1 Institute of Histology and
General Embryology and "Program in Neuroscience," University
of Fribourg, CH-1705 Fribourg, Switzerland;
2 Department of Anatomy, The
calcium-binding protein parvalbumin (PV) occurs at high concentrations
in fast-contracting vertebrate muscle fibers. Its putative role in
facilitating the rapid relaxation of mammalian fast-twitch muscle
fibers by acting as a temporary buffer for Ca2+ is still controversial. We
generated knockout mice for PV (PV
EF hand; calcium-binding protein; muscle relaxation; homologous
recombination
PARVALBUMIN (PV) is a low-molecular-weight,
high-affinity calcium-binding protein of the EF hand family found in a
limited number of vertebrate tissues, the most important being skeletal muscle and specific nerve cells (8, 15). The highest concentrations of
PV are found in the fast-contracting and -relaxing skeletal muscles,
whereas in the slow-twitch skeletal muscles, cardiac or smooth muscle,
there is little or no PV expressed (16). In lower vertebrates, up to
five isoforms classified into In mammals, the role of PV is less clear. Indeed, it has been reported
that this protein has little effect on relaxation rate (4) and
Ca2+ sequestration in fast-twitch
murine muscles (29). Nevertheless, the level of PV in various
vertebrates skeletal muscles (17) and the dissociation rates of
Ca2+ and
Mg2+ from this protein in the
temperature range 0-20°C in the frog (19) correlate fairly
well with muscle relaxation speed. Decay of
Ca2+ transients after electrical
stimulation of fast- and slow-twitch skeletal muscle fibers in the rat
(6) is proportional to the concentrations of PV in each. Furthermore,
induced expression of PV by injection of its cDNA into the slow-twitch
soleus muscle of rats leads to an increased relaxation rate (25).
To investigate the physiological role of PV in fast-twitch muscles of
mammals, we have created PV-deficient mice by homologous recombination.
These mice develop and breed normally, and no significant alterations
in their behavior or physical activity are observed under standard
housing conditions. Isometric contraction in the tibialis anterior
muscles and Ca2+ measurements in
isolated fibers of fast-twitch muscles revealed significant differences
in the PV Targeting
vector
construction. Two genomic clones for
PV were isolated from a 129Sv library (Stratagene) using a full-length mouse cDNA probe. From the 5' end of clone PV1.1, a 3.5-kb
fragment (Sal
I-Eco47 III), containing part of the
promoter and terminating at the Eco47
III site in exon 2, was used as the 5' fragment. For the
3'-flanking region we used a
Hind
III-EcoR I fragment from clone PV4.4,
which lies entirely within intron 4 and has a size of 8.5 kb. Within
the targeting vector pPVknock, the fragment from the
Eco47 III site in exon 2 up to the
Hind III one in intron 4 was replaced
by a neocassette (1.8 kb) containing the neomycin resistance gene
driven by the phosphoglycerate kinase (PGK) promoter and including the
PGK polyadenylation signal. At the 5' end of the targeting
vector, we inserted the herpes simplex virus-thymidine kinase. A scheme
of the targeting vector, pPVknock, is shown in Fig.
1A.
ABSTRACT
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
/
) and compared the
Ca2+ transients and the dynamics
of contraction of their muscles with those from heterozygous (PV
+/
) and wild-type (WT) mice. In the muscles of PV-deficient
mice, the decay of intracellular
Ca2+ concentration
([Ca2+]i)
after 20-ms stimulation was slower compared with WT mice and led to a
prolongation of the time required to attain peak twitch tension and to
an extension of the half-relaxation time. The integral [Ca2+]i
in muscle fibers of PV
/
mice was higher and consequently the force generated during a single twitch was ~40% greater than in
PV +/
and WT animals. Acceleration of the contraction-relaxation cycle of fast-twitch muscle fibers by PV may confer an advantage in the
performance of rapid, phasic movements.
INTRODUCTION
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
- and
-PVs have been detected,
whereas in adult rodents a single isoform of PV is expressed (3). A
second gene belonging to the
-PVs called oncomodulin (OM) is
expressed in the cytotrophoblasts of the fetal placenta, but in adult
mice OM expression is completely absent (3). The fact that PV is only
found in the cytosol and is not found in intracellular organelles or
associated with membranes makes it a strong candidate as a
physiological Ca2+ buffer as
opposed to Ca2+ sensors or
Ca2+ modulators like the
ubiquitous calmodulin. Suggestive evidence supporting a buffer role for
PV comes from structural studies in which it was demonstrated that rat
PV at 25°C does not undergo significant
Ca2+-dependent conformational
changes (31). PV contains two high-affinity Ca2+-binding sites (approximate
affinity 108
M
1) that are occupied by
Mg2+ under resting conditions
[intracellular Ca2+
concentration
([Ca2+]i) < 100 nM] (14). On cell activation
[Ca2+]i
rises to micromolar levels, and the
Mg2+ ions are displaced by these.
The net rate of Ca2+ uptake by PV
during contraction is determined by the rate of dissociation of
Mg2+ from this protein. The
Ca2+ association rate of PV is
slower than the rate of Ca2+
binding to troponin C (28). Hence,
Ca2+ binds preferentially to
troponin C during muscle activation, and the PV-buffering activity is
somewhat delayed. On the basis of these observations, PV was expected
to promote the relaxation of fast-contracting skeletal muscles (5, 12).
Since PV can deplete isolated myofibrils of
Ca2+, and isolated sarcoplasmic
reticulum can deplete PV of Ca2+
(12), the hierarchy of relative
Ca2+ affinities is a prerequisite
for this protein to act as a shuttle for
Ca2+ from the contractile
machinery to storage sites in the sarcoplasmic reticulum. Simulation
studies indicate that the exchange of
Ca2+ for
Mg2+ on PV can occur with
sufficient rapidity for it to contribute to the relaxation of frog
skeletal muscles maintained at 0°C (13), a prediction that has been
confirmed experimentally (19).
/
mice compared with wild-type (WT) animals.
Surprisingly, both the half-relaxation and the rise time to the peak
twitch tension were significantly increased, and concomitantly the
twitch force was higher than in heterozygous or WT animals.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
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Fig. 1.
Disruption of the parvalbumin (PV) gene by homologous recombination.
A: the targeting vector, pPVknock,
replaces the PV gene from the Eco47
III site in exon 2 to the Hind III one
in intron 4 with the phosphoglycerate kinase (PGK) neocassette. The 2 probes, P1 and P2, were used to check for homologous recombination.
B: Southern blot analysis of mouse
genomic DNA digested with Sca I. The
wild-type (WT) allele probed with P1 gives rise to a 13.7-kb fragment
(top arrow), the targeted
allele to a smaller one (11.4 kb;
bottom arrow). Samples: DNA
from heterozygous (+/ ) (lane
1), WT (+/+) (lane
2), and homozygous (
/
)
(lane 3) mice.
C: Western blot analysis of 3 different muscles tested with the antiserum PV-28. No signal is
apparent in the
/
mice, whereas a reduced intensity is
visible in the heterozygous (+/
) one. HSV-TK, herpes simplex
virus-thymidine kinase.
Generation
of PV-deficient
mice. Embryonic stem cells (E14/Ola), cultivated on
feeder cells in the presence of the leukemia inhibitory factor, were
electroporated with the targeting vector pPVknock and grown in the
presence of G418 and gancyclovir. From resistant clones, genomic DNA
was digested with either Sca I or Xho
I/Spe I and hybridized with two
probes, P1 [1.4-kb Hind III fragment derived from a third genomic clone upstream from PV1.1 (sites
not shown)] and P2 (2.3-kb EcoR
I fragment derived from PV4.4), respectively, both located outside the
targeting vector (Fig. 1A). The
absence of heterologous integration in the recombinant ES clones was
tested using a 0.7-kb Nco
I/Hind III fragment of the neomycin
resistance cassette as a probe (not shown). Of the five positive clones
(occurring at a frequency of ~), two were injected. The
highly chimeric mice from the 134 line were bred with C57/Bl6 WT ones,
which crossing resulted in germ-line transmission. Genomic DNA was
extracted from fresh mouse tail biopsies (2-3 mm in length) using
a commercial kit. Ten micrograms of
Sca I-digested genomic DNA were probed
with the P1 fragment in a Southern blot.
Immunohistochemistry. Immunohistochemical analysis of muscle tissue was performed as described by Celio and Heizmann (8), with the exception that the bound primary antibody was revealed by the avidin-biotin technique instead of the peroxidase-anti-peroxidase one.
Western blot detection of PV in various skeletal muscles. The panniculus carnosus, extensor digitorum longus (EDL), and abdominal muscle were removed from killed mice and homogenized, and the soluble proteins were separated by SDS-PAGE (12.5%) and transferred onto nylon membranes (Bio-Rad). After blocking [1% (wt/vol) bovine serum albumin and 10% (vol/vol) fetal calf serum], membranes were incubated with the PV-specific polyclonal antiserum PV-28 (1:1,000; Swant, Bellinzona, Switzerland) and were then further processed by the avidin-biotin method, using 4-chloro-1-naphthol/hydrogen peroxide as a chromogen.
Analysis of proteins from extracts of tibialis anterior muscles. Muscle samples were analyzed by SDS-PAGE (10%) in Tris-glycine buffer without added calcium. Muscle extracts were prepared in 10 volumes of 0.0625 M Tris (pH 6.8), 2% SDS, 2% 2-mercaptoethanol, and 0.001% bromophenol blue. The presence of different isoforms of troponin I was detected using the monoclonal antibody 42/25 that detects fast, slow, and cardiac troponin I. The presence of fast troponin T was analyzed by staining of the Western blots with antibody F24 as described before (11).
[Ca2+]i
measurements.
Methods for enzymatic dissociation and agarose suspension of PV
/
, PV +/
, and WT animal EDL fibers were essentially
the same as described previously for flexor digitorum brevis (FDB) fibers (7). Before agarose suspension, fibers were loaded with the
Ca2+ indicator dye indo 1-AM (cell
permeant form; 5 µM) for 40 min at 37°C and pipetted onto a
coverslip serving as the bottom of small culture dish placed on a Leica
TCS 4D confocal microscope. Indo 1 was excited using an ultraviolet
laser source set at <10% maximum output to minimize photobleaching
(bleaching was calculated to account for <5% of the total signal per
second). Fibers were stimulated by a train of 3-ms pulses (separated by
a 7-ms rest interval) for a total of 20- and 50-ms stimulation
durations from a programmable voltage source via platinum wires, and
the fluorescence signals were collected at 405 and 490 nm by
photomultiplier tubes using a fast line scan mode (13 ms/point). The
ratio of the indo 1 emission signals
(F405/F490)
was used to calculate the
[Ca2+]i
transients as described previously for fura 2 (6) using kinetic
corrections for the Ca2+-indo 1 reaction determined by Westerblad and Allen (30) in Xenopus fibers. Maximal binding was
determined from long stimulation durations (50-200 ms), which
clearly saturated the indicator with calcium, and minimum binding was
estimated by subtracting 15% of the mean resting ratio value. The
decay of the
[Ca2+]i
transients was determined by single exponential plus a constant fit as
described previously (6). Rate constants determined from WT mouse EDL
and FDB fibers (not shown) were consistent with values reported
previously (18, 24) and in agreement with the assumption that indo 1 does not significantly buffer
[Ca2+]i
in the fiber myoplasm (6). All fibers included in this analysis exhibited a "bracket" ratio record (>20-ms stimulation
duration) that was at least 94% the amplitude of the first ratio
record. Resting calcium, determined as described earlier (7), assuming Kd of 311 nM (30)
for the WT, PV +/
, and PV
/
mouse fibers was 117 ± 44 (±SE; n = 5), 140 ± 28 (±SE; n = 6), and 209 ± 66 nM (±SE; n = 4), respectively.
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RESULTS |
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PV-deficient mice are not distinguishable from WT
littermates under standard housing conditions. To
eliminate the functional gene for PV, a targeting vector (pPVknock) was
designed to replace the greater part of the coding sequence by a
neocassette (Fig. 1A). The
replacement begins at the Eco47 III
site in exon 2, which occurs 24 nucleotides after the initial A of the
start codon and terminates in intron 4. By this strategy, ~85% of
the coding sequence (from nucleotides 25 to 304) is replaced by the
neocassette. Its purpose is to ensure that no truncated protein
molecules, which could partially restore WT PV function, are produced.
Of the 100 G418-resistant ES clones analyzed, 5 had undergone
homologous recombination, as revealed by Southern blotting using two
external probes, P1 and P2 (Fig.
1A). Injection of clone 134 gave
rise to seven chimeric mice (>50% chimeric), which were mated with C57/Bl6 WT ones. Germ-line transmission could be demonstrated in
several cases. Crossing of the heterozygous animals yielded homozygous
mice at a frequency not statistically different from the expected
Mendelian transmission of an autosomal gene. This suggests that
elimination of the functional PV gene is neither lethal to embryos nor
significantly affects embryonic development. The three different
genotypes were characterized by Southern blotting of genomic DNA (Fig.
1B). The WT allele gave rise to a
fragment of ~13.7 kb, the targeted allele to one of only 11.4 kb. To
demonstrate the absence of the PV protein, we performed either Western
blotting or immunohistochemical analysis of various skeletal muscles.
Western blot analysis of soluble proteins extracted from three
different muscles revealed an intense signal in WT +/+ mice (Fig.
1C), one of intermediate intensity
in the heterozygous (+/) ones, and none whatsoever in PV
/
animals. For immunohistochemical analysis, we used
sections from the fast-twitch tibialis anterior muscle. No signal was
detected in the muscles of knockout animals, but the well-characterized
checkerboard staining pattern was manifested in fast-twitch muscle
fibers of WT and, less intensely, of heterozygous mice (Fig.
2).
|
Routine histological analysis of hematoxylin-eosin-stained sections of
different organs revealed the microscopic anatomy of PV /
animals to be normal. They grow and breed normally and have a normal
lifespan (of >1 yr). At 4 and 8 wk of age, their body weight does not
differ significantly from that of heterozygous and WT mice. The three
genotypes were indistinguishable with respect to behavior and physical
activity under standard housing conditions.
Lack of PV alters
Ca2+ transients
in single isolated EDL fibers.
Temporal as well as spatial aspects of
Ca2+ transients are modulated by
the presence of Ca2+ buffers such
as EF hand calcium-binding proteins (9). Depending on the intracellular
concentration and the kinetic parameters of metal binding of a
particular protein, different aspects of Ca2+ transients (e.g., amplitude,
duration) can be modified. To investigate the effect of PV in
fast-twitch muscles, Ca2+
measurements on isolated EDL fibers were carried out. A prolonged Ca2+ decay in the PV
/
mouse for the 20-ms stimulation period was observed,
whereas no difference existed between WT and PV
/
fibers
after 50-ms stimulation, when nearly all the PV
Ca2+-binding sites with a high
Mg2+ off rate are expected to be
saturated with Ca2+ (Fig.
3, A and
B). The rate constant of
Ca2+ decay was 33% lower in the
PV
/
compared with the WT animals (Fig.
3C). Although no conspicuous
differences existed between the peak
[Ca2+]i
values (Fig. 3D), there was a
twofold elevation of the integral [Ca2+]i
in the PV
/
compared with the WT mice (Fig.
3E).
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DISCUSSION |
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Since more than 20 years ago, PV has been predicted to promote
relaxation in fast-contracting skeletal muscle (5, 12). A major problem
in the debate on the role of PV in mammalian muscle relaxation has been
the purportedly slow rate of Mg2+
dissociation from this protein. It has been assumed that
Ca2+ was transported into the
sarcoplasmic reticulum via a
Ca2+-ATPase before PV had a chance
to contribute to the buffering of this ion. But evidence has now
accumulated that the dissociation rate of
Mg2+ from PV at temperatures more
physiological for mammals is significantly higher than that documented
for the frog and fish in the 0-20°C range. In addition, the
Mg2+ off rates are not identical
for the two metal-binding sites, one being almost one order of
magnitude faster than the other (20, 26). Values between 11 and 25 s1 have been estimated from
fura 2 and mag-fura 2 recordings, respectively, in fast-twitch muscle
of the rat at 26-28°C (6). Permiakov et al. (26) have reported
the Mg2+ dissociation rate of PV
to be as high as 33 s
1 at
30°C as against 4 s
1 at
10°C. Taken together, these results support the notion that the
Mg2+ dissociation rate of PV at
elevated temperatures higher than 30°C is sufficiently rapid for
this protein to act as a Ca2+
acceptor during a single twitch.
With increasing knowledge about the different EF hand calcium-binding proteins, it becomes evident that not only binding constants but the on/off rates for the different metal ions play a crucial role in understanding their functions. The spatial and temporal aspects of Ca2+ transients are regulated by the efficacy and geometry of the Ca2+-release and Ca2+-uptake/extrusion systems as well as by the concentration and kinetic parameters of soluble Ca2+ buffers such as EF hand calcium-binding proteins. It had been suggested that proteins with Ca2+-specific sites and high on rates would strongly affect the amplitude of an intracellular rise in [Ca2+]i, whereas "slow" Ca2+-binding proteins would mainly increase the decay rate of [Ca2+]i without much affecting the amplitude. For the protein calbindin-D28k (CB), which contains four Ca2+-specific sites, this was demonstrated in rat dorsal root ganglion neurons by Chard et al. (9). The addition of CB via patch clamp pipette lowered the amplitude of a Ca2+ transient induced by a brief depolarization stimulus and caused an eightfold decrease in the rate of rise in [Ca2+]i. Under the same conditions, the effect of PV was much less pronounced. It was postulated that because Mg2+ has to dissociate from PV before Ca2+ ions can be bound, the effect in reducing d[Ca2+]i/dt during the rapid rising phase would be significantly smaller for PV than for CB. A similar finding for CB was observed in CB-deficient mice, where the amplitude of Ca2+ transients in Purkinje cells evoked by extracellular stimulation of the afferent climbing fiber was increased by >80% in mice lacking CB (1).
In our calcium measurements in the EDL muscle fibers, the increase in
[Ca2+]i
after 20- or 50-ms stimulation was not significantly different in the
PV-deficient mice compared with the WT animals, whereas the rate
constant of
[Ca2+]i
decay was significantly smaller after a 20-ms stimulation in PV
/
mice. As a result, the integral
[Ca2+]i
was increased during both 20- and 50-ms stimulations. Hence, the
contraction-relaxation cycle in WT mouse muscles containing PV is
shorter, owing to the increased rate of decay of
[Ca2+]i
(6, 24), and maximal twitch force, although of lower amplitude, is more
rapidly attained than in PV-deficient ones. It should be noted that our
rate constants of calcium decay for the WT fibers are consistent with
values reported previously for mouse fast-twitch fibers (18, 24).
However, the amplitude of the calcium transient is significantly lower
than the value reported by Hollingworth et al. (18) for mouse EDL. This
difference is most likely to be due to differences in calcium
indicators and Kd
values chosen. We found that even after increasing the
Kd 10-fold (by
decreasing the on rate for indo 1 10-fold), which
increased the amplitude of the calcium transient by 10-fold, the
rate constants of calcium decay were unaffected, and the observed
differences between the WT and PV
/
mice fibers
were still valid.
An interesting finding in the PV-deficient mice is the observation that all the proteins of the contractile complex are isoforms found in fast-twitch muscles. It has been demonstrated before that fast-twitch muscles can be transformed into slower-contracting ones by chronic low-frequency stimulation (CLFS) (27). Whereas in rabbits the conversion of myosin heavy chain fast isoform MHCII(x) to slow isoform MHCIb is almost complete after 60 days, rats exhibit a restricted capacity for fast-to-slow conversion (21). Because the disappearance of PV during CLFS precedes the conversion of MHC isoforms, it has been hypothesized that the downregulation of PV might in part be linked to the switch from fast- to slow-twitch isoforms of contractile proteins. Our results strongly disfavor this theory, because all the parameters investigated suggest that besides the lack of PV the muscle constituents are the ones found in fast-twitch muscles.
The only existing in vivo study that has addressed the function of PV
in the rodent model is that reported by Müntener et al. (25).
These authors injected the cDNA for PV into the soleus muscle of rats
and observed an increase in its rate of relaxation. In this previous
study, the contraction time was not significantly affected, although a
decrease in this parameter from 43.5 ± 2.6 to 39.1 ± 7.1 ms was
documented for transfected muscles that contained the highest levels of
mRNA for PV. At first sight, these findings appear to be inconsistent
with our own data pertaining to PV-deficient mice, in which not only
the half-relaxation time but also that required to attain peak twitch
in the tibialis anterior muscle were significantly longer than in the
WT ones. During the early phase of contraction, it is principally the
kinetics of Ca2+ release from the
sarcoplasmic reticulum, the binding of the released ions to the
proteins of the contractile complex (troponin C), and the subtype of
myosin that determine those governing the rise in tension force. In the
soleus muscle, the isoforms of troponin C and myosin heavy chain as
well as those of other proteins are of the slow type, and, seemingly,
overexpression of PV does not overtly affect the time to attain peak
twitch force (albeit a trend toward shorter times), but only the
half-relaxation time. Furthermore, the levels of PV and the number of
PV-expressing fibers in the transfected rat soleus muscle are
considerably lower than in the murine tibialis anterior one. In the
latter, >95% of the fibers express this
Ca2+-binding protein (17), the
concentration of which has been estimated to be higher than 0.4 mM (4.8 g PV/kg wet wt). In PV-deficient mice, all components of the tibialis
anterior muscle thus far investigated (troponin C, troponin T, and
myosin heavy chain) are of the fast type and identical to those in WT
mice. That the myosin heavy chain isoforms remain unchanged in
PV-deficient mice also argues against an alteration in the rate of rise
in force during a single twitch. It indicates rather that the greater
length of time required to attain peak force in PV /
mice
is due to an increased duration of the active state of the tibialis
anterior muscle fibers. The results of Müntener et al. (25)
pertaining to the rat slow-twitch soleus muscle overexpressing
relatively low levels of PV cannot therefore be directly compared with
our results on the murine fast-twitch tibialis anterior muscle. It is
evident, however, that an investigation of the physiological effects of
PV during contraction is better addressed using the tibialis anterior
muscle, within which this
Ca2+-binding protein is normally
expressed, than the soleus one, in which it is absent.
In the tension experiments, tibialis anterior muscles of the three genotypes were stimulated at different frequencies (20, 40, and 80 Hz). At 80 Hz, no significant differences between the three genotypes were observed, the effect of PV deficiency being evident only when pulses were delivered at 20 Hz. In WT animals, the muscles stimulated at 20 Hz relaxed almost completely between successive stimuli, whereas in PV-deficient ones, this process was significantly compromised. These results qualitatively resemble those reported by Jiang et al. (22). These authors investigated the role of PV during the relaxation of frog skeletal muscle fibers at 10°C in which activity of the sarcoplasmic reticulum (SR) Ca2+-ATPase was inhibited by 2,5-di-tert-butyl-1,4-benzohydroquinone. After induction of a series of twitches (at a frequency that resulted in complete relaxation of control fibers) the rate of twitch relaxation decreased progressively and approached zero after the seventh pulse. As an explanation for this finding, the authors suggested that a soluble relaxation factor was lost in a time-dependent manner. After conducting additional experiments, they came to the conclusion that loss of the capacity to relax paralleled the saturation of PV with Ca2+ and that Mg2+-bound PV could induce relaxation at a rate that is defined by the Mg2+ off rate from this protein.
From the observation that PV is enriched in the I band region (2), and
from the present finding that the integral
[Ca2+]i
is about twofold elevated in the PV /
phenotype, an
additional role of PV may be suggested:
Ca2+ not associated with PV would
be more homogeneously distributed in the sarcoplasm and therefore less
accessible to the SR Ca2+-ATPase.
The evidence that PV-bound Ca2+ is
a substrate of the SR Ca2+-ATPase
has been demonstrated long ago (12). PV-bound
Ca2+ would thus be enriched in the
I band and the role of PV as a vehicle of
Ca2+-transport between the
contractile apparatus and the SR
Ca2+-ATPase would be similar to
that of other calcium-binding proteins that selectively shuttle calcium
signals to nearby sites of elevated Ca2+ (10).
It has been postulated that PV would have its greatest effect on the relaxation time in skeletal muscles of poikilotherms at low temperatures, when the activity of the Ca2+ pump of the sarcoplasmic reticulum is depressed (13). In view of our results, the very high concentrations of PV found in fish and amphibians could be considered as a mobile "emergency Ca2+ store" that would facilitate rapid contraction and relaxation at low temperatures. Because the body temperature in mammals is closely regulated and Ca2+ uptake into the sarcoplasmic reticulum via its Ca2+-ATPase is very efficient, we postulate that the advantage conferred by PV lies in the gain in speed to be achieved at the expense of force in a single twitch. The absence of PV under "wildlife" conditions may be a selective disadvantage for knockout mice.
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ACKNOWLEDGEMENTS |
---|
We thank Y. Lang and P. Buchwald from Hoffmann-LaRoche, Basel, and B. Belser, B. Herrmann, and C. Pythoud from the Institute of Histology, Fribourg, for their excellent technical help.
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FOOTNOTES |
---|
This work was supported by the Swiss National Science Foundation (Grant no. 3100-047291.96).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: B. Schwaller, Institute of Histology and General Embryology, University of Fribourg, CH-1705 Fribourg, Switzerland.
Received 2 July 1998; accepted in final form 11 November 1998.
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