Adaptive mechanisms of intracellular calcium homeostasis in mammalian hibernators
1 National Laboratory of Biomembrane and Membrane Biotechnology, College of
Life Sciences, Peking University, Beijing 100871, China
2 Laboratory of Cardiovascular Sciences, National Institute on Aging, NIH,
Baltimore, MD 21224, USA
* Author for correspondence (e-mail: wangsq{at}grc.nia.nih.gov)
Accepted 1 July 2002
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Summary |
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Key words: hibernation, Ca2+ metabolism, adaptation, excitationcontraction coupling, homeostasis
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Introduction |
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Two different strategies for probing calcium homeostatic mechanisms are: (1) the discovery of mechanisms of [Ca2+] dysregulation and development of methods to prevent or reverse the defects; and (2) investigation of wild natural models that show an extraordinary capability of handling intracellular Ca2+.
Hibernating mammals are one such special natural model. A mammalian
hibernator, like all other mammals, can maintain its body temperature
(Tb) at approximately 37°C during most of its
lifetime. But in winter, hibernators can actively regulate their
Tb down to only a few °C, entering into a distinct
state known as hibernation (for reviews, see
Lyman et al., 1982;
Wang, 1988
). During
hibernation, circulation and respiration are well maintained, although at much
lower rates than normal. Tb can be periodically,
temporarily restored during hibernation (the whole period of entry,
maintenance and arousal from a period of hibernation is termed `hibernation
bout'), indicating that neural regulation is still active, despite deep
hypothermia. A complete arousal occurs either upon external stimulation or as
`scheduled' by an internal clock, during which, in ground squirrels and
hedgehogs, normal Tb can be restored within 30 min by
internal heat production. In this hibernationarousal cycle, hibernators
have to survive a set of extreme conditions that are fatal to humans and other
non-hibernating mammals, including sustained deep hypothermia, violent shifts
in Tb, highly intensified sympathetic innervation (during
arousal), high viscosity and hypocoagulation of blood, and oxidative stress.
As an adaptation during evolution, hibernators exhibit distinct resistance to
hypothermia, arrhythmias (Johansson,
1996
) and hypoxia (for a review, see
Wang and Zhou, 1999a
).
In this brief review, we summarize the major known aspects of the adaptive mechanisms of intracellular Ca2+ homeostasis in hibernating mammals, and discuss their general significance and possible applications.
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Enhanced capability to maintain intracellular Ca2+ homeostasis |
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In contrast to [Ca2+] dysregulation in non-hibernating animals
and humans during hypothermia, [Ca2+] regulation in hibernator
cells is strikingly resistant to temperature change. At 30-10°C, resting
[Ca2+]i in heart cells from the ground squirrel
(Spermophilus dauricus, a well-characterized hibernating rodent)
changes very little (range 125±10 nmol Ca+ l-1)
(Wang et al., 1999;
Fig. 1). The dynamic amplitude
of Ca2+ transients following excitation is actually increased
during cooling (Wang et al.,
2000
), which may help to retain forceful contraction despite the
decreased Ca2+ sensitivity of myofilaments at low temperatures
(Khromov et al., 1990
;
Liu et al., 1993
). As a
result, cardiac muscle from the ground squirrel and the hedgehog (another
hibernating mammal) exhibited even higher contraction amplitudes at low
temperatures than at normal temperatures
(Liu et al., 1990
;
Wang et al., 1997b
), which is
an adaptive mechanism to ensure sufficient pumping pressure despite the
consequent increased blood viscosity and peripheral resistance.
The adaptive capability to maintain intracellular Ca2+
homeostasis in hibernator cells is associated with stable cell function
despite some pathological or stressful stimulii
(Johansson, 1996). It was
observed that, in hedgehog hearts, the epicardial application of aconitine,
administration of high concentrations of CaCl2, injection of
procaine after previous adrenaline treatment, or ligation of the left
descending coronary artery, each failed to induce the ventricular fibrillation
that usually occurs in guinea pig hearts in response to these perturbations
(Johansson, 1996
). During
experimental ischemiareperfusion paradigms, ground squirrel heart
showed significantly less injury, monitored by creatine kinase leakage, than
rat heart, suggesting that hibernator cells are resistant to the oxygen
paradox and calcium paradox (Gao et al.,
1996
). Brain cells of hibernating mammals are also protected
against a variety of insults that are detrimental to humans and other
nonhibernating species (for a review, see
Drew et al., 2001
), but the
relationship of the neural protection to [Ca2+]i
regulation still needs further study.
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Reduced Ca2+ entry through ion channels |
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The downregulation of sarcolemmal Ca2+ channels may help to
prevent excessive Ca2+ entry into cells during hypothermia. At low
temperatures, ion transport becomes slow, and the cell tends to become
depolarized owing to the loss of ionic gradients
(Wang et al., 1997a). If
depolarized to approximately -50 mV, a tonic, non-inactivating `window'
current of voltagegated Ca2+ channels usually becomes activated,
and may further depolarize the cell, leading to more influx of
Ca2+. As a result, cells become arrhythmic and calcium-overloaded.
Downregulation of voltage-gated Ca2+ channels during hibernation
decreases the chance of activation of the window current. Moreover, in
hibernating ground squirrels (S. undulatus) the activation threshold
of L-type Ca2+ channels shifts towards more positive potentials
(Alekseev et al., 1996
); the
cells in hibernators can also better maintain ionic gradients (for reviews,
see Willis, 1979
;
Wang, 1988
) and thereby
maintain their membrane potential (Liu et
al., 1991a
; Wang et al.,
1997a
) independently of temperature. These adaptive mechanisms
effectively prevent the excessive Ca2+ entry and intracellular
Ca2+ overload that would otherwise occur during hypothermia.
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Remodeling of cardiac excitationcontraction coupling |
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The enhanced contractility may be due either to increased Ca2+
transients or to an increase in myofiliment sensitivity to [Ca2+].
It was found that the myofiliment sensitivity to [Ca2+] decreases
as temperatures are lowered, in both hibernating and non-hibernating mammals
(Khromov et al., 1990;
Liu et al., 1993
). Although
myofiliments from hibernating ground squirrels (S. richardsonii)
exhibit a somewhat higher Ca2+ sensitivity at low temperature than
squirrels in the non-hibernating state, this still cannot fully explain the
observed enhanced contractility over a wide temperature range.
The size of the Ca2+ transients has not yet been compared in
animals in the hibernating and non-hibernating states, but all pharmacological
evidence to date supports the idea that SR Ca2+ release is
increased during hibernation. Blocking SR Ca2+ release by ryanodine
or caffeine caused greater inhibition of myocardial contraction in hibernating
chipmunks (Kondo and Shibata,
1984) and ground squirrels (S. richardsonii)
(Zhou et al., 1991
) than when
they were non-hibernating. This implies during hibernation, cardiac EC
coupling is remodeled so that a lower Ca2+ influx triggers a
greater Ca2+ release response.
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Enhanced Ca2+ uptake by intracellular Ca2+ store |
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A reduction in temperature leads to a reduction in the rate of
Ca2+ removal from the cytosol. Although the relaxation velocity of
myocardial contraction decreases monotonically as the temperature is lowered
in both rats and ground squirrels (S. dauricus), ground squirrel
myocardium shows a higher relaxation velocity at any temperature between
35°C and 10°C (Wang et al.,
1997b). [Ca2+]i measurements indicated that
the Ca2+ transient decays faster in ground squirrel cells than in
rat cells owing to a faster Ca2+ uptake rate by the SR
(Wang et al., 2000
). This
implies that the quantity and/or quality of SR and SERCA2 may be adaptively
modified in hibernators.
Indeed, ultrastructural analysis has revealed that the proportional volume
of SR in myocardium from hibernating ground squirrel
(Rosenquist, 1970;
Tang et al., 1995
) and hamster
(hibernator) (Skepper and Navaratnam,
1995
) is double or triple that of individuals that are
non-hibernating, and is mainly due to an increase in longitudinal SR, which
contains abundant Ca2+-ATPase and is responsible for
Ca2+ uptake. By contrast, the content of junctional SR, where
Ca2+-release channels are located, changes little
(Tang et al., 1995
; see
Table 1; Skepper and Navaratnam,
1995
).
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The increase in quantity of SR during hibernation is paralleled by an
increase in the Ca2+ uptake capacity of the SR. SR vesicles
isolated from winter-hibernating ground squirrels exhibit a faster rate of
Ca2+ uptake and a greater level of Ca2+ accumulation
than those from non-hibernating individuals either in winter or in other
seasons (Belke et al., 1991;
Tang et al., 1995
;
Fig. 4). Even in
non-hibernating ground squirrels, the Ca2+ uptake rate by the SR is
still higher than those in rats at temperatures between 35°C and 5°C
(Liu et al., 1997
).
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SERCA is the central protein involved in active SR/ER Ca2+
uptake against an electrochemical gradient. Since Ca2+ regulation
in the SR during hibernation is enhanced, it is surprising that the enzymatic
activity of SERCA is unchanged (Belke et
al., 1991). Although SERCA from ground squirrels (S.
richardsonii) is less temperature-sensitive than that from rats
(Liu et al., 1997
), the major
mechanism for enhanced SR Ca2+ uptake is reliant upon the increased
volume of SR present during hibernation.
Calsequestrin is a Ca2+-binding protein in the SR that binds
Ca2+ at a ratio of 40-50 Ca2+ per molecule, and greatly
increases the Ca2+ storage capacity of the SR, facilitating further
Ca2+ uptake by decreasing the free Ca2+ concentration in
the SR lumina. Calsequestrin also directly regulates the leakage and release
of Ca2+ via RyRs by the structural link between them
(Sitsapesan and Williams,
1997). In an early electron microscopic study, Rosenquist
(1970
) noticed that the
terminal cisternae of myocardial SR in hibernating ground squirrels exhibits a
higher electron density than those in non-hibernating animals, which suggests
an increased expression of calsequestrin during hibernation. A novel isoform
of calsequestrin has been identified in isolated cardiac SR from two species
of ground squirrels, with a molecular mass about 7% greater than that of
cardiac calsequestrin isolated from other mammals
(Milner et al., 1991
). The
increased molecular mass is partially due to its distinct glycosylation, which
appears to include an additional carbohydrate chain that is not present in
other isoforms. This molecular modification improves the binding of
Ca2+ to calsequestrin (Milner
et al., 1991
) and would thus be helpful in facilitating
Ca2+ uptake, suppressing Ca2+ leakage and increasing the
amount of Ca2+ available for release; therefore, this novel isoform
of calsequestrin enhances Ca2+-induced Ca2+ release in
cardiac cells during hibernation.
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The role of the Na+Ca2+ exchanger |
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Because the NCX does not consume ATP directly, it was hypothesized to be a
preferred Ca2+ transporting system during hibernation, when ATP
production is limited owing to the low temperature. The major evidence for
this idea is the observation that the cardiac action potential in hibernating
chipmunks (Kondo, 1987) and
ground squirrels (Wang et al.,
1995
) is characterized by a prolonged low-level plateau that is
sensitive to extracellular Ca2+ and Na+ concentrations.
However, this phenomenon may also be attributed to the enhanced amplitude or
prolonged duration of Ca2+ transient at low temperatures
(Wang et al., 2000
), which
drive NCX more quickly, but passively. Direct analysis of Ca2+
removal mechanisms in both ground squirrels (S. dauricus) and rats
has failed to establish a dominant role of NCX in removing Ca2+. On
the contrary, the fractional contribution of NCX to total Ca2+
removal is significantly less in ground squirrels than in rats
(Wang et al., 2000
). Moreover,
NCX contributes even less at lower temperatures in both species, consistent
with the observation that the temperature coefficient for NCX is even greater
than that for SERCA (Marengo et al.,
1997
). In addition, NCX is not efficient in terms of energy used.
By contrast to SERCA, which transports two Ca2+ per ATP molecule
consumed, NCX extrudes only one Ca2+ per ATP molecule (the three
Na+ that enter during the exchange are then pumped out by
Na+/K+-ATPase/Na+-pump at the expense of one
ATP molecule) (Bers, 2000
).
Therefore, the role of NCX in Ca2+ removal is decreased in
hibernator cells, possibly as an adaptive regulation; consequently,
SERCA-based Ca2+ uptake becomes more important during
hibernation.
Although the role of NCX is secondary in rapid Ca2+ removal from
the cell, it is still important because it brings Ca2+ regulation
within a global ionic homeostasis. Owing to the existence of NCX,
Ca2+ homeostasis is tightly linked to the homeostasis of
Na+ and K+ and to the ATP supply. An enhanced ability of
hibernating animals to produce ATP and to maintain Na+ and
K+ gradients by Na+/K+-ATPase (for reviews,
see Willis, 1979; Lymann et
al., 1982; Wang, 1988
;
Willis et al., 1992
) is thus
an essential part of intracellular Ca2+ homeostasis.
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Significance and general discussions |
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Based on current knowledge, the strategy used by hibernators to maintain
intracellular Ca2+ homeostasis is to reduce Ca2+ entry
into the cell while enhancing Ca2+ removal. If the SR function in
ground squirrel cardiomyocytes is partially inhibited by caffeine, its
resistance to hypothermia is lost, and the cells exhibit the same
manifestations of Ca2+ overload as those observed in nonhibernator
cells. Conversely, in the rat, which does not hibernate, partial blockade of
Ca2+ entry through L-type Ca2+ channels prevents many
effects of hypothermic Ca2+ overload in heart muscle
(Wang et al., 1997b). These
observations suggest that the Ca2+ homeostasis occurring under
hypothermia or other similar stressful conditions is not `patented' by
hibernators. The underlying regulatory mechanisms may rather also be employed
by non-hibernator and human cells to improve stress resistance. In this way,
extending our studies on the mechanisms of hibernation may provide strategies
for developing new therapies or designing new drugs, and thereby contribute to
human health.
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