Comparative analysis of the kinetic characteristics of L-type calcium channels in cardiac cells of hibernators.

An undefined property of L-type Ca2+ channels is believed to underlie the unique phenotype of hibernating hearts. Therefore, L-type Ca2+ channels in single cardiomyocytes isolated from hibernating versus awake ground-squirrels (Citellus undulatus) were compared using the perforated mode of the patch-clamp technique, and interpreted by way of a kinetic model of Ca2+ channel behavior based upon the concept of independence of the activation and inactivation processes. We find that, in hibernating ground-squirrels, the cardiac L-type Ca2+ current is lower in magnitude when compared to awake animals. Both in the awake or hibernating states, kinetics of L-type Ca2+ channels could be described by a d2f1(2)f2 model with an activation and two inactivation processes. The activation (or d) process relates to the movement of the gating charge. The slow (or f1) inactivation is associated with movement of gating charge and is current-dependent. The rapid (or f2) inactivation is a complex process which cannot be represented as a single-step conformational transition induced by the gating charge movement, and is regulated by beta-adrenoceptor stimulation. When compared to awake animals, the kinetic properties of Ca2+ channels from hibernating ground-squirrels differed in the following parameters: (1) pronounced shift (15-20 mV) toward depolarization in the normalized conductance of both inactivation components, and moderate shift in the activation component; (2) 1.5-2-fold greater time constants; and (3) two-fold greater activation gating charge. Thus, L-type Ca2+ channels apparently switch their phenotype during the hibernating transition. Stimulation of beta-adrenoceptors by isoproterenol, reversed the hibernating kinetic- (but not amplitude-) phenotype toward the awake type. Therefore, an aberrance in the beta-adrenergic system can not fully explain the observed changes in the L-type Ca2+ current. This suggests that during hibernation additional mechanisms may reduce the single Ca2+ channel-conductance and/or keep a fraction of the cardiac L-type Ca2+ channel population in a non-active state.     

Torpor and digestion in food-storing hibernators

Many species of hibernating mammals rely on hoarded food rather than body fat to support winter energy requirements. Here, we evaluate whether the associated ingestive and digestive requirements reduce the benefits that food-storing hibernators can accrue from torpor. Using a simple model, we predict (1) that digestive efficiency could either increase or decrease with increased use of torpor, depending on the Q(10) of digestion relative to the Q(10) of whole-animal metabolism and (2) that increased torpor will result in a linear decrease in energy consumption but an exponential increase in euthermic intake requirements. In 16 captive eastern chipmunks (Tamias striatus), the proportion of time that different individuals spent in torpor was highly variable (29.8%+/-5.9%; 0.0%-86.3%), positively correlated with dry matter digestibility (r2=0.53, P=0.02) and negatively correlated with energy consumption (r2=0.72, P=0.002). Thus, by both increasing conversion efficiency and reducing energy requirements, torpor appears to provide a double benefit for energy conservation by food-storing hibernators. Despite this, a comparative analysis shows that the euthermic intervals of food-storing rodents are four times as long and torpor intervals are half as long as that of fat-storing rodents. Given that required euthermic intake rates are expected to increase exponentially at high levels of torpor, the reduced torpor expression of food-storing species may result from constraints on their ability to load enough food into the gut when euthermic to cover the energy requirements of the subsequent torpor cycle.     

Ventricular fibrillation in hibernators and nonhibernators

Previous studies have shown that there are differences between hibernators and nonhibernators in the susceptibility to ventricular fibrillation. In an attempt to clarify these differences ventricular fibrillation was induced in isolated hearts of the hibernator, the woodchuck, Marmota monax by cooling, warming, puncture, and by norepinephrine administration. It was shown that the hearts of the winter animals were completely resistant toward the ventricular fibrillation inducing agents, which was not the case for the hearts from summer, active animals. Further, the hearts of another hibernator, the hedgehog, Erinaceus europaeus, and guinea pig, Cavia porcellus, were studied electrophysiologically in anesthetized animals with open chests and with bipolar electrodes attached to the epicardium. During pacing it was shown that the hedgehog had a higher stimulus threshold and a lower maximal following frequency than the guinea pig. The summer hedgehogs showed resistance toward both ventricular premature beats and ventricular fibrillation. Sixty percent of the summer hedgehogs and 100 percent of the winter hedgehogs and guinea pigs developed ventricular fibrillation. The threshold for ventricular fibrillation was highest for summer hedgehogs. The effective refractory period of papillary muscle of summer hedgehogs was shorter than that of guinea pigs. The force frequency relationship of the isolated papillary muscle showed a greater degree of independence in the hedgehog than in the guinea pig. Consequently, the results show that the heart of the hibernator is more arrhythmia resistant than the heart of the nonhibernator, although there are seasonal differences.     

Functions of fat in hibernators: Thermal aspects

1. 1.|A numerical, distributed parameter model of heat exchange is used to evaluate the thermal significance of the presence and placement of subcutaneous fat in hibernating marmots. The model is most sensitive to changes in conductivity and metabolic rate of muscle tissue, parameters which are known to greater precision than are others in the model.

2. 2.|Alternative models are developed for animals with fat located dorsally and with no fat at all.

3. 3.|A comparison of these three models shown that there is no difference in the metabolic output required to maintain body temperature among the three alternatives. Therefore, neither the presence nor the location of fat serves an insulative role in hibernators.

Comparative analysis of cell surface proteins in chronic and acute leukemia cell lines

This study was designed to identify the cell surface protein markers that can differentiate between chronic myeloid leukemia (CML) and acute promyelocytic leukemia cells (APL). The differentially expressed plasma membrane proteins were analyzed between CML cell line (K562) and APL cell line (NB4) using the comparative proteomic approach. The cell membrane proteins were enriched by labeling with a membrane-impermeable biotinylation reagent, sulfo-NHS-SS-Biotin, and subjected to liquid chromatography tandem mass spectrometry (LC-MS/MS). By comparative proteomic analysis of K562 and NB4 cells, we identified 25 membrane and 14 membrane-associated proteins. The result of LC-MS/MS combined with chemical tagging method was validated by confirming the expression and localization of one of the differentially expressed plasma membrane proteins, CD43, by FACS and confocal microscopy. Our results indicate that CD43 could be a potential candidate for differentiating CML from APL.     

Proteome-wide analysis of neural stem cell differentiation to facilitate transition to cell replacement therapies

Neurodegenerative diseases are devastating disorders and the demands on their treatment are set to rise in connection with higher disease incidence. Knowledge of the spatiotemporal profile of cellular protein expression during neural differentiation and definition of a set of markers highly specific for targeted neural populations is a key challenge. Intracellular proteins may be utilized as a readout for follow-up transplantation and cell surface proteins may facilitate isolation of the cell subpopulations, while secreted proteins could help unravel intercellular communication and immunomodulation. This review summarizes the potential of proteomics in revealing molecular mechanisms underlying neural differentiation of stem cells and presents novel candidate proteins of neural subpopulations, where understanding of their functionality may accelerate transition to cell replacement therapies.