L. Elaine Epperson

Quantitative Analysis of Liver Protein Expression During Hibernation in the Golden-mantled Ground Squirrel

Mammals that enter deep hibernation experience extreme reductions in body temperature and in metabolic, respiratory, and heart rates for several weeks at a time. Survival of these extremes likely entails a highly regulated network of tissue- and time-specific gene expression patterns that remain largely unknown. To date, studies to identify differentially-expressed genes have employed a candidate gene approach or in a few cases broader unbiased screens at the RNA level. Here we use a proteomic approach to compare and identify differentially expressed liver proteins from two seasonal stages in the golden-mantled ground squirrel (summer and entrance into torpor) using two-dimensional gels followed by MS/MS. Eighty-four two-dimensional gel spots were found that quantitatively alter with the hibernation season, 68 of which gave unambiguous identifications based on similarity to sequences in the available mammalian database. Based on what is known of these proteins from prior research, they are involved in a variety of cellular processes including protein turnover, detoxification, purine biosynthesis, gluconeogenesis, lipid metabolism and mobility, ketone body formation, cell structure, and redox balance. A number of the enzymes found to change seasonally are known to be either rate-limiting or first enzymes in a metabolic pathway, indicating key roles in metabolic control. Functional roles are proposed to explain the changes seen in protein levels and their potential influence on the phenotype of hibernation.

Seasonal proteomic changes reveal molecular adaptations to preserve and replenish liver proteins during ground squirrel hibernation

Hibernators are unique among mammals in their ability to survive extended periods of time with core body temperatures near freezing and with dramatically reduced heart, respiratory, and metabolic rates in a state known as torpor. To gain insight into the molecular events underlying this remarkable physiological phenotype, we applied a proteomic screening approach to identify liver proteins that differ between the summer active (SA) and the entrance (Ent) phase of winter hibernation in 13-lined ground squirrels. The relative abundance of 1,600 protein spots separated on two-dimensional gels was quantitatively determined using fluorescence difference gel electrophoresis, and 74 unique proteins exhibiting significant differences between the two states were identified using liquid chromatography followed by tandem mass spectrometry (LC-MS/MS). Proteins elevated in Ent hibernators included liver fatty acid-binding protein, fatty acid transporter, and 3-hydroxy-3-methylglutaryl-CoA synthase, which support the known metabolic fuel switch to lipid and ketone body utilization in winter. Several proteins involved in protein stability and protein folding were also elevated in the Ent phase, consistent with previous findings. In contrast to transcript screening results, there was a surprising increase in the abundance of proteins involved in protein synthesis during Ent hibernation, including several initiation and elongation factors. This finding, coupled with decreased abundance of numerous proteins involved in amino acid and nitrogen metabolism, supports the intriguing hypothesis that the mechanism of protein preservation and resynthesis is used by hibernating ground squirrels to help avoid nitrogen toxicity and ensure preservation of essential amino acids throughout the long winter fast.

Metabolic cycles in a circannual hibernator

Hibernation as manifested in ground squirrels is arguably the most plastic and extreme of physiological phenotypes in mammals. Homeostasis is challenged by prolonged fasting accompanied by heterothermy, yet must be facilitated for survival. We performed LC and GC-MS metabolomic profiling of plasma samples taken reproducibly during seven natural stages of the hibernators year, three in summer and four in winter (each n ≥ 5), employing a nontargeted approach to define the metabolite shifts associated with the phenotype. We quantified 231 named metabolites; 106 of these altered significantly, demarcating a cycle within a cycle where torpor-arousal cycles recur during the winter portion of the seasonal cycle. A number of robust hibernation biomarkers that alter with season and winter stage are identified, including specific free fatty acids, antioxidants, and previously unpublished modified amino acids that are likely to be associated with the fasting state. The major pattern in metabolite levels is one of either depletion or accrual during torpor, followed by reversal to an apparent homeostatic level by interbout arousal. This finding provides new data that strongly support the predictions of a long-standing hypothesis that periodic arousals are necessary to restore metabolic homeostasis.

Proteomic analysis of the winter-protected phenotype of hibernating ground squirrel intestine

The intestine of hibernating ground squirrels is protected against damage by ischemia-reperfusion (I/R) injury. This resistance does not depend on the low body temperature of torpor; rather, it is exhibited during natural interbout arousals that periodically return hibernating animals to euthermia. Here we use fluorescence two-dimensional difference gel electrophoresis (DIGE) to identify protein spot differences in intestines of 13-lined ground squirrels in the sensitive and protected phases of the circannual hibernation cycle, comparing sham-treated control animals with those exposed to I/R. Protein spot differences distinguished the sham-treated summer and hibernating samples, as well as the response to I/R between summer and hibernating intestines. The majority of protein changes among these groups were attributed to a seasonal difference between summer and winter hibernators. Many of the protein spots that differed were unambiguously identified by high-pressure liquid chromatography followed by tandem mass spectrometry of their constituent peptides. Western blot analysis confirmed significant upregulation for three of the proteins, albumin, apolipoprotein A-I, and ubiquitin hydrolase L1, that were identified in the DIGE analysis as increased in sham-treated hibernating squirrels compared with sham-treated summer squirrels. This study identifies several candidate proteins that may contribute to hibernation-induced protection of the gut during natural torpor-arousal cycles and experimental I/R injury. It also reveals the importance of enterocyte maturation in defining the hibernating gut proteome and the role of changing cell populations for the differences between sham and I/R-treated summer animals.

Skeletal muscle proteomics: carbohydrate metabolism oscillates with seasonal and torpor-arousal physiology of hibernation

The physiology of small mammalian hibernators shifts profoundly over a year, from summer homeothermy to winter heterothermy. Torpor-arousal cycles define high-amplitude tissue activity fluctuations in winter, particularly for skeletal muscle, which contributes to the energetically demanding rewarming process via shivering. To better understand the biochemistry underlying summer-winter and torpor-arousal transitions, we applied two-dimensional gel electrophoresis coupled with liquid chromatography/mass spectrometry/mas spectrometry to the soluble proteins from hindlimb muscle of 13-lined ground squirrels (Ictidomys tridecemlineatus) in two summer and six winter states. Two hundred sixteen protein spots differed by sampled state. Significantly, intrawinter protein adjustment was a minor component of the dataset despite large discrepancies in muscle activity level among winter states; rather, the bulk of differences (127/138 unequivocally identified proteins spots) occurred between summer and winter. We did not detect any proteomic signatures of skeletal muscle atrophy in this hibernator nor any differential seasonal regulation of protein metabolism. Instead, adjustments to metabolic substrate preferences dominated the detected proteomic differences. Pathways of carbohydrate metabolism (glycolysis and gluconeogenesis) were summer enriched, whereas the winter proteome was enriched for fatty acid β-oxidation. Nevertheless, our data suggest that some reliance on carbohydrate reserves is maintained during winter. Phosphoglucomutase (PGM1), which reversibly prepares glucose subunits for either glycolysis or glycogenesis, showed apparent winter state-specific phosphorylation. PGM1 was phosphorylated during rewarming and dephosphorylated by interbout arousal, implying that glucose supplements lipid fuels during rewarming. This, along with winter elevation of TCA cycle enzymes, suggests that hindlimb muscles are primed for rapid energy production and that carbohydrates are an important fuel for shivering thermogenesis.

Multistate proteomics analysis reveals novel strategies used by a hibernator to precondition the heart and conserve ATP for winter heterothermy

The hibernators heart functions continuously and avoids damage across the wide temperature range of winter heterothermy. To define the molecular basis of this phenotype, we quantified proteomic changes in the 13-lined ground squirrel heart among eight distinct physiological states encompassing the hibernators year. Unsupervised clustering revealed a prominent seasonal separation between the summer homeotherms and winter heterotherms, whereas within-season state separation was limited. Further, animals torpid in the fall were intermediate to summer and winter, consistent with the transitional nature of this phase. A seasonal analysis revealed that the relative abundances of protein spots were mainly winter-increased. The winter-elevated proteins were involved in fatty acid catabolism and protein folding, whereas the winter-depleted proteins included those that degrade branched-chain amino acids. To identify further state-dependent changes, protein spots were re-evaluated with respect to specific physiological state, confirming the predominance of seasonal differences. Additionally, chaperone and heat shock proteins increased in winter, including HSPA4, HSPB6, and HSP90AB1, which have known roles in protecting against ischemia-reperfusion injury and apoptosis. The most significant and greatest fold change observed was a disappearance of phospho-cofilin 2 at low body temperature, likely a strategy to preserve ATP. The robust summer-to-winter seasonal proteomic shift implies that a winter-protected state is orchestrated before prolonged torpor ensues. Additionally, the general preservation of the proteome during winter hibernation and an increase of stress response proteins, together with dephosphorylation of cofilin 2, highlight the importance of ATP-conserving mechanisms for winter cardioprotection.

Extensive use of torpor in 13-lined ground squirrels in the fall prior to cold exposure.

Mammalian hibernation is characterized by profound reductions in body temperature (Tb) and metabolic, heart and respiratory rates. These reductions are characteristic of torpor, which is temporally confined to winter. Hibernators including ground squirrels are heterothermic in winter, cycling between multiday periods of torpor with low Tb and brief periods of rewarming. In contrast, ground squirrels remain homeothermic during summer, like non-hibernating mammals. The transition between the homeothermic and heterothermic phases of the circannual rhythm of hibernation is often overlooked in hibernation studies. Here, we examined the use of torpor throughout the fall transition in laboratory-housed 13-lined ground squirrels by recording core body temperature with an implanted data logger. As is typical of laboratory-based hibernation studies, animals were kept in standard housing prior to being moved into a cold, dark room to simulate natural hibernation conditions. Significantly, the vast majority of both male and female ground squirrels expressed torpor in the fall while still housed conventionally and prior to cold exposure. The expression of torpor was not predicted by body weight or age, rather it appears to be preprogrammed in a time-dependent manner that is independent of, yet enhanced by, environmental cues. The timing and duration of these torpor bouts occurring prior to cold exposure were also remarkably sporadic. Thus, it is not possible to know with certainty which animals are torpor-naive before cold exposure in the absence of continuous measurement of body temperature. We conclude that fall animals encompass variable points in the transition between summer and winter phases of the circannual cycle of hibernation, thereby confounding studies in which they are used as non-hibernating controls. Conversely, these fall transition animals offer unique opportunities to define the molecular changes that accompany and enable hibernation.

Seasonal protein changes support rapid energy production in hibernator brainstem

During the torpor phase of mammalian hibernation when core body temperature is near 4°C, the autonomic system continues to maintain respiration, blood pressure and heartbeat despite drastic reductions in brain activity. In addition, the hibernator’s neuronal tissues enter into a protected state in which the potential for ischemia–reperfusion injury is markedly minimized. Evolutionary adaptations for continued function and neuroprotection throughout cycles of torpor and euthermia in winter are predicted to manifest themselves partly in changes in the brainstem proteome. Here, we compare the soluble brainstem protein complement from six summer active ground squirrels and six in the early torpor (ET) phase of hibernation. Thirteen percent of the ~1,500 quantifiable 2D gel spots alter significantly from summer to ET; the proteins identified in these differing spots are known to play roles in energy homeostasis via the tricarboxylic acid cycle (8 proteins), cytoarchitecture and cell motility (14 proteins), anabolic protein processes (13 proteins), redox control (11 proteins) and numerous other categories including protein catabolism, oxidative phosphorylation, signal transduction, glycolysis, intracellular protein trafficking and antiapoptotic function. These protein changes represent, at least in part, the molecular bases for restructuring of cells in the brainstem, a shift away from glucose as the primary fuel source for brain in the winter, and the generation of a streamlined mechanism capable of efficient and rapid energy production and utilization during the torpor and arousal cycles of hibernation.

Metabolic changes associated with the long winter fast dominate the liver proteome in 13-lined ground squirrels

Small-bodied hibernators partition the year between active homeothermy and hibernating heterothermy accompanied by fasting. To define molecular events underlying hibernation that are both dependent and independent of fasting, we analyzed the liver proteome among two active and four hibernation states in 13-lined ground squirrels. We also examined fall animals transitioning between fed homeothermy and fasting heterothermy. Significantly enriched pathways differing between activity and hibernation were biased toward metabolic enzymes, concordant with the fuel shifts accompanying fasting physiology. Although metabolic reprogramming to support fasting dominated these data, arousing (rewarming) animals had the most distinct proteome among the hibernation states. Instead of a dominant metabolic enzyme signature, torpor-arousal cycles featured differences in plasma proteins and intracellular membrane traffic and its regulation. Phosphorylated NSFL1C, a membrane regulator, exhibited this torpor-arousal cycle pattern; its role in autophagosome formation may promote utilization of local substrates upon metabolic reactivation in arousal. Fall animals transitioning to hibernation lagged in their proteomic adjustment, indicating that the liver is more responsive than preparatory to the metabolic reprogramming of hibernation. Specifically, torpor use had little impact on the fall liver proteome, consistent with a dominant role of nutritional status. In contrast to our prediction of reprogramming the transition between activity and hibernation by gene expression and then within-hibernation transitions by posttranslational modification (PTM), we found extremely limited evidence of reversible PTMs within torpor-arousal cycles. Rather, acetylation contributed to seasonal differences, being highest in winter (specifically in torpor), consistent with fasting physiology and decreased abundance of the mitochondrial deacetylase, SIRT3.

Therapeutic Molecular Phenotype of β-Blocker–Associated Reverse-Remodeling in Nonischemic Dilated Cardiomyopathy

Background—When &bgr;-blockers produce reverse-remodeling in idiopathic dilated cardiomyopathy, they partially reverse changes in fetal–adult/contractile protein, natriuretic peptide, SR-Ca2+-ATPase gene program constituents. The objective of the current study was to further test the hypothesis that reverse-remodeling is associated with favorable changes in myocardial gene expression by measuring additional contractile, signaling, and metabolic genes that exhibit a fetal/adult expression predominance, are thyroid hormone-responsive, and are regulated by &bgr;1-adrenergic receptor signaling. A secondary objective was to identify which of these putative regulatory networks is most closely associated with observed changes. Methods and Results—Forty-seven patients with idiopathic dilated cardiomyopathy (left ventricular ejection fraction, 0.24±0.09) were randomized to the adrenergic-receptor blockers metoprolol (&bgr;1-selective), metoprolol+doxazosin (&bgr;1/&agr;1), or carvedilol (&bgr;1/&bgr;2/&agr;1). Serial radionuclide ventriculography and endomyocardial biopsies were performed at baseline, 3, and 12 months. Expression of 50 mRNA gene products was measured by quantitative polymerase chain reaction. Thirty-one patients achieved left ventricular ejection fraction reverse-remodeling response defined as improvement by ≥0.08 at 12 months or by ≥0.05 at 3 months (&Dgr; left ventricular ejection fraction, 0.21±0.10). Changes in gene expression in responders versus nonresponders were decreases in NPPA and NPPB and increases in MYH6, ATP2A2, PLN, RYR2, ADRA1A, ADRB1, MYL3, PDFKM, PDHX, and CPT1B. All except PDHX involved increase in adult or decrease in fetal cardiac genes, but 100% were concordant with changes predicted by inhibition of &bgr;1-adrenergic signaling. Conclusions—In addition to known gene expression changes, additional calcium-handling, sarcomeric, adrenergic signaling, and metabolic genes were associated with reverse-remodeling. The pattern suggests a fetal–adult paradigm but may be because of reversal of gene expression controlled by a &bgr;1-adrenergic receptor gene network. Clinical Trial Registration—URL: www.clinicaltrials.gov. Unique Identifier: NCT01798992.