The behavior of titin and the proteins of its family from skeletal muscles of ground squirrel (Citellus undulatus) during hibernation and rats under conditions of simulated microgravity

By the use of SDS PAGE, the behavior of titin and MyBP-C in fast (m. psoas) as well as titin and MyBP-X in slow (m. soleus) muscles of ground squirrels (Citellus undulatus) during hibernation was compared with the behavior of titin and MyBP-X in rat m. soleus under conditions of simulated microgravity. A decrease in the amount of titin 1 and MyBP-C relative to that of myosin heavy chains by approximately 30% and approximately 40%, correspondingly, in muscles of hibernating and arousing ground squirrels was revealed in comparison with active animals. No differences in the relative amount of MyBP-X in m. soleus of hibernating, arousing and active ground squirrels were found. Under conditions of simulated microgravity, a decrease in the amount of titin 1 by approximately 2 times and MyBP-X by approximately1.5 times relative to that of myosin heavy chains in rat m. soleus was observed. By the method of SDS PAGE modified by us, an almost twofold decrease in the amount of short isovariants of the titin N2A isoform relative to that of myosin heavy chains was shown in muscles of hibernating and arousing ground squirrels, whereas no changes were found in the amount of long titin isovariants. The conditions of simulated microgravity resulted in a twofold decrease in the relative amount of both short and long titin isovariants in rat m. soleus. The results indicate that hibernating ground squirrels have an evolutionarily determined adaptive mechanism of selective degradation of fast muscle fibers and preservation or increase of slow fibers, as the most economic and energetically advantageous, with proteins typical of them. The microgravitation of nonhibernating animals (rats) leads to a non-selective degradation of MyBP-X and titin isovariants, which contributes to considerable atrophy of soleus fibers.     

Properties of C protein from skeletal and cardiac muscles of the ground squirrel Citellus undulatus at various stages of hibernation

Changes in the molecular weight and functional properties of the C and X proteins from skeletal muscles and the C protein from the cardiac muscle of hibernating ground squirrels Citellus undulatus at different stages of the hibernation were studied. A decrease in the molecular weight of the C protein from fast fibers of skeletal muscles of hibernating ground squirrels compared with awakening and active animals was revealed. The appearance of shorter molecules of the C protein upon hibernation was accompanied by a lowering of its capacity to enhance the actin-activated ATPase activity of control myosin and by the inhibition of its Ca(2+)-sensitivity. No similar changes were observed for the skeletal X protein and the cardiac C protein. The influence of the skeletal C protein on the main functional properties of myosin allows one to draw a conclusion about its contribution to the inhibition of contractile activity of skeletal muscles upon hibernation. The physiological significance of the changes in the C protein upon hibernation is discussed in connection with similar changes in some cardiomyopathies.     

Composition of titin isoforms of skeletal and cardiac muscles in pathologies

An electophoretic study of changes in composition of titin isoforms in human and rat skeletal and cardiac muscles is carried out. A more considerable decrease in the content of intact titin isoforms was observed than in the content of N2A-titin in the dorsal muscle of patients with the “stiff-person syndrome” and in m. soleus of humans and rats during development of “muscle hypogravity syndrome” and than in the content of N2BA- and N2B-titins in hypertrophic heart of spontaneously hypertensive rats. The relation between reduction of titin content in m. soleus and the increase of time the rats were in conditions of simulated microgravity is revealed. On electrophoregrams of left ventricle myocardium of patients with terminal stage of dilated cardiomyopathy the intact titin and N2BA-titin were absent and a considerable decrease in the content of N2B-titin was observed. This could be the consequence of the terminal stage of pathology. It follows that development of the diseases is accompanied by a greater destruction of intact titin than of its other forms which may be important for diagnostics of pathological processes.     

Seasonal changes in the composition of titin isoforms in muscles of hibernating ground squirrels

An electophoretic study of changes in the content of intact titin isoforms, N2B-, N2BA-, N2A-titins and T2 in skeletal and cardiac muscles of ground squirrel (Spermophillus undulatus) is made in different periods: summer activity, autumnal activity, hibernation, arousal, and winter activity. In atria and ventricles of ground squirrels in the period of autumnal activity an increase (by ～1.5 times) in the N2BA to N2B ratio was observed, in comparison with that in cardiac muscle in summer activity. During hibernation, the decrease in the relative content of N2B-, N2BA-titins and T2 in cardiac muscle as well as of N2A-titin and T2 in skeletal muscles was determined against the background of preservation of the relative amount of intact titin isoforms. At waking of ground squirrels and in a short period of winter activity, a rapid restoration of the content of N2B-, N2BA-, N2A-titisns and T2 in muscles was observed. In the myocardium of hibernating, waking ground squirrels and of those during winter activity the increased N2BA to N2B ratio was retained. The changes in the titin content are discussed in the aspect of adaptation of ground squirrels to hibernation.     

Myosin light chains of skeletal and cardiac muscles of ground squirrel Citillus undulatus in different periods of hibernation

The isoform composition of myosin light chains and the extent of their phosphorylation in skeletal and cardiac muscles of ground squirrel Citellus undulatus in different periods of hibernation were studied. Regulatory myosin light chains of skeletal muscles of hibernating ground squirrels were completely dephosphorylated, while 25% of these light chains in active animals were phosphorylated. During hibernation, a shift of isoform composition of essential and regulatory skeletal muscle myosin light chains toward slower isoforms was observed, which is evidenced by the data obtained on m. psoas and on the totality of all skeletal muscles. In the atrial myocardium of hibernating ground squirrels, ventricular myosin light chains 1 (up to 60%) were registered. In contrast, during arousal of ground squirrels, in ventricular myocardium the appearance of atrial myosin light chains 1 (up to 30%) was revealed. A possible role of posttranslation changes in myosin light chains and their isoform shifts in the hibernation scenario is discussed.     

Production of tumor necrosis factor in cells of hibernating ground squirrels Citellus undulatus during annual cycle

TNF production has been studied in peritoneal macrophages and splenic T cells of Arctic Yakutian ground squirrel (Citellus Undulatus Pallas) in hibernating and awake animals in winter and in prehibernating autumn as well as in active euthermic spring-summer animals. A high level of TNF production in macrophages of ground squirrel is observed over the active period and during arousals in winter. There are no significant season variations in TNF production in splenic T lymphocytes of ground squirrels. This suggests the major role of activated macrophages in the arousals of hibernating animals. T lymphocyte proliferation in ground squirrels in the active period is higher than in winter, and the most significant seasonal variations are found in T cell mitogenic response, which increases in spring-summer period. Evidence is presented that functional activity of macrophages of squirrel in autumn has much in common with that in winter rather than in spring-summer period.     

On the growth of incisors in ground squirrels (Spermophilus) during hibernation

The growth of incisors was studied in two Spermophilus parryii and two S. undulatus ground squirrels with DS-1922L temperature data loggers implanted in the peritoneal cavity, which were kept under laboratory conditions. Daily increments on the incisors surface were similar to those in other species of ground squirrels, but they were less distinct and regular than in wild-living conspecific individuals from the same region. Two S. parryii and one S. undulatus ground squirrels entered hibernation and successfully overwintered. Despite some anomalies in their incisors, changes in body temperature during hibernation (recorded by the data loggers) had an effect on the pattern of their growth, resulting in the formation of a “hibernation zone” on the incisor surface. The number of narrow increments within this zone roughly corresponded to the number of alternating periods of torpor and euthermia during hibernation. This could be regarded as evidence that the incisors of the animals studied continued growing throughout hibernation, including the period of deep hibernation, with the rhythm of their growth coinciding with the rhythm of changes in body temperature. The effect of spontaneous trauma of an upper incisor on the growth of other incisors is described.     

Regulation of the rate of respiration and oxidative phosphorylation in liver mitochondria from hibernating ground squirrels, Citellus undulatus

1. The rates of oxidation of various substrates (beta-hydroxybutyrate, succinate, ascorbate + TMPD) and the rate of ATP synthesis in liver mitochondria from active and hibernating ground squirrels were measured. 2. It was shown that the rate of mitochondrial respiration is significantly lower in hibernating animals than in active animals. 3. The degree of inhibition of mitochondrial respiration in hibernating ground squirrels was found to correlate with the length of the respiratory chain fragment involved in the oxidation of a given substrate. 4. The inhibition of mitochondrial respiration in hibernating animals was accompanied by a decrease in the rate of ATP synthesis. 5. The activity of phospholipase A2 in liver mitochondria from hibernating ground squirrels was found to be decreased. The activation of phospholipase A2 by Ca2+ ions eliminated the inhibition of respiration almost completely. 6. It was assumed that the inhibition of mitochondrial respiration during hibernation is (a) related to the suppression of phospholipase A2 activity and (b) caused by the reduced rates of electron transport through the respiratory chain and/or of substrate transport across the mitochondrial membrane.     

Summer hibernation induced in ground squirrels (Citellus tridecemlineatus) by urine or plasma from hibernating bats (Myotis lucifugus or Eptesicus fuscus)

Summer hibernation induced in ground squirrels (Citellus tridecemlineatus) by urine or plasma from hibernating bats (Myotis lucifugus or Eptesicus fuscus). Summer hibernation in the thirteen-lined ground squirrel can be induced by intravenous injection of urine or blood plasma previously isolated from winter hibernating little brown bats (M. lucifugus) or big brown bats (E. fuscus). Urine- and plasma-injected ground squirrels kept at 8 °C hibernated earlier, longer, and deeper (as indicated by core temperature and respiratory rate measurements) than control ground squirrels injected with saline. This successful cross-order induction of hibernation demonstrates that the hibernation-inducing trigger (HIT) may be present in nonrodent mammals.     

Changes of activity of the protein-synthesizing system of brain neurons of the ground squirrel Citellus undulatus during hibernation and hypothermia

Using fluorescent and electron microscopy a comparative analysis was performed of components of the protein-synthesizing system of hippocampal neurons both in ground squirrels in various phases of the torpor-activity cycle and in rats cooled under the hypoxia-hypercapnia conditions. Results of the study have shown that in hippocampal neurons of the ground squirrels entering the natural torpor state and of rats under conditions of artificial hypothermia to 17°C, similar mechanisms might be possible to function, one of their obligatory components being a generalized decrease of activity of the protein-synthesizing system with its subsequent restoration at the exit from hypothermia. Cessation of hypoxia-hypercapnia even under conditions of a further temperature decrease restored the rat neuronal protein-synthesizing activity, which seems to indicate the presence of a potential possibility of adaptation of brain neurons in vivo to low temperatures, at which the integral organism of non-hibernating homoeothermic animals does not survive.     

Regulation of Akt during hibernation in Richardson's ground squirrels

Akt (or protein kinase B) plays a central role in coordinating growth, survival and anti-apoptotic responses in cells and we hypothesized that changes in Akt activity and properties would aid the reprioritization of metabolic functions that occurs during mammalian hibernation. Akt was analyzed in skeletal muscle and liver of Richardson's ground squirrels, Spermophilus richardsonii, comparing the enzyme from euthermic and hibernating states. Akt activity, measured with a synthetic peptide substrate, decreased by 60–65% in both organs during hibernation. Western blotting showed that total Akt protein did not change in hibernation but active, phosphorylated Akt (Ser 473) was reduced by 40% in muscle compared with euthermic controls and was almost undetectable in liver. Kinetic analysis of muscle Akt showed that S_0.5 values for Akt peptide were 28% lower during hibernation, compared with the euthermic enzyme, whereas S_0.5 ATP increased by 330%. Assay at 10 °C also elevated S_0.5 ATP of euthermic Akt by 350%. Changes in ATP affinity would limit Akt function in the hibernator since the muscle adenylate pool size is also strongly suppressed during cold torpor. Other parameters of euthermic and hibernator Akt were the same including activation energy calculated from Arrhenius plots and sensitivity to urea denaturation. DEAE Sephadex chromatography of muscle extracts revealed three peaks of Akt activity in euthermia but only two during hibernation suggesting isozymes are differentially dephosphorylated during torpor. Altered enzyme properties and suppression of Akt activity would contribute to the coordinated suppression of energy-expensive anabolic and growth processes that is needed to maintain viability during over weeks of winter torpor.     

Carboxyatractylate-sensitive uncoupling in liver mitochondria from ground squirrels during hibernation and arousal

Energy coupling parameters of liver mitochondria from hibernating and arousing ground squirrels have been studied. In the oligomycin-treated mitochondria, carboxyatractylate, an inhibitor of the ATP/ADP-antiporter, is shown to decrease the respiration rate, to increase the membrane potential and to lower the rate of the membrane-potential discharge after the addition of cyanide to liver mitochondria from hibernating and arousing animals. BSA effectively substitutes for carboxyactactylate so that carboxyactactylate, added after BSA, has no effect. In mitochondria from hibernating animals, the maximal respiration rate in the presence of DNP and the rate of the membrane potential discharge in its absence are much lower than in those from arousing animals. It has been concluded that upon arousal of the animals from hibernation, the uncoupling of oxidative phosphorylation, mediated by free fatty acids and ATP/ADP-antiporter, parallels the respiratory chain activation.     

Regulation of Akt during hibernation in Richardson's ground squirrels

Akt (or protein kinase B) plays a central role in coordinating growth, survival and anti-apoptotic responses in cells and we hypothesized that changes in Akt activity and properties would aid the reprioritization of metabolic functions that occurs during mammalian hibernation. Akt was analyzed in skeletal muscle and liver of Richardson's ground squirrels, Spermophilus richardsonii, comparing the enzyme from euthermic and hibernating states. Akt activity, measured with a synthetic peptide substrate, decreased by 60-65% in both organs during hibernation. Western blotting showed that total Akt protein did not change in hibernation but active, phosphorylated Akt (Ser 473) was reduced by 40% in muscle compared with euthermic controls and was almost undetectable in liver. Kinetic analysis of muscle Akt showed that S(0.5) values for Akt peptide were 28% lower during hibernation, compared with the euthermic enzyme, whereas S(0.5) ATP increased by 330%. Assay at 10 degrees C also elevated S(0.5) ATP of euthermic Akt by 350%. Changes in ATP affinity would limit Akt function in the hibernator since the muscle adenylate pool size is also strongly suppressed during cold torpor. Other parameters of euthermic and hibernator Akt were the same including activation energy calculated from Arrhenius plots and sensitivity to urea denaturation. DEAE Sephadex chromatography of muscle extracts revealed three peaks of Akt activity in euthermia but only two during hibernation suggesting isozymes are differentially dephosphorylated during torpor. Altered enzyme properties and suppression of Akt activity would contribute to the coordinated suppression of energy-expensive anabolic and growth processes that is needed to maintain viability during over weeks of winter torpor.     

The hypothalamo-hypophysial system in the ground squirrels, Citellus erythrogenys Brandt and Citellus undulatus Pallas. I. Microanatomy and cytomorphology of the gomori-positive neurosecretory system with special reference to its state during hibernation.

Microanatomy of the peptidergic hypothalamo-hypophysial neurosecretory system in the ground squirrels, C. erythrogenys and C. undulatus, is similar to that of other rodents, but differs in details. Bundles of neurosecretory fibres with single neurosecretory cells along their course connect the hypothalamic neurosecretory centers into the one system. The dorso-caudal portions of the NPV reach the level of the columna fornix. The NPO is a special indepedent formation and appears to have some specific functions in the HHNS. The infundibular recess penetrates deeply ihto the hypophysial stem, but does not reach the posterior pituitary. The glandular epithelium of the tuberal portions of the adenohypophysis surrounds the median eminence and a considerable part of the lateral surface of the tuber cinereum. Different functional types of neurosecretory cells reflect certain phases of their secretory cycle. Correlations between their size, structure and the content of NSM are found. A low activity of the HHNS is observed in both C. erythrogenys and C. undulatus during December-January. A higher activity of the HHNS in torpid C. undulatus than in C. erythrogenys is established. This correlates with a difference in deepness of torpor in C. erythrogenys and C. undulatus. An important role of the HHNS in hibernation, i.e. in realization of adaption of species in ontogenesis is suggested.     

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.     

Myocyte enhancer factor-2 and cardiac muscle gene expression during hibernation in thirteen-lined ground squirrels

Long non-coding RNA urothelial carcinoma associated 1 (UCA1) promotes human bladder cancer cell proliferation, but the underlying mechanism remains unknown. After knocking down of UCA1 in BLZ-211 cells, several cell cycle-related genes (CDKN2B, EP300 and TGFβ-2) were screened by microarray assay and validated by real-time PCR. Interestingly, in western blot analysis, p300 (encoded by EP300) and its coactivator cAMP response element-binding protein (CREB) level were significantly down-regulated. Both suppression of UCA1 expression by shRNA in BLZ-211 cells and ectopic expression of UCA1 in UMUC-2 cells showed that UCA1 alteration paralleled to the expression and phosphorylation of CREB, and UCA1 obviously influenced AKT expression and activity. Furthermore, in BLZ-211 cells, cell cycle progression was greatly reduced after PI3-K pathway was blocked by LY294002, indicating that UCA1 affected cell cycle progression through CREB. Taken together, we concluded that UCA1 regulated cell cycle through CREB via PI3K-AKT dependent pathway in bladder cancer.     

Heat production in the ground squirrel Citellus pygmaeus during arousal from hibernation]

Heat production has been both measured experimentally (Qcal) and calculated from oxygen consumption (QO2) in arousing ground squirrels during the rise of their body temperature. Studies were made on total heat production during all the period of their warming as well as on the heat production at various stages of arousal. Qcal was evaluated by changes in body temperature and those in heat losses via convection and irradiation (calorimetrically). During arousal of animals, their body temperature, heat losses, Qcal and QO2 Gradually increase. However, the increase in heat losses is 3-4 times lower as compared to the intrinsic heat production measured both calorimetrically and by oxygen consumption. Limitation of heat losses (due to the constriction of subcutaneous blood vessels) together with activation of the metabolism in muscles and other tissues provide for significant heat accumulation and the increase in body temperature of arousing ground squirrels.     

Seasonal changes in the isoform composition of the myosin heavy chains in skeletal muscles of hibernating ground squirrels Spermophilus undulatus

Seasonal changes of the isoform composition of myosin heavy chains in skeletal muscles (m. triceps, m. longissimus dorsi, m. soleus, m. gastrocnemius, m. vastus lateralis) of hibernating ground squirrels Spermophilus undulatus were studied. Functional properties of myosin (the actin-activated ATPase activity and its Ca²⁺-sensitivity in vitro) were also examined. It was observed that the content of slow myosin heavy chain I isoform increased and the content of fast IIx/d isoform decreased in muscles of torpid ground squirrels and animals which are active in autumn and winter. In muscles of these animals the content of N2A-titin isoform decreased although the relative content of NT-titin isoform, observed in striated muscles of mammals in our previous experimental works, increased. Actin-activated ATPase activity and Ca²⁺-sensitivity of myosin isolated from skeletal muscles of torpid and interbout ground squirrels were found to reduce. The changes observed are discussed in the context of adaptation of skeletal muscles of ground squirrels to hibernation conditions.