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* Department of Physiology and Pharmacology, Karolinska Institutet, S-171 77 Stockholm, Sweden; and
Department of Cell Biology and Histology, Faculty of Medical Sciences, University of Nijmegen, 6500 HB Nijmegen, The Netherlands
1Correspondence: Department of Physiology and Pharmacology, von Eulers väg 4, Karolinska Institutet, 171 77 Stockholm, Sweden. E-mail: Hakan.Westerblad{at}fyfa.ki.se
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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Key Words: calcium • excitation-contraction coupling • inorganic phosphate • low-frequency fatigue • muscle metabolism
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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. A key enzyme for maintaining a constant ATP/ADP ratio
during rapid energy turnover is creatine kinase (CK) (2)
,
which catalyzes phosphate exchange between the high free energy
phosphates ATP and phosphocreatine (PCr) via the reaction: PCr + ADP +
H+ 
creatine (Cr) + ATP. In skeletal muscle
there are two major forms of CK: one that is found in the cytosol
(M-CK) and one associated with mitochondria (ScCKmit). M-CK dominates
in fast-twitch muscles and is considered important for energy
utilization at sites of high-energy turnover, i.e., cross-bridges,
sarcoplasmic reticulum (SR) Ca2+ pumps, and
sarcolemma Na+-K+ pumps.
ScCKmit, on the other hand, is considered important for efficient
transport and conversion of high-energy phosphates in the mitochondria
(2)
.
CK is considered to minimize changes of ATP/ADP during periods of
intense activity, i.e., act as a temporal energy buffer. CK may also be
important for the communication between intracellular sites of ATP
production and consumption, acting as a spatial energy buffer (2
, 3)
. If changes in high-energy phosphates are involved in muscle
fatigue, it may be postulated that fatigue will develop more rapidly in
muscles devoid of CK. In line with this, medial gastrocnemius muscle of
mice completely deficient in CK (CK-/- mice)
shows a markedly faster force decline during highly intense stimulation
(170 ms tetanic stimulation every 250 ms, giving a duty cycle of 68%)
than muscles from wild-type mice (4)
. Diaphragm muscle
strips from CK-/- mice also fatigue more
rapidly during repeated tetanic stimulation (33% duty cycle) under
both isometric (5)
and isotonic (6)
conditions. Thus, CK appears important for avoiding fatigue during
intense muscle activity.
However, the CK reaction may also contribute to fatigue development.
The net reaction that takes place when high-energy phosphates are
transferred from PCr to ATP is that PCr breaks down, and Cr and
inorganic phosphate ions (Pi) accumulate. Cr is
believed to have little effect on contractile function, whereas
Pi reduces both the maximum force that
cross-bridges can produce and the myofibrillar
Ca2+ sensitivity (7
, 8)
.
Pi may also affect activation of the contractile
machinery by acting on the SR Ca2+ release. In
this context, Pi can exert opposite effects.
Pi may bind to SR Ca2+
release channels, the ryanodine receptors, and increase their open
probability (9)
, hence causing an increased SR
Ca2+ release. On the other hand,
Pi may enter the SR and precipitate with
Ca2+, thus reducing the
Ca2+ available for release (10
, 11)
.
The aim of the present study was to directly test the role of CK in fatigue by using isolated whole muscles and single muscle fibers from CK-/- mice. It appeared likely that CK is most important during high-intensity activation, and therefore single muscle fibers were fatigued both with high-intensity (67% duty cycle) and less intense (initial duty cycle of 14%) repeated tetanic stimulation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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. Adult female
mice were used; they were killed by rapid neck disarticulation and
thereafter muscles were isolated. All procedures were approved by the
local ethical committee.
Solutions
Experiments were performed at room temperature (24°C).
Isolated fibers or muscles were bathed in a tyrode solution of the
following composition (mM): NaCl, 121; KCl, 5.0;
CaCl2, 1.8; MgCl2, 0.5;
NaH2PO4, 0.4;
NaHCO3, 24.0; EDTA, 0.1; glucose, 5.5; 0.2%
fetal calf serum was added to the solution to improve survival of
single fibers (12)
. The solution was bubbled with 5%
CO2/95% O2, which gives a
pH of 7.4.
Single fiber dissection, mounting, and stimulation
Intact, single fibers were dissected from the flexor brevis
muscle of the hind limb as described elsewhere (12)
. The
isolated fiber was mounted between an Akers 801 force transducer
(SensoNor, Horten, Norway) and an adjustable holder in a chamber that
was continuously superfused by the tyrode solution. The fiber length
was adjusted to that giving maximum tetanic force and the diameter was
measured. Tetanic stimulation was achieved by supramaximum current
pulses (duration 0.5 ms) delivered at 70 Hz via platinum plate
electrodes lying parallel to the fibers. The duration of tetani was 350
ms except during the high-intensity fatiguing protocol (see below).
[Ca2+]i and force measurements
[Ca2+]i was
measured with the fluorescent Ca2+ indicator
indo-1 (Molecular Probes Europe B.V., Leiden, The Netherlands); the
pentapotassium salt of indo-1 was microinjected into fibers, which
avoids problems with loading of organelles. The fluorescence of indo-1
was measured with a system consisting of a Xenon lamp, a monochromator,
and two photomultiplier tubes (PTI, Photo Med GmbH, Wedel, Germany).
The excitation light was set to 360 ± 5 nm and the light emitted
at 405 ± 5 and 495 ± 5 was measured. The ratio of the light
emitted at 405 nm to that at 495 nm was translated to
[Ca2+]i as described
elsewhere (13)
.
After injection of indo-1, fibers were allowed to rest for at least 60 min. Thereafter some tetanic contractions were produced at 1 min intervals to ensure that force and [Ca2+]i were stable. Fibers that produced a force markedly lower than that before injections were not used. During tetani, fluorescence and force signals were sampled on-line and stored on a desktop computer for subsequent data analysis. The mean fluorescence ratio during tetanic stimulation was measured and translated to [Ca2+]i. Force is expressed as the peak tetanic force divided by the cross sectional area.
Fatiguing protocols in single fiber experiments
Fatigue was produced by repeated tetanic stimulation. Two
protocols with different intensities were used and these will be
referred to as high-intensity and low-intensity fatiguing stimulation,
respectively. The high-intensity stimulation protocol was designed to
mimic that used previously on CK-/- muscle
(4)
and consisted of 20 cycles of 200 ms tetanic
stimulation given every 300 ms, i.e., with a duty cycle (tetanic
duration divided by tetanic interval) of 0.67. After this series of
tetani, fibers were allowed to rest for at least 30 min, by which time
tetanic force and [Ca2+]i
had fully recovered. The low-intensity fatiguing stimulation was
similar to that normally used in our laboratory and consisted of 350 ms
tetani repeated until force was reduced to 30% of the original. It
started with a tetanic interval of 2.5 s, with a duty cycle of
0.14. In experiments on wild-type fibers, the tetanic interval was kept
constant, and this brought peak tetanic force down to 30% of the
original within 100 tetani. Fibers from CK-/-
mice were markedly more fatigue resistant; to produce fatigue in these,
the tetanic interval was reduced to 1.5 s after 100 tetani (duty
cycle = 0.23), and in fibers that still produced more than 30%
tetanic force, to 1 s after an additional 100 tetani (duty
cycle = 0.35). Recovery after the low-intensity fatiguing
stimulation was tested by producing a single tetanus at 2, 5, 10, and
20 min after the end of the stimulation period.
Experiments on whole muscles
Intact extensor digitorum longus (EDL) and soleus muscles were
dissected from both legs. For each pair of muscles, one muscle was
fatigued while the other served as an unstimulated control. Small
stainless steel loops were tied, using thin nylon thread, to the
tendons of the muscle. The muscle was then mounted between a force
transducer and an adjustable hook, which allowed the muscle to be
stretched to the length giving maximum tetanic force. The muscle was
bathed in continuously stirred, oxygenated tyrode solution (see above).
Tetanic stimulation was produced by applying supramaximal current
pulses (0.5 ms duration) via plate electrodes lying on each side of the
muscle. The stimulation frequency and tetanic duration were 70 Hz and
300 ms for EDL and 50 Hz and 600 ms for soleus.
After being mounted, muscles were allowed to rest for 30 min before fatiguing stimulation was started. Fatigue was produced by giving a tetanus every 2 s, and the total number of tetani given was 50 for EDL and 100 for soleus. These fatiguing protocols were designed to give a force reduction in wild-type muscles similar to that obtained in the single fiber experiments. Immediately after the end of fatiguing stimulation, muscles were removed from the stimulation chamber and frozen within ~10 s in liquid nitrogen. Thereafter the control muscle, which had been kept under the same conditions without being stimulated, was frozen in the same way. Muscles were kept in liquid nitrogen until analyzed.
Measurements of metabolites
Muscles were freeze-dried, cleaned from connective tissue,
extracted with ice-cold 0.5 M perchloric acid, and centrifuged. The
supernatant was neutralized with 2.2 M KHCO3 and
centrifuged; the latter supernatant was stored at -80°C. Metabolites
were analyzed with enzymatic techniques adapted for fluorometry,
measuring changes in NADH or NADPH as described in ref 14
.
The concentrations of metabolites were adjusted to a mean total Cr (sum
of PCr and Cr) of 61.7 ± 2.4 µmol/g dry wt in solei
(n=20) and 83.3 ± 2.2 µmol/g dry wt in EDL muscles
(n=20). There were no significant differences in total Cr
between muscles from CK-/- and wild-type
animals.
Statistics
Values are presented as means ± SEM. When
repeated measurements were performed in the same fiber during fatiguing
stimulation, statistically significant differences were determined
using one-way repeated measures analysis of variance, followed by
Student-Newman-Keuls test for multiple comparisons. Paired t
tests were used when only two measurements were performed in the same
fiber and when the concentrations of metabolites in rested and fatigued
muscles were compared. Unpaired t tests were used when
differences between two groups were analyzed. The significance level
(P) was set at 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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High-intensity fatiguing stimulation
Representative original records from single fibers stimulated for
200 ms every 300 ms are shown in Fig. 1
and the results are summarized in Fig. 2
. With this type of fatiguing stimulation, wild-type fibers showed an
increase of tetanic
[Ca2+]i and no
significant change of force, whereas CK-/-
fibers displayed a transient decline of both tetanic
[Ca2+]i and force. The
maximum decline in CK-/- fibers was obtained in
the fourth stimulation train, which started 900 ms after the onset of
fatiguing stimulation; thereafter, both tetanic
[Ca2+]i and force
recovered.
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Low-intensity fatiguing stimulation
Figure 3
shows representative original
[Ca2+]i and force records
obtained during low-intensity fatiguing stimulation of single fibers
from a wild-type mouse and a CK-/- mouse.
Tetanic force had fallen to 30% of control and fatiguing stimulation
was stopped after 88 tetani in the wild-type fiber, whereas force was
virtually unaffected after 100 tetani in the
CK-/- fiber. In the latter fiber, the tetanic
interval was then reduced to 1.5 s and after an additional 100
tetani to 1.0 s; after 304 tetani, force had fallen to 30% of
control. A major difference between the two fibers can be observed
early during fatiguing stimulation. In the wild-type fiber there was a
clear decline of tetanic force (~10%) and an increase of
[Ca2+]i during the first
10 tetani, which did not occur in the CK-/-
fiber.
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Figure 4
summarizes the results from all fibers tested (n=6 in each
group). During the 10th fatiguing tetanus,
[Ca2+]i had increased
significantly to 135 ± 4% of the control and force had decreased
significantly to 88 ± 3% in the wild-type fibers. Such changes
in early fatigue have been consistently observed in mouse fibers and
are referred to as phase 1 (15)
. At this time no
significant changes were observed in the CK-/-
fibers. However, in accordance with the results from the high-intensity
fatiguing stimulation, CK-/- fibers showed a
transient reduction of tetanic
[Ca2+]i to 93 ± 2%
in the second tetanus of the low-intensity protocol and a minor (not
significant) decrease of tetanic force to 96 ± 2% (data not
shown). Thereafter, both tetanic
[Ca2+]i and force
increased, and both had recovered fully in tetanus three to five.
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After the initial rapid decline of tetanic force in wild-type fibers,
there was a period with more stable force production (phase 2; see Fig. 4A
). Thereafter, tetanic force again started to fall rapidly
(phase 3) and had fallen to 30% of the unfatigued value after 82 ± 12 tetani were given at 2.5 s intervals (duty cycle =
0.14). After the initial increase, tetanic
[Ca2+]i started to fall
in the wild-type fibers, reaching 68 ± 4% of the control when
fatiguing stimulation was stopped. Tetanic
[Ca2+]i decreased
significantly during phase 2, whereas tetanic force showed relatively
little change (Fig. 4A
). This indicates that tetanic
[Ca2+]i was close to
saturation during phase 2, and hence a reduction had little effect on
force production (i.e., flat part of the
force-[Ca2+]i
relationship). The accelerated force decline during phase 3 would then
represent the situation when tetanic
[Ca2+]i was declining on
the steep part of the
force-[Ca2+]i
relationship (see Fig. 3
of ref 16
).
CK-/- fibers showed no significant change in
either tetanic [Ca2+]i or
force after the initial 100 tetani given at 2.5 s intervals (Fig. 4B
). Consequently, the stimulation pattern was intensified
to produce fatigue. Eventually force also started to decline in the
CK-/- fibers: these fibers entered a state
similar to phase 3 in wild-type fibers. However, one important
difference is that in the CK-/- fibers neither
tetanic force nor [Ca2+]i
displayed any significant changes before this state was reached. The
number of tetani required to bring tetanic force down to 30% of the
control in the CK-/- fibers was 267 ± 52;
in fact, to produce this force reduction, four of the six fibers (e.g.,
the fiber shown in Fig. 3B
) had to be stimulated at a
tetanic interval of 1 s (duty cycle = 0.35). In the fatigued
state, tetanic [Ca2+]i
had fallen significantly to 69 ± 6%, which is similar to the
reduction observed in wild-type fibers.
A comparison of the force records in fatigue in Fig. 3
shows that
development of tetanic force was markedly delayed in the fatigued
CK-/- fiber, which means that force was still
increasing rapidly at the end of the tetanus. To investigate whether
this was a general feature, we measured the rate of force increase
during the last 50 ms of tetanic stimulation in fatigued fibers, and
the results showed a significantly higher rate in
CK-/- fibers (179±31 kPa
s-1) as compared to wild-type fibers
(83±29 kPa s-1). This rate of force increase
showed no significant difference between the two groups at the start of
fatiguing stimulation or at 2 min of recovery (data not shown). The
slow force increase in fatigued CK-/- fibers
was not accompanied by a markedly reduced rate of rise of tetanic
[Ca2+]i (see Fig. 3B
).
Tetanic [Ca2+]i depends
on both the SR Ca2+ release and uptake. That
tetanic [Ca2+]i remained
high for a longer time in CK-/- fibers may be
due to a slowing of SR Ca2+ uptake rather than
preserved Ca2+ release. An indication of slowed
uptake can be obtained by measurements of the
[Ca2+]i at rest, which
increases if the rate of uptake is markedly reduced (17)
.
Before fatiguing stimulation, there was no significant difference in
the resting [Ca2+]i of
wild-type fibers (89±15 nM) and CK-/- fibers
(85±5 nM). In wild-type fibers, resting
[Ca2+]i measured
immediately before the last fatiguing tetanus had increased to 229 ± 12 nM. In CK-/- fibers, on the other hand,
resting [Ca2+]i was only
increased to 108 ± 9 nM immediately before the 100th tetanus.
When the CK-/- fibers eventually were fatigued,
resting [Ca2+]i had
increased to 263 ± 27 nM, similar to the value in fatigued
wild-type fibers. Thus, the preserved tetanic
[Ca2+]i during fatigue of
CK-/- fibers does not seem to be due to a
larger slowing of the SR Ca2+ uptake.
Two minutes after the end of fatiguing stimulation, tetanic [Ca2+]i had recovered substantially in both wild-type and CK-/- fibers, being 93 ± 3% and 95 ± 6% of control, respectively. However, at this time the recovery of tetanic force was significantly different in the two groups, with wild-type fibers producing 67 ± 3% and CK-/- fibers 83 ± 4% of the control tetanic force. Thereafter, tetanic [Ca2+]i displayed some decrease in both wild-type and CK-/- fibers, reaching 76 ± 2% and 77 ± 4% of control at 20 min of recovery. During the same period, tetanic force increased in wild-type fibers to 78 ± 6% of control whereas force remained virtually constant in CK-/- fibers, being 85 ± 2% of control at 20 min recovery.
Metabolites and fatigue in whole EDL and soleus muscles
In these experiments EDL muscles (with predominantly fast-twitch
fibers) and soleus muscles (with predominantly slow-twitch fibers; ref
18
) were used. An indication of the fiber type composition
can be obtained from the twitch contraction time, i.e., the time from
the onset of contraction until maximum force is produced in response to
a single stimulus pulse. The twitch contraction time was not
significantly different in wild-type and CK-/-
muscles but, as expected, was markedly shorter in EDL as compared to
soleus. Mean values in wild-type and CK-/-
muscles were 24.4 ± 1.0 and 25.8 ± 1.4 ms in EDL, and
56.2 ± 6.2 and 56.0 ± 7.9 ms in soleus (n=5 in
both groups). As judged from the twitch contraction time, the single
fibers described above would resemble the fast-twitch fibers of the
EDL, because the mean twitch contraction time was 23.6 ± 1.0 ms
in single fibers from wild-type animals and 22.9 ± 1.4 ms in
CK-/- fibers (n=6 in both groups).
Whole muscles were fatigued with repeated tetani using protocols with a
fixed number of tetani (see Materials and Methods for details). Mean
data of the force produced during fatigue are shown in Fig. 5
. It can be seen that whereas EDL muscles of
CK-/- animals were more fatigue resistant, the
opposite was true for soleus muscles. There was a significant reduction
of force in the second fatiguing tetanus in EDL muscles from
CK-/- mice (mean reduction 9%), and force
thereafter increased again (data not shown). This pattern is similar to
that in single fibers from CK-/- mice and was
not observed in wild-type muscles or in CK-/-
soleus muscles.
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The concentrations of metabolites at rest and immediately after
fatiguing stimulation in EDL and soleus muscles are shown in
Table 1
and Table 2
, respectively. The major difference between wild-type and
CK-/- muscles was that during fatigue PCr did
not break down in CK-/- muscles, and
consequently there was no accumulation of Pi in
these muscles. Another notable result was that lactate accumulation was
smaller in the CK-/- muscles as compared to the
wild-type muscles.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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The tetanic force produced per cross sectional area was significantly
lower in CK-/- fibers as compared to wild-type
fibers, which agrees with previous results (4)
. The lower
tetanic force in CK-/- muscle is not due to a
major difference in Ca2+ activation of
contractile proteins since tetanic
[Ca2+]i was not different
in CK-/- and wild-type fibers. Possible
explanations for the lower force in CK-/-
muscle include cytoarchitectural abnormalities and a higher proportion
of the cell volume being occupied by mitochondria (4
, 18
, 19)
, leading to a reduced concentration of myofibrils.
Furthermore, the higher Pi concentration in
rested CK-/- fast-twitch muscle (Table 1
; see
also ref 4
) may contribute to the lower force production,
since Pi is known to reduce both the maximum
force production and the myofibrillar Ca2+
sensitivity (7
, 8)
.
High-intensity fatiguing stimulation
We observed a transient decline of tetanic
[Ca2+]i and force during
high-intensity fatiguing stimulation of CK-/-
fibers. In CK-/- fibers we also observed a
transient decrease of tetanic
[Ca2+]i in the second
tetanus of low-intensity fatiguing stimulation. Furthermore,
CK-/- EDL muscles showed a transient decrease
of tetanic force in the second fatiguing tetanus. Thus, in most
situations, CK-deficient muscle exhibit transient inhibition of
Ca2+ release early during repeated tetanic
stimulation. Since this decline was never observed in wild-type muscle,
it is likely that it is caused by a transient change in high-energy
phosphates occurring in the absence of PCr energy buffering. Possible
mechanisms include inhibition of SR Ca2+ release
channels due to reduced ATP (20)
, increased free
myoplasmic [Mg2+]
([Mg2+]i)
(21)
, or both (22)
. The hypothesized changes
of ATP and/or Mg2+ may also be localized to the
sites of SR Ca2+ release, i.e., in the triads
(23)
. Therefore, CK may be important for shuttling of
high-energy phosphates between triads and mitochondria (2
, 3)
. In muscles with a high mitochondrial content, the distance
between mitochondria and sites of ATP consumption will be smaller and
therefore PCr energy shuttling may be less important (24)
.
In line with this, the transient decrease in force was never observed
in soleus muscles.
The time course of the transient decrease of tetanic
[Ca2+]i and force can be
analyzed in the experiments with high-intensity stimulation (Fig. 2)
.
There the maximum depression occurred ~1 s after the onset of
stimulation. This may reflect the time it takes for mitochondrial ATP
production to start reversing the hypothesized changes in high-energy
phosphates in triads, including the time required for transportation.
In a recent study by Steeghs et al. (4)
, high-intensity
tetanic stimulation of the medial gastrocnemius muscle caused a rapid
decline of force that did not show any recovery. This contrasts with
our experiments where a marked recovery was observed. This may be
explained by unrestricted oxygen supply in our experiments, whereas in
the experiments of Steeghs et al. the blood supply to the activated
muscle was probably compromised (25)
leading to a limited
oxygen availability. Other factors may also contribute to the
difference; for instance, the higher temperature used in the study of
Steeghs et al. (4)
.
One conspicuous result with high-intensity fatiguing stimulation is
that the increase in
[Ca2+]i in wild-type
fibers had little effect on force, whereas the decline in
[Ca2+]i in
CK-/- resulted in a substantial force reduction
(Figs. 1
and 2)
. These results can be explained by the fact that 70 Hz
stimulation gave forces close to the maximum, i.e., close to saturation
of the force-[Ca2+]i
relationship. This means that an increase of
[Ca2+]i will have little
effect on force, whereas a reduction will have a great impact since it
moves force to the steep part of the
force-[Ca2+]i
relationship (see Fig. 3
in ref 16
). In wild-type fibers
there was a reduction of maximum force at the onset of low-intensity
fatiguing stimulation, possibly due to Pi
accumulation (see below), which will counteract any force enhancement
due to increased [Ca2+]i.
Also, fast-twitch CK-/- muscle has a higher
Pi concentration at rest (Table 1)
, which may
exaggerate the force decline with high-intensity stimulation due to a
reduced myofibrillar Ca2+ sensitivity (7
, 8)
.
Low-intensity fatiguing stimulation
During low-intensity fatiguing stimulation, the fast-twitch
CK-/- fibers and EDL muscles showed an
increased fatigue resistance whereas the slow-twitch soleus muscles
fatigued faster. Skeletal muscles of CK-/- mice
display marked adaptations, with an increased mitochondrial content and
oxidative capacity (4
, 18
, 19)
. This, together with an
unrestricted oxygen supply under our experimental conditions, is likely
to contribute to the increased fatigue resistance in fast-twitch
CK-/- muscle. Thus an effective high-energy
phosphate transfer can exist at sites of rapid energy consumption and
at the mitochondria even in the absence of PCr buffering. Also, the
transport of high-energy phosphates between mitochondria and sites of
energy consumption can be effective without the proposed PCr shuttle
(2
, 3)
. Another factor that may contribute to the
increased fatigue resistance in fast-twitch
CK-/- muscle is the lack of large increases of
Pi during fatiguing stimulation (see below).
Soleus muscles, on the other hand, were less fatigue resistant in
CK-/- mice. This highly oxidative, slow-twitch
muscle also shows increased oxidative capacity in
CK-/- mice, but the adaptation is smaller than
in fast-twitch muscle (19)
. Our fatigue results,
therefore, suggest that the extra oxidative capacity cannot be fully
utilized in highly oxidative CK-/- muscles.
This could possibly be due to the absence of ScCKmit and, hence,
inefficient transport and conversion of high-energy phosphates in the
mitochondria.
Our measurements of metabolites in whole muscles show that
CK-/- muscles have a normal total Cr
concentration (4)
. PCr breakdown cannot be used to
regenerate ATP in CK-/- muscles, which is
clearly shown by the fact that neither phosphocreatine, creatine, nor
Pi was significantly different in rested and
fatigued CK-/- muscles (see Tables 1
and 2
).
Lactate accumulation during fatigue was smaller in
CK-/- muscles than in wild-type muscles. Still,
fatigue occurred more slowly in CK-/- EDL
muscles and more rapidly in CK-/- soleus
muscles as compared to wild-type muscles, which supports the idea that
lactate accumulation (and the associated acidosis) is not a major
factor causing fatigue (ref 26
and references therein). It
should be noted that PCr breakdown results in consumption of
H+ (27)
. There will therefore be an
additional acid load during fatigue in CK-/-
muscles. However, this additional acid load is likely balanced by the
reduced lactate accumulation in these muscles and the observed change
in pH with prolonged exercise is not different in wild-type and
CK-/- muscle (4)
.
With low-intensity fatiguing stimulation, we had previously observed a
10–20% reduction of tetanic force that is completed within 20 tetani
and associated with an increase of tetanic
[Ca2+]i. This has been
named phase 1 and was observed in isolated fibers of various fatigue
resistance and in Xenopus and mouse fibers (15
, 28
, 29)
. The underlying mechanism has not been established, but
Pi accumulation has been suggested to be a strong
candidate (1)
. The present results support this suggestion
since muscle cells from CK-/- mice did not show
the changes associated with phase 1. The force decrease during phase 1
is then likely due to a direct effect of Pi on
cross-bridge cycling with a reduced fraction of cross-bridges in
strong, force-generating states (8)
. Furthermore, there
are several mechanisms by which increased myoplasmic
Pi may cause increased tetanic
[Ca2+]i: 1)
decreased myoplasmic Ca2+ buffering due to
Pi reducing the number of strong,
force-generating cross-bridges interacting with the thin filament
(30)
; 2) Pi-induced
inhibition of the SR Ca2+ pumps
(31)
; and 3) Pi binding
to SR Ca2+ release channels resulting in an
increased rate of Ca2+ release (9)
.
During the first 2 min of recovery after fatiguing stimulation, tetanic
[Ca2+]i increased up to
approximately the prefatigue level in both wild-type and
CK-/- fibers. However, tetanic force was still
markedly reduced, especially in wild-type fibers where the mean force
was only 67% of the control (vs. 83% in CK-/-
fibers). The difference between the two groups of fibers might be
explained by a markedly higher myoplasmic Pi
concentration in the wild-type fibers, since Pi
is known to decrease both myofibrillar Ca2+
sensitivity and maximum cross-bridge force (7
, 8)
. A
similar difference would also be expected at the end of fatiguing
stimulation, but at this stage the measured tetanic
[Ca2+]i and force were
equally decreased in wild-type and CK-/-
fibers. However, tetanic force development was delayed in fatigued
CK-/- fibers (see Fig. 3
) and hence force was
still increasing rather rapidly at the end of tetanic stimulation in
these fibers. This means that force would have been less reduced in
fatigued CK-/- fibers if the tetanic duration
was increased, again suggesting a greater decrease in myofibrillar
Ca2+ sensitivity and maximum cross-bridge force
in wild-type fibers. Furthermore, the slow force development in
fatigued CK-/- fibers could not be explained by
a reduced rate of [Ca2+]i
increase at the onset of tetanic contractions. This indicates a major
decrease in the rate of cross-bridge cycling in fatigued
CK-/- fibers, possibly due to ADP accumulation
in the vicinity of cross-bridges (32)
, which might become
more prominent in the absence of CK energy buffering.
Tetanic [Ca2+]i was
markedly more fatigue resistant in CK-/- than
in wild-type fibers (except for the second fatiguing tetanus; see
above). The mechanism behind the decrease in tetanic
[Ca2+]i with
low-intensity fatiguing stimulation is not clear. It has been shown to
coincide with an increase of
[Mg2+]i that would stem
from a net breakdown of ATP (33)
. Both reduced ATP and
increased [Mg2+]i may
impair SR Ca2+ release (20
21
22)
, and
such changes are likely delayed in fibers with a higher oxidative
capacity. This is one possible explanation for the increased fatigue
resistance in CK-/- fibers. Another mechanism
behind the decrease of tetanic
[Ca2+]i in fatigue might
be that Pi enters the SR and precipitates with
Ca2+, leading to a reduced amount of
Ca2+ available for release (10
, 11
, 34)
. This mechanism would require an increase of
Pi during fatiguing stimulation that does not
occur in CK-/- muscle, and hence this offers
another feasible explanation for the delayed decrease of tetanic
[Ca2+]i in
CK-/- fibers.
After the initial 2 min of recovery, tetanic
[Ca2+]i fell somewhat in
both groups, reaching ~75% of the control at 20 min of recovery.
This decrease of tetanic
[Ca2+]i is a normal
feature after fatigue in mouse fibers and is the mechanism underlying
low-frequency fatigue (LFF; ref 35
). Tetanic
[Ca2+]i is reduced at all
stimulation frequencies in LFF whereas force is decreased mainly at low
stimulation frequencies, because tetanic
[Ca2+]i then falls on the
steep part of the
force-[Ca2+]i curve. The
amount of LFF has been shown to correlate with the time integral of
increased [Ca2+]i
(36)
. CK-/- fibers would then be
expected to display a more profound LFF, since these fibers fatigued
much more slowly and in this way were exposed to more
Ca2+. However, this was not the situation;
therefore, some mechanism must protect CK-/-
fibers from the potentially damaging effects of increased
Ca2+.
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CONCLUSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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=
CK-/-; error bars in most instances smaller than
symbols.