SDS-PAGE, and blotted with antibodies for quantification. The pSer23/24 to total troponin ratios were higher in summer compared to torpor (1.22±0.75 vs 0.32±0.19, p=0.003) and IBA (0.57±0.19, p=0.03), but not different between IBA and torpor. No significant season-dependence was observed for the serine 43 cTnI phosphorylation to total troponin ratio (torpor 1.45±0.69, summer 1.05±0.34, IBA 1.15±0.84). No significant season-dependence was observed in HSP27 (normalized to GAPDH) (summer 0.09±0.02, IBA 0.14±0.10, torpor 0.16±0.08). Nor was there a significant season-dependence observed in HSP90 (normalized to GAPDH) (summer 1.57±0.66, IBA 1.13±0.19, torpor 1.22±0.21). Conclusions: None of these mechanisms fully explain the rapid change in contractility observed during the hibernating periods of 13-lined ground squirrels. Other post-translational modifications of troponin or modifiers of HSP activity may modify the reported results. Such regulation may identify new treatments for humans where rapid changes in heart rate or contractility occur.

Mitochondrial microRNA profiles are altered in thirteen-lined ground squirrels (Ictidomys tridecemlineatus) during hibernation.

Thirteen-lined ground squirrels (TLGSs) are obligate hibernators that cycle between torpor (low metabolic rate and body temperature) and interbout euthermia (IBE; typical euthermic body temperature and metabolism) from late autumn to spring. Many physiological changes occur throughout hibernation, including a reduction in liver mitochondrial metabolism during torpor, which is reversed during arousal to interbout euthermia. Nuclear-encoded microRNA (miRNA, small posttranscriptional regulator molecules) differ in abundance throughout TLGS hibernation and have been shown to regulate mitochondrial gene expression in mammalian cell culture (where they are referred to as mitomiRs). This study characterized differences in mitomiR profiles from TLGS liver mitochondria isolated during summer, torpor, and IBE, and predicted their mitochondrial targets. Using small RNA sequencing, differentially abundant mitomiRs were identified between hibernation states, and using quantitative PCR analysis, we quantified the expression of predicted mitochondrial mRNA targets. Most differences in mitomiR abundances were seasonal (i.e., between summer and winter) with only one mitomiR differentially abundant between IBE and torpor. Multiple factor analysis (MFA) revealed three clusters divided by hibernation states, where clustering was predominantly driven by mitomiR abundances. Nine of these differentially abundant mitomiRs had predicted mitochondrial RNA targets, including subunits of electron transfer system complexes I and IV, 12S rRNA, and two tRNAs. Overall, mitomiRs were predicted to suppress the expression of their mitochondrial targets and may have some involvement in regulating protein translation in mitochondria. This study found differences in mitomiR abundances between seasons and hibernation states of TLGS and suggests potential mechanisms for regulating the mitochondrial electron transfer system.NEW & NOTEWORTHY During the hibernation season, thirteen-lined ground squirrels periodically increase metabolism remarkably between torpor and interbout euthermia (IBE). This process involves rapid reactivation of mitochondrial respiration. We predicted that mitochondrial microRNA (mitomiRs) might be altered during this response. We found that the abundance of 38 liver mitomiRs differs based on hibernation state (summer, IBE, and torpor). Small RNA sequencing identified mitomiR profiles, including some mitomiRs that are predicted to bind to mitochondrial RNAs.

Hypothalamic hormone deficiency enables physiological anorexia in ground squirrels during hibernation

Mammalian hibernators survive prolonged periods of cold and resource scarcity by temporarily modulating normal physiological functions, but the mechanisms underlying these adaptations are poorly understood. The hibernation cycle of thirteen-lined ground squirrels (Ictidomys tridecemlineatus) lasts for 5–7 months and comprises weeks of hypometabolic, hypothermic torpor interspersed with 24–48-h periods of an active-like interbout arousal (IBA) state. We show that ground squirrels, who endure the entire hibernation season without food, have negligible hunger during IBAs. These squirrels exhibit reversible inhibition of the hypothalamic feeding center, such that hypothalamic arcuate nucleus neurons exhibit reduced sensitivity to the orexigenic and anorexigenic effects of ghrelin and leptin, respectively. However, hypothalamic infusion of thyroid hormone during an IBA is sufficient to rescue hibernation anorexia. Our results reveal that thyroid hormone deficiency underlies hibernation anorexia and demonstrate the functional flexibility of the hypothalamic feeding center.

Frequency-modulated timer regulates torpor–arousal cycles during hibernation in distinct small mammalian hibernators

Hibernation allows mammals to endure harsh seasons by reducing their basal metabolism and body temperature (Tb) to minimize energy expenditure. During hibernation in small animals such as Syrian hamsters and 13-lined ground squirrels, Tb decreases to an ambient level ( < 5 °C) and remains constant for days to weeks in a physiological condition termed deep torpor. Torpor is interrupted by periods of arousal, during which Tb recovers to a euthermic level (approximately 37 °C), and these torpor–arousal cycles are repeated multiple times during hibernation. However, little is known about the mechanisms governing Tb fluctuations during hibernation. In this study, we employed an unbiased model selection approach to Tb data and revealed that a model incorporating frequency modulation quantitatively reproduced Tb fluctuation during hibernation in Syrian hamsters. We found that an unexpectedly long period of 120–430 days modulates a shorter period of several days. In addition, the aforementioned model reproduced Tb fluctuation in 13-lined ground squirrels, which can undergo repeated hibernation according to intrinsic circannual rhythms in constant laboratory conditions. This is the first quantitative study to demonstrate the concerted action of two endogenous periods, one lasting a few days and the other lasting a year, in the torpor–arousal cycles of distinct mammalian hibernators. We anticipate that our theoretical analysis of Tb fluctuation will be a starting point for quantitative comparisons of hibernation patterns across various hibernating species. Furthermore, quantification of Tb data using models will foster our understanding of the molecular mechanisms of hibernation by revealing the biological processes operating within these periods.

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.

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.

Changes in microRNA expression related to ischemia-reperfusion injury in the kidney of the thirteen-lined ground squirrel during torpor

During the hibernation season, the thirteen-lined ground squirrel undergoes cyclical torpor and arousal periods. The decrease and restoration of metabolic rate and oxygen delivery during torpor and arousal, respectively, may cause reperfusion-ischemia injury in the kidneys. In order to maintain the structural integrity of the kidneys necessary for renal function resumption during arousal, the thirteen-lined ground squirrel has developed adaptive methods to prevent and repair kidney injury. In this present study, computational methods were used to clean and analyze sequenced kidney RNA samples. Significantly differentially expressed microRNAs and enriched gene sets were also determined. From the gene set analysis, the results showed an increase in ubiquitin-related processes and p53 signaling pathways which suggested the occurrence of kidney damage during torpor. There was also an observed increase in cell cycle processes and the anchoring junction cellular compartment which may lend to the prevention of kidney injury. From the differentially expressed microRNAs, miR-27a (log2FC = 1.639; p-value = 0.023), miR-129 (log2FC = 2.516; p-value = 0.023), miR-let-7b (log2FC = 2.360; p-value = 0.025), miR-let-7c (log2FC = 2.291; p-value = 0.037) and miR-let-7i (log2FC = 1.564; p-value = 0.039) were found to be significantly upregulated. These biochemical adaptations may allow the thirteen-lined ground squirrel to maintain kidney structure and function during hibernation.

Paper towel shredding as a novel, affordable, noninvasive method for detecting arousals in hibernating rodents.

Many research groups explore the regulation of hibernation or compare the physiology of heterothermic mammals between the torpid and aroused, euthermic states. Current methods for monitoring torpor (for example, infrared cameras, body temperature or heart-rate telemetry, and motion sensing) are costly, require specialized techniques, and can be invasive. Here we present an alternate method for determining torpor-bout duration that is cost-effective, noninvasive and accurate: paper towel shredding. In the winter, euthermic thirteen-lined ground squirrels will shred paper towels placed in the cage, but torpid animals will not. The presence of a shredded paper towel, indicating an arousal from torpor, is easily evaluated during routine daily monitoring. In 12 animals over 52 days, this simple technique detected 59 arousals with 100% accuracy when compared with the body temperature telemetry of the same animals. Moreover, this novel method avoids some of the drawbacks of other cheap monitoring systems such as the sawdust technique.

Vegetation diversity and structure influence small‐mammal communities in native and restored northern mixed grasslands

AbstractCurrent grassland restoration strategies aim to recreate grassland vegetation communities, and often rely on high‐diversity native seeding to promote vegetation diversity. Questions remain concerning the influence of vegetation richness and diversity on grassland fauna. Small‐mammal communities are integral parts of grassland ecosystems, but their responses to restoration are often mixed or overlooked. During July 2014 to 2016, we used Sherman live traps to survey grassland small‐mammal communities of 24 study sites in northeastern South Dakota and southeastern North Dakota, USA, to better understand their responses to vegetation cover type, diversity, richness, and site‐specific vegetation structure. Sites represented a vegetation species richness gradient and 3 vegetation cover types including low‐diversity restorations planted with dense nesting cover (DNC) seed mix, high‐diversity seeded restorations, and unseeded reference grasslands. Small‐mammal abundance was highest at low‐diversity DNC restoration sites and lowest in reference grassland. Small‐mammal diversity was highest at high‐diversity restoration sites and lowest at low‐diversity DNC restoration sites. Models assessing the influence of vegetation structure on the abundance of focal taxa differed. Deer mice (Peromyscus spp.) were negatively influenced by percent native vegetation cover, and voles (Microtus spp.) showed yearly variation and were influenced positively by litter depth and negatively by vegetation richness. Small‐mammal communities of low‐diversity DNC restorations differed from reference sites, but high‐diversity restorations were not different from reference or low‐diversity DNC sites. Thirteen‐lined ground squirrel (Ictidomys tridecemlineatus) abundance was higher at reference and high‐diversity restored sites, while low‐diversity DNC sites had higher deer mice abundance. Results indicate small mammals are unlikely to respond uniformly to vegetation characteristics, and diversity of seed mixes used in grassland restoration is likely to influence grassland small‐mammal communities.

Abstract 1661 Alterations of DNA methylation as an epigenetic regulatory mechanism in thirteen-lined ground squirrels' cardiac muscle during hibernation

Abstract 1661 Alterations of DNA methylation as an epigenetic regulatory mechanism in thirteen-lined ground squirrels' cardiac muscle during hibernation

Human iPSC-derived photoreceptor transplantation in the cone dominant 13-lined ground squirrel

Several retinal degenerations affect the human central retina, which is primarily comprised of cones and is essential for high acuity and color vision. Transplanting cone photoreceptors is a promising strategy to replace degenerated cones in this region. Although this approach has been investigated in a handful of animal models, commonly used rodent models lack a cone-rich region and larger models can be expensive and inaccessible, impeding the translation of therapies. Here, we transplanted dissociated GFP-expressing photoreceptors from retinal organoids differentiated from human induced pluripotent stem cells into the subretinal space of damaged and undamaged cone-dominant 13-lined ground squirrel eyes. Transplanted cell survival was documented via noninvasive high-resolution imaging and immunohistochemistry to confirm the presence of human donor photoreceptors for up to 4months posttransplantation. These results demonstrate the utility of a cone-dominant rodent model for advancing the clinical translation of cell replacement therapies.

Neural control of fluid homeostasis is engaged below 10°C in hibernation.

Thirteen-lined ground squirrels (Ictidomys tridecemlineatus) hibernate for several months each winter without access to water,1 but the mechanisms that maintain fluid homeostasis during hibernation are poorly understood. In torpor, when body temperature (TB) reaches 4°C, squirrels decrease metabolism, slow heart rate, and reduce plasma levels of the antidiuretic hormones arginine vasopressin (AVP) and oxytocin (OXT).1 Squirrels spontaneously undergo interbout arousal (IBA) every 2weeks, temporarily recovering an active-like metabolism and a TB of 37°C for up to 48 h.1,2 Despite the low levels of AVP and OXT during torpor, profound increases in blood pressure and heart rate during the torpor-IBA transition are not associated with massive fluid loss, suggesting the existence of a mechanism that protects against diuresis at a low TB. Here, we demonstrate that the antidiuretic hormone release pathway is activated by hypothalamic supraoptic nucleus (SON) neurons early in the torpor-arousal transition. SON neuron activity, dense-core vesicle release from the posterior pituitary, and plasma hormone levels all begin to increase before TB reaches 10°C. Invivo fiber photometry of SON neurons from hibernating squirrels, together with RNA sequencing and c-FOS immunohistochemistry, confirms that SON is electrically, transcriptionally, and translationally active to monitor blood osmolality throughout the dynamic torpor-arousal transition. Our work emphasizes the importance of the antidiuretic pathway during the torpor-arousal transition and reveals that the neurophysiological mechanism that coordinates the hormonal response to retain fluid is active at an extremely low TB, which is prohibitive for these processes in non-hibernators.

Chemically induced cone degeneration in the 13-lined ground squirrel.

Animal models of retinal degeneration are critical for understanding disease and testing potential therapies. Inducing degeneration commonly involves the administration of chemicals that kill photoreceptors by disrupting metabolic pathways, signaling pathways, or protein synthesis. While chemically induced degeneration has been demonstrated in a variety of animals (mice, rats, rabbits, felines, 13-lined ground squirrels (13-LGS), pigs, chicks), few studies have used noninvasive high-resolution retinal imaging to monitor the in vivo cellular effects. Here, we used longitudinal scanning light ophthalmoscopy (SLO), optical coherence tomography, and adaptive optics SLO imaging in the euthermic, cone-dominant 13-LGS (46 animals, 52 eyes) to examine retinal structure following intravitreal injections of chemicals, which were previously shown to induce photoreceptor degeneration, throughout the active season of 2019 and 2020. We found that iodoacetic acid induced severe pan-retinal damage in all but one eye, which received the lowest concentration. While sodium nitroprusside successfully induced degeneration of the outer retinal layers, the results were variable, and damage was also observed in 50% of contralateral control eyes. Adenosine triphosphate and tunicamycin induced outer retinal specific damage with varying results, while eyes injected with thapsigargin did not show signs of degeneration. Given the variability of damage we observed, follow-up studies examining the possible physiological origins of this variability are critical. These additional studies should further advance the utility of chemically induced photoreceptor degeneration models in the cone-dominant 13-LGS.

Proteomic Identification of Seasonally Expressed Proteins Contributing to Heart Function and the Avoidance of Skeletal Muscle Disuse Atrophy in a Hibernating Mammal.

Hibernation in the thirteen-lined ground squirrel (Ictidomys tridecemlineatus) takes place over 4-6 months and is characterized by multiday bouts of hypothermic torpor (5-7 °C core body temperature) that are regularly interrupted every 1-2 weeks by brief (12-24 h) normothermic active periods called interbout arousals. Our goal was to gain insight into the molecular mechanisms that underlie the hibernator's ability to preserve heart function and avoid the deleterious effects of skeletal muscle disuse atrophy over prolonged periods of inactivity, starvation, and near-freezing body temperatures. To achieve this goal, we performed organelle enrichment of heart and skeletal muscle at five seasonal time points followed by LC-MS-based label-free quantitative proteomics. In both organs, we saw an increase in the levels of many proteins as ground squirrels transition from an active state to a prehibernation state in the fall. Interestingly, seasonal abundance patterns identified DHRS7C, SRL, TRIM72, RTN2, and MPZ as potential protein candidates for mitigating disuse atrophy in skeletal muscle, and ex vivo contractile mechanics analysis revealed no deleterious effects in the ground squirrel's muscles despite prolonged sedentary activity. Overall, an increased understanding of protein abundance in hibernators may enable novel therapeutic strategies to treat muscle disuse atrophy and heart disease in humans.

Electron transport system supercomplexes affect reactive-oxygen species production and respiration in both a hibernator (Ictidomys tridecemlineatus) and a nonhibernator (Rattus norvegicus).

Across many taxa, the complexes of the electron transport system associate with each other within the inner mitochondrial membrane to form supercomplexes (SCs). These SCs are thought to confer some selective advantage, such as increasing cellular respiratory capacity or decreasing the production of damaging reactive oxygen species (ROS). In this study, we investigate the relationship between supercomplex abundance and performance of liver mitochondria isolated from rats that do not hibernate and hibernating ground squirrels in which metabolism fluctuates substantially. We quantified the abundance of SCs (respirasomes (SCs containing CI, CIII, and CIV) or SCs containing CIII and CIV) and examined the relationship with state 3 (OXPHOS) and state 4 (LEAK) respiration rate, as well as net ROS production. We found that, in rats, state 3 and 4 respiration rate correlated negatively with respirasome abundance, but positively with CIII/CIV SC abundance. Despite the greater range of respiration rates in different hibernation stages, these relationships were similar in ground squirrels. This is, to our knowledge, the first report of differential effects of supercomplex types on mitochondrial respiration and ROS production.

Characterizing the rod pathway in cone-dominated thirteen-lined ground squirrels.

AII-amacrine cells (AIIs) are widely accepted as a critical element of scotopic pathways mediating night vision in the mammalian retina and have been well-characterized in rod-dominant mice, rabbits, and non-human primates. The rod pathway is characteristic of all mammalian eyes, however, the anatomic and physiologic role of AIIs and the rod pathways in cone dominant thirteen-lined ground squirrels (TLGS) is limited. Here, we employed both immunohistochemistry and electrophysiological approaches to investigate the morphology of AIIs and functional aspects of the rod pathway in TLGS. In all TLGS retinas examined, putative AIIs were calretinin-positive and exhibited connections to rod bipolar cells with decreased cell density and expanded arborization. Notably, AIIs retained connections with each other via gap junctions labeled with Connexin36. Comparisons between single photoreceptor recordings and full-field electroretinograms revealed scotopic ERG responses were mediated by both rods and cones. Thus, the components of the rod pathway are conserved in TLGS and rod signals traverse the retina in these cone-dominant animals. AIIs are sparsely populated, matching the diminished rod and rod bipolar cell populations compared to rod-dominant species. The infrequent distribution and lateral spacing of AII's indicate that they probably do not play a significant role in cone signaling pathways that encode information at a finer spatial scale. This contrasts with the mouse retina, where they significantly contribute to cone signaling pathways. Therefore, the AII's original function is likely that of a 'rod' amacrine cell, and its role in cone pathways in the mouse retina might be an adaptive feature stemming from its rod dominance.