Deficiency in parvalbumin increases fatigue resistance in
fast-twitch muscle and upregulates mitochondria
Gaoping
Chen1,
Stefanie
Carroll2,
Peter
Racay3,
Jim
Dick4,
Dirk
Pette5,
Irmtrud
Traub5,
Gerta
Vrbova4,
Peter
Eggli1,
Marco
Celio3, and
Beat
Schwaller3
1 Institute of Anatomy, University of Bern, CH-3012 Bern,
and 3 Program in Neuroscience, Institute of Histology and
General Embryology, University of Fribourg, CH-1705 Fribourg,
Switzerland; 2 National Institutes of Health, Bethesda, Maryland
20814; 4 Deptartment of Anatomy, University College, London WC1E
6BT, United Kingdom; and 5 Faculty of Biology, University of
Constance, D-78457 Constance, Germany
 |
ABSTRACT |
The soluble
Ca2+-binding protein parvalbumin (PV) is expressed at high
levels in fast-twitch muscles of mice. Deficiency of PV in knockout
mice (PV
/
) slows down the speed of twitch relaxation, while
maximum force generated during tetanic contraction is unaltered. We
observed that PV-deficient fast-twitch muscles were significantly more
resistant to fatigue than were the wild type. Thus components involved
in Ca2+ homeostasis during the contraction-relaxation cycle
were analyzed. No upregulation of another cytosolic
Ca2+-binding protein was found. Mitochondria are thought to
play a physiological role during muscle relaxation and were thus
analyzed. The fractional volume of mitochondria in the fast-twitch
muscle extensor digitorum longus (EDL) was almost doubled in PV
/
mice, and this was reflected in an increase of cytochrome c
oxidase. A faster removal of intracellular Ca2+
concentration ([Ca2+]i) 200-700 ms after
fast-twitch muscle stimulation observed in PV
/
muscles supports
the role for mitochondria in late [Ca2+]i
removal. The present results also show a significant increase of the
density of capillaries in EDL muscles of PV
/
mice. Thus alterations in the dynamics of Ca2+ transients detected in
fast-twitch muscles of PV
/
mice might be linked to the increase in
mitochondria volume and capillary density, which contribute to the
greater fatigue resistance of these muscles.
muscle fatigue; calcium-binding protein; EF hand; compensation
 |
INTRODUCTION |
MOST MAMMALIAN SKELETAL
MUSCLES are classified as either fast or slow twitch, depending
on the time course of the excitation-contraction-relaxation (ECR) cycle
caused by brief neural stimulation. The different phases of this cycle
in fibers after propagation of an action potential along the membrane
consist of 1) recognition of the depolarization signal by
the T tubule system and transmission of this signal to sarcoplasmic
reticulum (SR) membranes, 2) release of Ca2+
from SR resulting in increased intracellular Ca2+
concentration ([Ca2+]i) in the myoplasm,
3) activation of Ca2+-dependant regulatory
systems and binding of Ca2+ to the contractile machinery
(troponin C) resulting in fiber contraction, and 4) removal
of Ca2+ by cytoplasmic proteins and reuptake into SR by SR
Ca2+-ATPase (muscle relaxation phase). In each of these
steps, homologous molecular components (isoforms) are found in the
various types of muscle fibers, resulting in differences in the
kinetics of the ECR cycle and in the force generated during a twitch.
The fatigability of the different muscle fibers is linked to variations in the fiber type-specific components (for a review, see Ref. 34). In addition, not all steps of the ECR cycle display
the same sensitivity to fatigue. Various methods have been used to dissect the phases of the ECR cycle and to investigate the roles of
fiber-specific components in the ECR cycle and their contribution to
fatigue sensitivity or resistance. A marked increase in resistance to
fatigue was detected in fast-twitch fibers that were chronically stimulated at low frequency (CLFS) (Ref. 17; for a review,
see Ref. 27). During CLFS, several changes at the
molecular level occur, including changes in myosin heavy chain (MHC)
and troponin isoforms. Increases in mitochondrial volume and enzyme
activities of aerobic oxidative metabolism are observed as well as
changes in activities of Ca2+ release and uptake
(7) and also myoplasmic Ca2+-binding sites
(14, 16, 26).
Parvalbumin (PV), the main soluble Ca2+-binding protein
present only in fast-twitch fibers, is rapidly downregulated after CLFS (16). This protein was demonstrated to act as a relaxation
factor in amphibians (18, 37), and, recently, also in
mammals in vitro (25) and in vivo (31). The
recently generated PV knockout mice (PV
/
) (31)
represent an independent model to investigate the role of PV during
muscle contraction and relaxation. In these mice, it was shown that PV
is responsible for the fast initial drop of
[Ca2+]i, which helps to considerably shorten
the duration of a single-twitch contraction and relaxation. In PV
/
mice, the time-to-peak twitch force, and, in particular, the
half-relaxation time, is increased compared with wild-type (WT) mice.
In this report, we investigated how fast-twitch muscles cope with the
lack of the myoplasmic Ca2+ buffer PV, specifically whether
PV deficiency is compensated by upregulation of another
Ca2+ buffer, and how this might affect the fatigability of
fast-twitch muscles of PV
/
mice.
 |
MATERIALS AND METHODS |
Animals.
PV-deficient (PV
/
), heterozygous (PV +/
), or WT (PV +/+) mice
generated on a mixed 129/Ola × C57BL/6J genetic background (31) were used in this study. Tail biopsies from
all mice were used to genotype animals by PCR, and the genotype of all
mice was masked from the experimenters until data had been evaluated.
Muscle contraction.
Experiments on the contractile properties of a fast-twitch muscle, the
tibialis anterior (TA), were carried out essentially as described
previously (31). The distal tendons of the TA muscles of
anaesthetized mice (intraperitoneal injection of a 4.5% solution of
chloral hydrate; 1 ml/100 g of body wt) were dissected on both sides
from the surrounding tissue. They were cut, attached to a silk thread,
and the sciatic nerve was dissected. All of its branches, except for
the common peroneal nerve, were sectioned. The nerve was cut and its
distal stump prepared for stimulation. The lower limbs were secured to
a rigid table by pins through the knee and ankle joints. The muscle was
then connected by the silk thread to strain gauges. Contractions were
elicited by supramaximal stimulation of the distal stump of the sciatic
nerve using bipolar silver electrodes. The length of the muscles was
adjusted to obtain maximum twitch tension in response to a single
stimulus to the nerve. Isometric contractions were displayed on a
storage oscilloscope screen, the images photographed, and values of
single and tetanic contractions were calculated from these photographs.
Muscle fatigue was tested by subjecting the muscles to trains of
tetanic contractions at 40 Hz with a 250-ms duration every second for 5 min, displayed on a devices pen recorder. A fatigue index was
calculated by dividing the force produced at the end of the 5-min
stimulation by that produced by the muscle at the beginning of the
experiment. This fatigue test was chosen because it does not cause
failure of neuromuscular transmission (4). All experiments
were carried out in air-conditioned laboratories (20°C). With the use
of a rectal thermometer (thermocouple), the body temperatures of the
anaesthetized mice were monitored and varied between 30 and 33°C.
[Ca2+]i measurements.
Methods for enzymatic dissociation, agarose suspension, and loading of
indo 1-AM into extensor digitorum longus (EDL) fibers and flexor
digitorum brevis (FDB) fibers isolated from PV
/
and WT animals
were the same as described previously (31), and [Ca2+]i-measurements were carried out at room
temperature. 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. Indo 1 was excited with an ultraviolet laser source, and indo 1 fluorescence emission signals at 405 and 490 nm were collected by
photomultiplier tubes, as described previously, using a Leica confocal
setup with a fast line scan mode (13 ms/point) (31). The
ratio (R) of the indo 1 emission signals
(F405/F490) was used to calculate the
[Ca2+]i transients, as described previously,
for fura 2 (8) using the following equation
|
(1)
|
The rate constants for binding (kon) and
dissociation (koff) between Ca2+ and
indo 1 have been determined by Westerblad and Allen (36) in Xenopus fibers. Rmax was determined from long
stimulation durations (50-200 ms), which clearly saturated the
indicator with Ca2+, and Rmin was estimated by
subtracting 15% of the mean resting ratio value. During the
stimulation period (t = 100-300 ms) when the slow
dissociation of Ca2+ from indo 1 would distort the
Ca2+ transient, the derivative (dR/dt) was
calculated for each individual point of the fluorescence ratio using
the following equation
|
(2)
|
Before stimulation and after stimulation when indo 1 is expected
to be in more equilibrium with Ca2+, the derivative value
was set to zero. A "bracket" pulse of two or more pulses was
applied after the last pulse train of the experiment to insure that the
fiber remained viable throughout the experiment and had not run down.
All fibers included in this analysis exhibited a bracket ratio record
that was at least 94% the amplitude of the first ratio record.
[Ca2+]i elevation (Ca
)
values were calculated using the following equation
|
(3)
|
where the Ca2+ concentration after stimulation
(Ca
) was determined by fitting a constant
function from 200 to 700 ms after stimulation and
Ca
was the basal value, determined from a constant
function fit of the first 100 ms before stimulation.
ELISA.
The sandwich ELISA for PV is very similar to the one published in
detail for calretinin (30). ELISA plates were pretreated with the monoclonal mouse anti-PV antibody PV235 (0.2 mg/ml in bidistilled water, diluted 1:50 in 100 mM NaHCO3, pH 8.0;
Swant, Bellinzona, Switzerland) for 16-24 h at room temperature.
After two washes with water, the additional protein binding sites were blocked with 200 µl of 0.3 M Tris/acetate (pH 7.5) containing 1% BSA
and 0.02% Kathon (Christ Chemie, Aesch, Switzerland) for 24 h.
Standard curves (0-5 ng/ml of PV) were established with purified
recombinant PV (Swant; 1 µg/ml of PV in a solution that contained 0.1 M Tris · HCl buffer, pH 6.5, 0.5% BSA, and 0.02% Kathon) and
diluted in buffer A (0.3 M sodium acetate, pH 7.5, containing 10% FCS, 0.1% phenol, and 0.04% Kathon). For the
isolation of soluble proteins, dissected muscles were homogenized in
buffer (10 mM Tris and 2 mM EDTA, pH 7.5, containing a protease
inhibitor cocktail from Roche, Mannheim, Germany) using a Polytron
homogenizer (Kinematica, Luzern, Switzerland). The suspension was
centrifuged (18,000 g, 4°C, 30 min), and the supernatant
was used for ELISA. Protein concentrations were determined by the
method of Bradford using reagents from Bio-Rad. Muscle extracts
containing soluble proteins were diluted in the same solution as the
purified PV and added together with the detection antibody rabbit
anti-PV antiserum PV28 (Swant), diluted 1:800 with buffer A.
After incubation of the samples for 24 h at room temperature,
wells were washed twice with 0.05% (vol/vol) Tween 20 and twice with
water. A peroxidase-conjugated goat anti-rabbit IgG (Sigma, Buchs,
Switzerland; 1:500 in buffer A) was added to each well and
incubated for 2 h at room temperature, followed by washing four
times with water. To detect the bound peroxidase, 200 µl of a
3,3',5,5'-tetramethylbenzidine (TMB)-hydrogen peroxide solution (20 mM
TMB and 50 mM hydrogen peroxide in acetone/ethanol; 10:90) was added to
each well, and the development of the blue reaction product was blocked
after 10-30 min by adding 100 µl of 1 M sulfuric acid.
Absorbance was measured photometrically at 450 nm.
Morphometric analysis of mitochondrial fractional volume and
capillaries in muscles from PV-deficient mice.
For stereological analysis, dissected muscles [EDL and soleus (SOL)]
were fixed in a solution of 80 mM sodium cacodylate, pH 7.3, containing
2% (wt/vol) paraformaldehyde, 2.5% (vol/vol) glutaraldehyde, and 0.2 mM CaCl2, and was embedded in epon. Mitochondrial volume
density of two different muscles (EDL and SOL) from three different
animals per genotype was measured and statistically compared. Four
tissue blocks per muscle were sectioned for electron microscopy. The
orientation of the sections was transverse or slightly oblique with
regard to the fiber axis. The volumes of mitochondria, myofibrils, and
residual sarcoplasmic components per unit volume of muscle fiber were
estimated on high-power electron micrographs at a final magnification
of ×24,000. Systematic sampling with a random starting point was done
in consecutive frames of 200 square mesh grids. Ten micrographs per
section from all four blocks per muscle were taken and analyzed by
point counting with grid C (144 test points). A second set of
micrographs recorded at a final magnification of ×1,900 was used for
estimation of capillary density and capillary-to-fiber ratio. Four
micrographs per section were taken in consecutive frames of slotted
grids. Point counting was done on the test system A 100 (100 test
points). All stereological variables were calculated by applying
standard procedures (35) as previously established
(15).
45Ca2+ overlay blot of
soluble proteins extracted from TA, EDL, and SOL.
Adult mice (PV
/
and WT) were anaesthetized by inhalation of carbon
dioxide and briefly perfused by ice-cold phosphate-buffered saline
solution. After decapitation, the TA, EDL, and SOL muscles were
excised. Muscles were homogenized in homogenization buffer (10 mM
Tris · HCl and 1 mM EDTA, pH 7.4) using a Polytron homogenizer (Kinematica), and two different fractions, soluble and insoluble particulate, were prepared by centrifugation (30,000 g for
40 min).
Expression patterns of Ca2+-binding proteins (including EF
hand CaBP) were investigated by the Ca2+ overlay technique
according to Maruyama et al. (24). Soluble proteins were
separated by one-dimensional polyacrylamide gel electrophoresis
(SDS-PAGE) on 12.5% polyacrylamide and then transferred onto a
nitrocellulose membrane using a semi-dry transfer protocol. After
transfer, membranes were washed three times for 20 min in freshly
prepared solution (10 mM imidazole-HCl, 5 mM MgCl2, and 60 mM KCl, pH 6.8) and then incubated for 10 min in solution that contained 40 kBq/ml of 45CaCl2. Finally,
membranes were washed for 5 min in 50% ethanol, dried, and exposed to
a Molecular Imager screen for 24 h. The bands corresponding to the
EF hand CaBPs with bound 45Ca2+ were visualized
by a Molecular Imager system (Bio-Rad).
Quantitative measurement of cytochrome c oxidase subunit I by
Western blot analysis.
The insoluble membrane protein/cytoskeleton fractions, isolated from
both TA and EDL, were dissolved in 5% SDS, clarified by
centrifugation, and protein concentrations in supernatants were
determined by DC protein assay (Bio-Rad) using the protocol supplied by the manufacturer. After dilution with 6× Laemmli sample buffer, 25 µg of proteins were separated by SDS-PAGE on a 10% polyacrylamide gel and transferred on nitrocellulose membranes using
the semi-dry transfer protocol. Cytochrome c oxidase subunit I was detected using mouse monoclonal antibodies (clone 1D6-E1-A8) against human cytochrome c oxidase (Molecular Probes,
Eugene, OR; A-6403) using the protocol according to Capaldi et al.
(5) with slight modifications. Membranes were first
blocked for 60 min at room temperature with 10% nonfat milk in TBS-T
(10 mM Tris · HCl, pH 7.4, 500 mM NaCl, and 0.05% Tween 20)
and then processed by the avidin-biotin blocking method using the
manufacturer's protocol (Vector, Burlingame, CA). Incubation with
primary antibodies (1 µg/ml, diluted in 0.1% BSA in TBS-T) and with
biotinylated horse anti-mouse antibodies (Vector; diluted 1:10,000 in
TBS-T) was 90 min at room temperature for both steps. The membrane was then incubated with peroxidase complex solution (Vectastain ABC kit;
Vector) in TBS-T for 30 min, followed by extensive washing. Membranes
were preincubated with enhanced chemiluminescence solution (Pierce,
Rockford, IL) for 2 min and exposed for 5 min to a chemiluminescent screen (Bio-Rad). The bands corresponding to the cytochrome
c oxidase subunit I were visualized and quantified using
Molecular Imager hardware and software from the same manufacturer.
Statistical analysis.
For all morphometric and force measurements and for the quantification
of cytochrome c oxidase, values from PV +/
and PV
/
mice were compared with WT mice using the Student's t-test (unpaired, two-tailed). The values are expressed as means ± SD. Differences were considered significant at P < 0.05.
 |
RESULTS |
Increased resistance to muscle fatigue in fast-twitch muscles of PV
knockout mice.
We previously demonstrated that compared with muscles from WT mice,
twitch duration is prolonged in fast-twitch muscles of PV
/
mice,
while tension during maximal tetanic contraction is unaltered
(31). Here, we have investigated the effects of PV
deficiency on muscle fatigue. For this purpose, TA muscles were
subjected to trains of tetanic contractions (40 Hz) of 250-ms durations
every second for 5 min. This stimulation does not produce maximum
force. Traces recorded from the three genotypes are shown in Fig.
1. A fatigue index was calculated as
described in MATERIALS AND METHODS. The values were
0.32 ± 0.03 for WT (n = 10) and 0.44 ± 0.019 (n = 6) for PV
/
. Thus the PV-deficient
muscles were significantly more resistant to fatigue than the WT
muscles. However, the fatigue index of PV +/
mice was 0.228 ± 0.029 (n = 6), which showed them to be more fatigable
then those from either PV
/
or WT mice. Factor(s) that may explain
this difference in fatigability were studied next.

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Fig. 1.
Records of contractions elicited from the tibialis
anterior (TA) muscles by repeated stimulation of the motor nerve at 40 Hz for 250 ms every second from a wild type (WT; A), a
parvalbumin-deficient (PV / ; B), and a heterozygous (PV
+/ ; C) mouse. A fatigue index was calculated from these
recordings by dividing the force produced by the muscle at the end of
the stimulation (5 min) by the force produced at the beginning of the
experiment.
|
|
Basal [Ca2+]i is not
affected in PV knockout fast-twitch muscles, but kinetics of
Ca2+ transients and
Ca2+ clearance are altered.
Consistent with our previous report, where we demonstrated that the
resting [Ca2+]i level in the PV
/
fast-twitch muscle EDL was not significantly different from the WT one
[209 ± 66 nM vs. 117 ± 44 nM, respectively (31)], the FDB fibers analyzed in this study also showed
no significant differences (190 ± 48 nM vs. 189 ± 49 nM in
WT). On the other hand, the rate constant of
[Ca2+]i decay after 20 ms of stimulation was
significantly decreased (P < 0.05) in mice lacking PV
compared with WT animals in both EDL (31) and FDB fibers
(183 + 29 s
1 in WT vs. 76 + 23 s
1
in PV
/
). Close analysis of the previous data on EDL fibers (31) and current data on FDB fibers revealed that not only
the initial phase of decay of [Ca2+]i was
affected in PV
/
, but the kinetics of Ca2+ transients
at later times were altered. As demonstrated in inhibitory PV-expressing hippocampal neurons (21) or in PV-loaded
chromaffin cells (22), the kinetics of
[Ca2+]i decay in the presence of PV is
biphasic. PV initially increases the rate of Ca2+ decay but
then prolongs the transients by the release of Ca2+ from
PV. In this study, the difference in [Ca2+]i
levels maintained from 200 to 700 ms after stimulation compared with
the basal [Ca2+]i maintained ~100 ms before
stimulation was analyzed in both EDL and FDB fibers (Fig.
2). In WT mice, the net elevation of Ca2+ (mean of Ca
=
[Ca2+]i) was 10 nM for both EDL and FDB
after 20 ms of stimulation (Fig. 2, B and C,
respectively, open bars). Values were even higher after 50 ms of
stimulation, 15 and 20 nM for EDL and FDB, respectively (Fig. 2,
B and C, open bars), and reflect the higher
degree of saturation of PV and thus an increased release of
Ca2+ at the later phase of the transient. Interestingly,
the degree of Ca2+ elevation was similar to levels of
elevation observed in fast-twitch frog fibers stimulated for different
durations (19). The amount of PV detected in PV +/
fibers of EDL and TA was between 40 and 50%, compared with WT (see
below). Therefore, we hypothesized that the Ca2+ elevation
due to Ca2+ release from PV should be smaller in PV +/
fibers 200 ms after stimulation compared with WT muscles. In both FDB
and EDL fibers,
[Ca2+] values were clearly
smaller in PV +/
fibers stimulated for 20 or 50 ms (Fig. 2,
B and C, hatched bars). This effect is
not due to a reduced release of Ca2+ in PV +/
or PV
/
fibers, because we have shown previously that Ca2+ release
in EDL fibers is not different among the three genotypes, and the time
integral of the Ca2+ transient is actually slightly higher
in PV +/
and PV
/
fibers (31). The results obtained
on FDB fibers in this study were consistent with the previous findings,
especially regarding the increased time integral of the
Ca2+ transient determined in PV
/
fibers. Thus
[Ca2+] values for PV
/
fibers were expected to be
zero in PV
/
due to the complete lack of the slow Ca2+
buffer PV contributing to the Ca2+ elevation. However, in
PV
/
FDB and EDL fibers, [Ca2+]i 200 ms
after stimulation was lower than before stimulation, resulting in
negative
[Ca2+] values of
40 nM in FDB at 50 ms of
stimulation (Fig. 2C, filled bars). Although these values
for FDB at 20 ms of stimulation and EDL (20 and 50 ms) also changed in
the same direction (
[Ca2+] < 0), they did not reach
statistical significance. These data suggested that system(s)
contributing to Ca2+ clearance were more efficient or
upregulated in fast-twitch muscles of PV
/
mice. The main mechanism
for Ca2+ extrusion is the SR Ca2+-ATPase, the
activity of which differs between fast- and slow-twitch muscles
(12). However, this activity was found not to be different between WT and PV
/
mice in four fast-twitch muscles (TA, EDL, psoas, and gastrocnemius) (28), and thus other systems
implicated in Ca2+ clearance were investigated.

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Fig. 2.
Ca2+ transients in PV / and WT mice. A:
4 Ca2+ transients recorded from 50-ms stimulation durations
were averaged for both WT (thin line) and PV / (thick line) flexor
digitorum brevis (FDB) fibers and fit with a constant function from 200 to 700 ms after stimulation (time = 0.3 and 0.8 s,
respectively, see arrows). Stimulation protocol is shown in the lower
graph. To reveal differences in intracellular Ca2+
concentration ([Ca2+]i), following the
initial decay more clearly, the axis has been changed in the
inset. Ca2+ levels were determined 200 ms after
stimulation (A) for all Ca2+ transients recorded
from all 3 genotypes of extensor digitorum longus (EDL; B)
and FDB (C) fibers. The mean differences
( [Ca2+]i) in WT (open bars), PV +/
(hatched bars), and PV / (filled bars) fibers for 20 and 50 ms
demonstrate that significant differences were observed between WT and
PV / FDB fibers after a 50-ms stimulation duration (*WT vs. PV
/ , P < 0.05; #WT vs. PV +/ ,
P < 0.05). The differences between WT and PV /
were not significant for EDL for 50 ms (P = 0.087) and
FDB for 20 ms (P = 0.066) stimulation. The number
(n) of fibers tested for EDL of WT, PV +/ , and PV /
mice was 5, 6, and 4, respectively, and for FDB of WT, PV +/ , and PV
/ animals was 8, 8, and 5, respectively.
|
|
Cytosolic Ca2+-binding proteins and proteins of the
contractile complex are not altered in fast-twitch muscles of
PV-deficient mice.
Another possibility leading to the observed effects would be an
upregulation of a soluble Ca2+ buffer protein similar to PV
with a higher dissociation constant and even slower binding kinetics
than PV. To test this possibility, total soluble protein extracts from
TA, EDL, and the slow-twitch muscle SOL were isolated and analyzed by
45Ca2+ overlay blots. The major band in WT
muscles is PV (Fig. 3), which is clearly
reduced in PV +/
and absent in PV
/
muscles. The amounts of PV
were additionally quantified by ELISA and are in good agreement with
the qualitative aspects of the 45Ca2+
overlay blots (Table 1). In SOL muscle,
the signals for PV are much weaker, because the PV content of type IIa
fibers, which make up ~50% of total fibers, is very low, but the
relative proportions in the three genotypes are maintained. The amount
of PV in the three analyzed muscles (TA, EDL, and SOL; 2 mice/genotype)
of heterozygous (PV +/
) mice is ~40-50% compared with WT
animals (Table 1). A weaker band at ~20 kDa, most likely representing the ubiquitous Ca2+-binding protein calmodulin (CaM), was
observed in practically all samples, but considerable variations of
signal intensities were observed between membranes. No other band
representing a CaBP was detected in PV
/
muscles. Previously, we
have shown that, in addition, all the other proteins of the contractile
machinery [MHC isoforms (28) and troponins C, T, and I
(31)] were not affected in fast-twitch muscles of PV
/
mice.

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Fig. 3.
45Ca2+ overlay blot of soluble
proteins from TA, EDL, and soleus (SOL). Soluble proteins isolated from
TA, EDL, and SOL of 3 genotypes were heated in the presence of 1 mM
CaCl2 at 60°C for 3 min. Thermal stable proteins (50 µg
SOL, 40 µg TA, and 25 µg EDL) were separated by SDS-PAGE,
transferred onto a nitrocellulose membrane, and incubated with a
solution of 45CaCl2. The most intense single
band corresponds to PV. The positions of corresponding molecular weight
standards are indicated by arrowheads.
|
|
Increased mitochondrial fractional volume and increased
capillarization in fast-twitch muscle of PV
/
mice: link to fatigue
resistance?
In recent years, the role of mitochondria as a Ca2+ buffer
or store has often been investigated, but the physiological relevance is still under debate. The fractional volume of mitochondria was determined by stereological analysis in EDL and SOL of PV
/
and WT
mice. The quality of the fixed tissue used for the evaluation is shown
in Fig. 4 and demonstrates the integrity
of the cellular structures including mitochondria. In EDL, the
fractional volume was almost doubled in PV
/
(15.66 ± 0.54%)
compared with WT (8.46 ± 0.61%) but only slightly (20%)
increased in PV +/
(Table 2). A smaller
increase (39 and 14%) was also detected in SOL of PV
/
and PV +/
mice, respectively. Using a biochemical assay, cytochrome c
oxidase as a mitochondrial marker was determined by quantitative
Western blot analysis. Its higher levels in samples from PV
/
were
consistent with the results obtained by stereological analysis (Table
3). Cytochrome c oxidase in
muscles from PV
/
mice was higher by ~55 and 38% in TA and EDL,
respectively (Table 3). Interestingly, the number of capillaries per
mm2 surface [NA(c,f)] was
significantly higher (64% increase) in EDL of PV
/
, while values
in WT and PV +/
mice were similar (Table
4). On the other hand, the average
surface area of an EDL fiber was larger in PV +/
animals (1,638 ± 191 µm2) compared with both other genotypes,
1,199 ± 197 µm2 and 1,229 ± 125 µm2 for PV +/+ and PV
/
, respectively. The increase
in the density of capillaries (64%) in PV
/
EDL fibers paralleled
the values determined for the mitochondria parameters (85% in PV
/
EDL fibers, Table 2). Thus it is evident that both mitochondrial volumes and capillary densities are higher in PV
/
mice, and this
is most prominent in fast-twitch muscles lacking PV.

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Fig. 4.
Electron micrographs of cross-sectioned EDL myofibrils
from a wild-type (A) and a PV-deficient (B)
mouse. Scale bar: 0.5 µm.
|
|
 |
DISCUSSION |
Ca2+ plays a crucial role as a second messenger in
many biological processes, including gene regulation. Its role is
exerted either indirectly via Ca2+/CaM-dependant pathways
(e.g., CaM kinases leading to the phosphorylation of cAMP-responsive
element binding protein) (2, 13) or it can act directly
with specific Ca2+-dependant transcriptional proteins, such
as DREAM, which represses transcription from the early
response gene c-fos (6).
Ca2+-induced alterations in gene expression are also
observed in skeletal myofibers. Application of Ca2+
ionophore to cultured myotubes, which develop the adult pattern of fast
myosin light and heavy chains, alters the expression pattern toward
slow isoforms. This process is paralleled by an increase of the citrate
synthase activity and is reversible after withdrawal of the ionophore
(20). Cytochrome c expression was also shown to
be upregulated in myotubes after ionophore treatment (10). However, these experimental models do not reflect the physiological situation, because the increase in [Ca2+]i is
permanent, whereas during activity, the changes of
[Ca2+]i are transient and the amplitude as
well as the spatial and temporal distribution of these transients may
determine which pathways are activated. Thus specific information is
contained in the amplitude as well as the frequency of Ca2+
signals (1), and highly specialized systems for
Ca2+ release, intracellular buffering, and Ca2+
extrusion determine the shape of the transients. In the myoplasm, the
frequency, magnitude, and kinetics of elevations and decay of
[Ca2+]i depend on the frequency of
stimulation by the motor neuron, the kinetics of release from the SR,
soluble Ca2+ buffers, and the Ca2+ uptake
system, including SR and mitochondria. In fast-twitch muscles of
PV-deficient mice, Ca2+ transients induced by electrical
stimulation of isolated EDL fibers have the same rate of rise and peak
amplitude, which is reflected by the unchanged kinetics of contraction
during the initial phase (31). This indicates that systems
that contribute to the rise of [Ca2+]i are
not affected by the lack of PV. On the other hand, the initial rate of
[Ca2+]i decay is significantly lower compared
with WT fibers (31) and is consistent with results
obtained either on inhibitory hippocampal neurons (21) or
PV-injected chromaffin cells (22) in which the rate of
[Ca2+]i decay was significantly increased by
PV. In the late phase of [Ca2+]i decay,
however, PV is expected to prolong the transient due to the release of
Ca2+ from PV (22). Analyses of the
Ca2+ transients of EDL and FDB fibers from PV
/
mice
show that although the initial decay of
[Ca2+]i is slower, this is followed by a
period with even lower [Ca2+]i than before
the stimulation. This "negative elevation" was seen only in PV
/
fast-twitch muscles and was more prominent during stimulation of
longer duration (50-ms vs. 20-ms stimulation), indicative of an
additional or enhanced system of Ca2+ buffering or
Ca2+ removal. In gel overlay assays, no evidence for
upregulation of another Ca2+-binding protein was obtained,
especially not in the PV
/
mice. Only one relatively weak signal
compared with PV (with a relative molecular mass ~20 kDa, most likely
representing CaM) was observed, but it was not different in the three
genotypes. Even if another as yet unidentified CaBP existed, the
properties of this protein would have to include slower
Ca2+-binding kinetics than PV and no refolding of the
Ca2+-binding domains in the presence of Ca2+ on
the nitrocellulose membranes, two characteristics that have not been
observed in another EF hand CaBP. The activity of the SR
Ca2+-ATPase, the most effective system of Ca2+
reuptake in fast-twitch muscles, was unaffected in all investigated muscles of PV
/
mice when tested in vitro (28).
However, it could be that the increase in oxidative enzymes seen in
this study led to a faster rephosphorylation of ATP so that the ratio
of ATP:ADP was higher and the phosphorylation potential greater. This
would lead to a more efficient Ca2+ pump (38)
and may account for the "overshoot" of Ca2+ removal
that was particularly obvious after prolonged stimulation. Another
possibility is that the increased number of mitochondria not only
provides the higher levels of oxidative enzymes but also plays a direct
role in Ca2+ sequestration. The possible role of
mitochondria in Ca2+ sequestration during relaxation of
slow-twitch and cardiac muscles has previously been discussed in detail
by Lehninger (23). According to this author, the
Ca2+ sequestration by mitochondria encompasses two separate
processes. The process involved in fast Ca2+ sequestration
most likely relates to its binding to the mitochondrial membrane
("membrane loading"), whereas "matrix loading" represents the
slower component. The role of mitochondria in Ca2+
signaling, their role as a Ca2+ store or sink, and their
involvement in muscle fatigue have gained much attention in recent
years (9, 11, 32), and development of new techniques has
allowed the investigation of the dynamics of mitochondrial
Ca2+ ([Ca2+]mit)
(29). Earlier experiments have demonstrated that
45Ca2+ uptake is significantly faster in
mitochondria from slow-twitch, oxidative fibers (STR), compared with
fast-twitch, glycolytic fibers (32). It was hypothesized
that mitochondrial Ca2+ uptake in STR could account for up
to 100% contributing to the relaxation rate at low-frequency
stimulation. Evidence for an involvement of mitochondria in the
relaxation of slow-twitch fibers was provided by Gillis
(11) using the mitochondrial Ca2+ uptake
inhibitor ruthenium red. The rate of relaxation in the presence of the
inhibitor was significantly decreased in slow-twitch muscles, while not
affecting the contraction-relaxation cycle of fast-twitch muscles.
As demonstrated previously (31) and in this study either
directly or indirectly, proteins of the contractile apparatus [MHC isoforms (28), troponins T and I], SR
Ca2+-ATPase activity, and Ca2+ release from the
SR were not affected by the lack of PV. The present results show that
in addition to the slower time course of contraction and relaxation,
the only differences that were clearly detectable in PV
/
fast-twitch fibers were the increase in mitochondria (evidenced by the
increased fractional volume and cytochrome c oxidase) and
the increased capillarization. The changes of contractile properties
are evident, because removal of Ca2+ by mitochondria sets
in with a delay, thus the kinetics of a twitch in a PV
/
fast-twitch muscle is still slower than in a WT mouse, despite an
almost doubling of mitochondrial fractional volume. On the other hand,
the increases in mitochondria and capillarization provide the most
likely explanation for the enhanced resistance of PV
/
fast-twitch
muscles to fatigue. In PV +/
EDL fibers in which the PV content is
~50-60% smaller than in WT ones, the mitochondrial volume is
only slightly elevated, the capillary density is similar to WT fibers,
and the average fiber size is larger. Interestingly, these changes
together result in a higher sensitivity to fatigue compared with WT muscles.
From our results, several concepts and hypotheses can be put forward.
1) The alteration in the shape of a Ca2+
transient (slower Ca2+ decay) is sufficient to induce
mitochondrial biogenesis and does not require sustained
Ca2+ elevations. The mitochondrial volume in EDL of PV
/
mice (15.66 ± 0.54%) is similar to that found in the
slow-twitch muscle SOL of WT animals (15.42 ± 1.35%).
2) The increased volume of mitochondria in PV
/
is not
sufficient to revert twitch parameters to those of the WT, due to the
slow kinetics of Ca2+ uptake compared with PV. Thus
mitochondria cannot accurately compensate for the deficiency of PV.
Nevertheless, their increase in the fast muscles is most likely
responsible for the change in the resistance to fatigue. Whether the
altered Ca2+ transients are directly linked to enhanced
capillarization or if the signal is transmitted through the increased
mitochondrial volume remains to be investigated. Findings that
increases in capillarization precede the increases in key enzymes of
mitochondrial energy metabolism in low-frequency stimulated rabbit TA
(3, 33) argue against the possibility that the increase in
mitochondria is responsible for the growth of capillaries. In rats and
rabbits, chronic low-frequency stimulation induces, in addition to the downregulation of PV and elevations in capillary and mitochondrial densities, fiber type transitions. These transitions encompass multiple
exchanges of fast-type with slow-type isoforms of thick and thin
filament proteins, as well as of the SR Ca2+-ATPase and
several other membrane proteins related to Ca2+ release and
sequestration (26, 27). In the mouse, low-frequency electrical stimulation does not induce changes of fiber types, and this
is consistent with the present finding on the lack of fiber type
transitions in PV
/
mouse muscles. This makes it much easier to
relate the observed changes in the physiological properties to the
altered Ca2+ transients and the increase in mitochondrial
density. 3) The possibility exists that the transition of
fast-twitch to the slow-twitch fiber isoforms (MHC, troponins, SR
Ca2+-ATPases) fails to occur in PV
/
mice because
increases in mitochondria and oxidative capacity of the muscle fibers
reduce [Ca2+]i after the contraction. Thus
the signal, i.e., prolonged increased levels of
[Ca2+]i, is not maintained long enough to
drive the system all the way toward a slow muscle fiber. The lack of
isoform conversion of proteins involved in muscle contraction in mice
after CLFS indicates that the relatively high content and rapid
increase in mitochondria in this species may attenuate the signals
involved in fiber type transitions.
 |
ACKNOWLEDGEMENTS |
We thank P. Nicotera, University of Konstanz, for use of the
confocal imaging system. The excellent technical help of I. Marquardt (Konstanz), C. Pythoud and V. Neuhaus (Fribourg), and W. Graber (Bern)
is greatly appreciated. H. Hoppeler's group was of great assistance in
establishing the morphometric measurements. We are grateful to Drs.
Merdol Ibrahim and Jean-Marie Gillis for critical reading of the manuscript.
 |
FOOTNOTES |
The project was supported by Swiss National Science Foundation Grant
3100-047291.96 (to M. Celio) and Novartis.
Address for reprint requests and other correspondence: B. Schwaller, Institute of Histology and General Embryology, Univ. of
Fribourg, CH-1705 Fribourg, Switzerland (E-mail:
beat.schwaller{at}unifr.ch).
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. Section 1734 solely to indicate this fact.
Received 19 July 2000; accepted in final form 5 February 2001.
 |
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