Summer-active Ground Squirrels Research Articles

Sodium current preserves electrical excitability in the heart of hibernating ground squirrel (Citellus undulatus)

Hibernating mammals are capable of maintaining normal cardiac function at low temperatures. Excitability of cardiac myocytes crucially depends on the fast sodium current (INa), which is decreased in hypothermia due to both depolarization of resting membrane potential and direct negative effect of low temperature. Therefore, INa in hibernating mammals should have specific features allowing to maintain excitability of myocardium at low temperatures. The current-voltage dependence of INa, its steady-state inactivation and activation and recovery from inactivation were studied in winter hibernating (WH) and summer active (SA) ground squirrels and in rats using whole-cell patch clamp at 10 °C and 20 °C. INa peak amplitude and the parameters of steady-state activation and inactivation curves did not differ between SA and WH ground squirrels at both temperatures. However, at both temperatures strong positive shift of activation and inactivation curves by 5–12 mV was observed in both WH and SA ground squirrels if compared to rats. This peculiarity of cardiac INa in ground squirrels helps to maintain excitability in conditions of depolarized resting membrane potential. The time course of INa recovery from inactivation at 10 °C was faster in WH than in SA ground squirrels, which could ensure normal activation of myocardium during hibernation.

Hypothermia-Induced Postrepolarization Refractoriness Is the Reason of the Atrial Myocardium Tolerance to the Bioelectrical Activity Disorders in the Hibernating and Active Ground Squirrel Citellus undulatus.

The electrophysiological mechanism of the atrial myocardium resistance to the cold-induced arrhythmias was studied in the hibernating ground squirrel Citellus undulatus. The atrial action potentials (APs) and refractoriness were recorded with microelectrodes in isolated multicellular preparations of the atrial myocardium taken from the hibernating and summer active ground squirrels (HS and SAS, respectively) at 37, 27, and 17°С to estimate the AP and refractoriness durations. In both HS and SAS, hypothermia increased the duration of the AP and refractoriness period (APD and RD, respectively), and in both animal groups RD was longer than APD under hypothermia but not at 37°С. This last observation can be a result of the postrepolarization refractoriness (PRR), which seems to contribute substantially to the atrial myocardium tolerance of the hibernating animals to the hypothermia-induced arrhythmias because it prevents afterdepolarizations.

Investigating the effect of temperature on large aggregate conversion of surfactant from ground squirrels

During hibernation, ground squirrels’ body temperature is 5°C. Our research focuses on pulmonary surfactant during hibernation. Surfactant is a phospholipid‐rich material which regulates surface tension at the alveolar surface. A change in surface‐area during respiration leads to conversion of the large aggregate form of surfactant to the small aggregate form. Since temperature affects aggregate conversion, we tested the hypothesis that hibernation led to less conversion of large to small aggregates. Lungs of hibernating and summer active ground squirrels were lavaged to obtain large and small aggregates which were measured by a phospholipid‐phosphorous assay. Surface‐area cycling was performed at 37°C and 4°C to determine large aggregate conversion. Total amounts of large aggregate were higher in hibernating squirrels compared to summer active group. The large aggregates from all groups underwent a conversion to a greater extent at 37°C than at 4°C. Changes in large aggregate pool sizes occur during hibernation, in vitro aggregate conversion data suggests this may be as a result of reduced aggregate conversion at low temperatures.Funding: Canadian Institutes for Health Research.

Seasonal protein changes support rapid energy production in hibernator brainstem

During the torpor phase of mammalian hibernation when core body temperature is near 4 degrees 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 approximately 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.

Electrophysiological mechanisms of antiarrhythmic protection during hypothermia in winter hibernating versus nonhibernating mammals

Robust cell-to-cell coupling is critically important in the safety of cardiac conduction and protection against ventricular fibrillation (VF). Hibernating mammals have evolved naturally protective mechanisms against VF induced by hypothermia and reperfusion injury. We hypothesized that this protection strategy involves a dynamic maintenance of conduction and repolarization patterns through the improvement of gap junction functions. We optically mapped the hearts of summer-active (SA) and winter-hibernating (WH) ground squirrels Spermophilus undulatus from Siberia and nonhibernating rabbits during different temperatures (+3 degrees C to +37 degrees C). Midhypothermia (+17 degrees C) resulted in nonuniform conduction slowing, increased dispersion of repolarization, shortened wavelength, and consequently enhanced VF induction in SA ground squirrels and rabbits. In contrast, wavelength was increased during hypothermia in WH hearts in which VF was not inducible at any temperature. In SA and rabbit hearts, but not in WH, conduction anisotropy was significantly increased by pacing acceleration, thus promoting VF induction during hypothermia. WH hearts maintained the same rate-independent anisotropic propagation pattern even at 3 degrees C. connexin 43 (Cx43) had more homogenous transmural distribution in WH ventricles as compared to SA. Moreover, Cx43 and N-cadherins (N-cad) densities as well as the percentage of their colocalization were significantly higher in WH compared to SA epicardium. Rate-independent conduction anisotropy ratio, low dispersion of repolarization, and long wavelength-these are the main electrophysiological mechanisms of antiarrhythmic protection in hibernating mammalian species during hypothermia. This strategy includes the improved gap junction function, which is due to overexpression and enhanced colocalization of Cx43 and N-cad.

Mechanical overload increases muscle mass but does not change myosin isoform expression in a rodent hibernator, Spermophilus lateralis

Hibernating ground squirrels do not show slow to fast myosin heavy‐chain (MyHC) isoform transitions with atrophy, and we test whether increased muscle loading would also fail to produce typical MyHC isoform shifts. Field‐caught, Summer‐active ground squirrels (Spermophilus lateralis) were assessed for the responsiveness of hindlimb plantaris muscles to mechanical overload through synergist surgical ablation. Plantaris muscles of surgical animals were 40% larger than controls after 14 days, but did not show any of the expected shifts in MyHC isoform expression. We also measured MyHC isoform mRNA expression, citrate synthase activity, and the expression of a suite of genes involved in the control of skeletal muscle mass and MyHC isoform expression, including MAFbx, MuRF1, FOXO1, IGF1 and IGFBP5, and myostatin. Our results show an unusual insensitivity of hibernator muscles to mechanical overload induced isoform shifts, yet muscle mass was increased, which has implications for isoform expression and muscle adaptations during hibernation. Funding sources: NIH SCORE, NIH RISE, CSULB.

Delta opioid peptide (D-Ala 2, D-Leu 5) enkephalin: linking hibernation and neuroprotection.

Hibernation is a potential protective strategy for the peripheral, as well as for the central nervous system. A protein factor termed hibernation induction trigger (HIT) was found to induce hibernation in summer-active ground squirrels. Purification of HIT yielded an 88-kD peptide that is enriched in winter hibernators. Partial sequence of the 88-kD protein indicates that it may be related to the inhibitor of metalloproteinase. Using opioid receptor antagonists to elucidate the mechanisms of HIT, it was found that HIT targeted the delta opioid receptors. Indeed, delta opioid (D-Ala 2, D-Leu 5) enkephalin (DADLE) was shown to induce hibernation. Specifically, HIT and DADLE were found to prolong survival of peripheral organs, such as the lung, the heart, liver, and kidney preserved en bloc or as a single preparation. In addition, DADLE has been recently demonstrated to promote survival of neurons in the central nervous system. Exposure to DADLE dose-dependently enhanced cell viability of cultured primary rat fetal dopaminergic cells. Subsequent transplantation of these DADLE-treated dopaminergic cells into the Parkinsonian rat brain resulted in a two-fold increase in surviving grafted cells. Interestingly, delivery of DADLE alone protected against dopaminergic depletion in a rodent model of Parkinson s disease. Similarly, DADLE blocked and reversed the dopaminergic terminal damage induced by methamphetamine (METH). Such neuroprotective effects of DADLE against METH neurotoxicity was accompanied by attenuation of mRNA expressions of a tumor necrosis factor p53 and an immediate early gene c-fos. In parallel to these beneficial effects of DADLE on the dopaminergic system, DADLE also ameliorated the neuronal damage induced by ischemia-reperfusion following a transient middle cerebral artery occlusion. In vitro replication of this ischemia cell death by serum-deprivation of PC12 cells revealed that DADLE exerted neuroprotection in a naltrexone-sensitive manner. These results taken together suggest that DADLE stands as a novel therapeutic agent. In this review paper, we present laboratory evidence supporting the use of DADLE for protection of peripheral and central nervous system.

Role of lipids in the assembly of endoplasmic reticulum and dictyosomes in neuronal cells from the cerebral cortex of Yakutian ground squirrel (Citellus undulatus) during hibernation.

Phospholipids and cholesterol were assayed in homogenates and microsomal fractions from the cerebral cortex of summer-active, winter-torpid, and winter-active Yakutian ground squirrels (Citellus undulatus). Ultrastructural analysis of both microsomal fraction and intact neurons was performed by serial ultramicrotomy. The levels of sphingomyelin (SM), phosphatidylserine (PS), and phosphatidylethanolamine (PEA) were decreased in homogenates from the cerebral cortex of winter ground squirrels compared with the summer-active animals, while the levels of phosphatidylcholine (PC) and cardiolipin (CL) were increased. The level of cholesterol was decreased in the cerebral cortex of winter-torpid animals compared with both winter-active and summer-active animals, and the level of total phospholipids was decreased in comparison to the summer-active animals. Three-dimensional reconstruction of serial membrane profiles displayed the microsomal fraction to be an interconnected system of cisterns and vesicles, which corresponds to endoplasmic reticulum and dictyosomes (Golgi stacks) of intact neurons. In winter the content of PC was increased in the microsomal fraction, while the contents of lysophosphatidylcholine (LPC), PS, phosphatidylinositol (PI), and SM were decreased. In winter-torpid animals compared with the winter-active ones the contents of total phospholipids, PEA, LPC, and cholesterol were decreased. As for the winter-active ground squirrels, their lipid contents did not differ from those in the summer-active animals, but LPC content was decreased. The changes in microsomal lipid contents in intact pyramidal neurons throughout the hibernation were accompanied by disassembly of dictyosomes and endoplasmic reticulum (ER), including the decomposition of polyribosomes to monosomes. The ultrastructural analysis of nucleoli, ER, and dictyosomes of both winter-active and torpid ground squirrels showed a direct correlation between the increasing contents of both cholesterol and total phospholipids (mainly PEA and LPC) in microsomes and the structural recovery of endoplasmic reticulum, Golgi stacks, and nucleoli in intact pyramidal neurons. A role of seasonal variations in lipid contents of brain cells in their adaptation to low temperature is discussed. We also propose an involvement of cholesterol in the activation of protein-synthesizing function of endoplasmic reticulum and Golgi stacks in intact neurons.

Delta opioid peptide[D- Ala(2),D-Leu(5)]enkephalin promotes cell survival.

By studying the hibernation in ground squirrels, a protein factor termed hibernation induction trigger (HIT) was found to induce hibernation in summer-active ground squirrels. Further purification of HIT yielded an 88-kD peptide that is enriched in winter hibernator. Partial sequence of the 88-kD protein indicates that it may be related to the inhibitor of metalloproteinase. Delta opioid [D-Ala(2),D-Leu(5)]enkephalin (DADLE) also induced hibernation. HIT and DADLE were found to prolong survival of peripheral organs preserved en bloc or as a single preparation. These organs include the lung, the heart, liver and kidney. DADLE also promotes survival of neurons in the central nervous system. Methamphetamine (METH) is known to cause destruction of dopaminergic (DA) terminals in the brain. DADLE blocked and reversed the DA terminal damage induced by METH. DADLE acted against this effect of METH at least in part by attenuating the mRNA expressions of a tumor necrosis factor p53 and an immediate early gene c-fos. DADLE also blocked the neuronal damage induced by ischemia-reperfusion following a transient middle cerebral artery occlusion. In PC12 cells, DADLE blocked the cell death caused by serum deprivation in a naltrexone-sensitive manner. Thus, DADLE, and by extension the endogenous delta opioid peptides and delta opioid receptors, may play an important role in organ and neuronal survival. Here, critical developments concerning these fascinating cell protective properties of DADLE are reviewed.

Isolation and partial characterization of an opioid-like 88 kDa hibernation-related protein

Previous studies show that infusion of hibernating woodchuck albumin (HWA) induces hibernation in summer-active ground squirrels and results in profound behavioral and physiological depression in primates. These effects are reversed by the administration of opiate antagonists, suggesting that the putative hibernation induction trigger (HIT) may act through opioid receptors. We have demonstrated that both HIT-containing plasma and the synthetic α opioid d-Ala 2- d-Leu 5-enkephalin (DADLE), which mimics the activity of HIT in hibernators, extend tissue survival time of a multi-organ autoperfusion system by 3-fold. In this study we present the first data showing biological activity with a much more highly purified plasma fraction from hibernating woodchucks, identified as the hibernation-related factor (HRF). Both the HRF and DADLE show opiate-like contractile inhibition in the mouse vas deferens (Mvd) bioassay. We also have preliminary evidence in an isolated rabbit heart preparation indicating that the HRF and DADLE act similarly to restore left ventricular function following global myocardial ischemia. Furthermore, we have partially sequenced an α 1-glycoprotein-like 88 kDa hibernation-related protein (p88 HRP) present in this fraction, which may prove to be the blood-borne HIT molecule.

Further studies on opioids and hibernation: Delta opioid receptor ligand selectively induced hibernation in summer-active ground squirrels

To examine the possible involvement of multiple opioid receptors in animal hibernation, we infused opioids selective for mu , kappa , and delta opioid receptors into summer-active ground squirrels ( Citellus tridecemlineatus ). The effects of those opioid treatments on the hibernation induced by HIT (Hibernation Induction Trigger) were also examined. Mu opioids morphine (1.50 mg/kg/day) and morphiceptin (0.82 mg/kg/day) and kappa opioid peptide dynorphin A (0.82 mg/kg/day) did not induce hibernation. On the contrary, morphine, morphiceptin and dynorphin A antagonized HIT-induced hibernation in summer-active ground squirrels. Infusion of delta opioid DADLE (D-Ala 2-D-Leu 5 enkephalin; 1.50 mg/kg/day), however, induced summer hibernation in a manner comparable to that induced by HIT. It is concluded therefore that delta opioid receptor and its ligand may be intimately involved in animal hibernation. In view of the fact that HIT was obtained from winter hibernating animals and might therefore be responsible for natural hibernation, our results also suggest that naturally occuring mu and kappa opioids may play an important role in the arousal state of hibernation.

Opioids and hibernation. II. Effects of kappa opioid U69593 on induction of hibernation in summer-active ground squirrels by “hibernation induction trigger” (HIT)

A “Hibernation Induction Trigger” (HIT) isolated from plasma of winter-hibernating woodchucks induced hibernation in summer-active ground squirrels ( Citellus tridecemlineatus ). Effects of kappa opioid U69593 on the HIT-induced hibernation were examined. U69593 alone did not elicit marked behavioral alteration or hibernation in summer-active ground squirrels. U69593, however, antagonized hibernation induced by HIT in summer active ground squirrels. In the guinea pig ileum myenteric plexus-longitudinal muscle preparation, woodchuck HIT depressed the electrically-induced contraction. The depression was, however, neither reversed nor blocked by naloxone even when naloxone was used at high doses. This study demonstrates that kappa opioid, at least in the case of U69593, was unable to induce hibernation in the summer-active ground squirrels. The results also demonstrate that woodchuck HIT, like the bear HIT, did not act directly at opioid receptors. Together with our previous observation that naloxone blocked summer hibernation induced by HIT (Bruce et al. , Life Sci., this issue), it is tempting to suggest that HIT may not mediate its effects through kappa opioid receptors but may do so through other types of opioid receptors such as mu or delta . U69593 may antagonize HIT-induced hibernation as a mu or delta receptor antagonist.

Seasonal changes in mitochondrial succinate dehydrogenase activity in a hibernator,Spermophilus richardsonii

1. Liver mitochondrial functions were evaluated in Richardson's ground squirrels during different phases of the annual hibernation cycle. 2. Arrhenius plots of succinate oxidase-linked H+ ejection, Ca2+ uptake and O2 consumption exhibited discontinuities (Tc) near 21 °C in summer-active (July) ground squirrels and at 11–13 °C in warm (20 °C)- or cold (4 °C)-acclimated winter animals. No discontinuities were detected in some warm- and cold-acclimated individuals which had not yet exhibited torpor. 3. Arrhenius plots of animals killed during hibernation (Tb=4 °C) or 12–14 h after arousal from hibernation (Tb=37 °C) were linear between 4–37 °C. The rates of mitochondrial O2 consumption were markedly depressed in hibernating animals. 4. Spring (April, emerged from hibernation) animals showed a return to discontinuous Arrhenius plots with aTc near 12 °C. 5. The results indicate seasonal differences in the thermal behaviour of mitochondrial membrane functions commensurate with hibernation. The lowering of aTc in euthermic animals between summer and winter and the absence of aTc in hibernating and aroused animals suggest these seasonal changes are independent of ambient and body temperature.

Absence of evidence for a hibernation “trigger” in blood dialyzate of Richardson's ground squirrel

In order to examine evidence for a blood-borne “trigger” for mammalian hibernation, serum dialyzate from hibernating Richardson's ground squirrels (Spermophilus richardsonii) was injected into summer-active ground squirrels of the same species. Four independent trials involving 52 animals were performed. In all trials, no effect of the dialyzate was seen on nest building, weight gain or loss, or on occurrence of hibernation.

Isolation of a hibernation inducing trigger(s) from the plasma of hibernating woodchucks.

Plasma from hibernating woodchucks was desalted utilizing a hollow fiber device having a M. W. cut-off of 5,000. This preparation was fractionated by isoelectric focusing (IEF) in a pH gradient extending from 3.5 to 10.0 resulting in protein components having isoelectric points (pIs) of 4.5, 5.2, 5.5, 6.3, and 7.0. Fraction I (comprised of proteins having pIs of 4.5 and 5.2) induced hibernation within 2 to 6 days in 8 out of 10 summer-active ground squirrels. Fraction II (pI 5.5) and Fraction III (pI 6.3 and 7.0) failed to induce any summer hibernation in 10 animal test groups at identical sample concentrations. Polyacrylamide gel electrophoresis of Fraction I indicated that albumin was a major constituent of this still heterogeneous preparation. Thus, in order to more clearly define the plasma locus of this hibernation inducing trigger(s) (HIT) molecule, whole plasma and/or Fraction I was fractionated by 3 distinct resolving techniques. These included sub-fractionation of Fraction I by isoelectric focusing utilizing a narrower pH gradient extending from 3.5 to 6.0, isotachophoresis of whole plasma and affinity chromatography of Fraction I and whole plasma. A total of 40 summer-active ground squirrels were injected and assayed for HIT activity with fractionated preparations derived by the three previously cited separation techniques. A total of 18 of these summer-active ground squirrels hibernated. However, a much more impressive figure is that 16 out of 21 animals hibernated when injected with resolved hibernating plasma fractions in which albumin was the predominant plasma protein. A total of 8 control animals were injected with vehicle and none of these hibernated.