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1 School of Exercise and Sport
Science and 3 Rehabilitation Research Centre, ABSTRACT Booth, John, Michael J. McKenna, Patricia A. Ruell, Tom H. Gwinn, Glen M. Davis, Martin W. Thompson, Alison R. Harmer, Sandra K. Hunter, and John R. Sutton. Impaired calcium pump function does
not slow relaxation in human skeletal muscle after prolonged exercise.
J. Appl. Physiol. 83(2): 511-521, 1997.
fatigue; muscle contractile function; sarcoplasmic reticulum; calcium uptake; calcium adenosinetriphosphatase activity
INTRODUCTION FATIGUE IN HUMAN SKELETAL MUSCLE can be accompanied by
both a decline in maximal tension and a prolongation of half relaxation time (RT1/2) (24). Although the mechanisms of
fatigue are multifactorial in origin, one contribution to fatigue may
be a slowed rate of Ca2+ uptake by
the sarcoplasmic reticulum (SR) (6, 7, 24). The SR is the primary
regulator of intracellular
[Ca2+] (where brackets
denote concentration) in mammalian skeletal muscle, and hence the
contractile process, and reduced
Ca2+ uptake during fatigue could
alter muscle contractile function in two ways. A reduced rate of
intracellular Ca2+ removal could,
first, slow myofilament dissociation, thereby prolonging muscular
relaxation, and, second, affect the stoichiometry between
Ca2+ uptake and
Ca2+ release, resulting in reduced
Ca2+ release and a consequent
decline in tension. With the development of reliable methods to measure
the maximum rate of Ca2+ uptake
from human muscle homogenates (47), the relationship between in vitro
SR Ca2+ uptake in whole muscle
homogenates and in vivo muscle contractile function can be assessed. In
human skeletal muscle, short-term high-intensity exercise reduced
muscle homogenate SR Ca2+ uptake
by 42%, which was associated with a greater than twofold prolongation
of twitch RT1/2 (24).
However, definition of the relationship between impaired SR
Ca2+ regulation and muscle
contractile function after short-term intense exercise is complicated
by the accompanying acidosis, which can impair both SR
Ca2+ uptake (35) and cross-bridge
cycling (42). To avoid the deleterious effects of an exercise-induced
acidosis, the relationship between muscle contractile function in vivo
and Ca2+ uptake in muscle
homogenates was assessed at rest and after prolonged submaximal
exercise. The effect of prolonged exhaustive exercise on SR
Ca2+ handling and contractile
function has not been previously determined in humans. The
aim of the study was to determine whether a slowing of
Ca2+ uptake occurred after
prolonged exercise and whether this was associated with a prolongation
of RT1/2, as reported previously after short-term intense
exercise (24).
MATERIALS AND METHODS
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
ACKNOWLEDGEMENTS
FOOTNOTES
REFERENCES
This study examined the effects of prolonged exercise on human
quadriceps muscle contractile function and homogenate sarcoplasmic
reticulum Ca2+ uptake and
Ca2+-adenosinetriphosphatase
activity. Ten untrained men cycled at 75 ± 2% (SE) peak oxygen
consumption until exhaustion. Biopsies were taken from the
right vastus lateralis muscle at rest, exhaustion, and 20 and 60 min
postexercise. Peak tension and half relaxation time of the left
quadriceps muscle were measured during electrically evoked twitch and
tetanic contractions and a maximal voluntary isometric contraction at
rest, exhaustion, and 10, 20, and 60 min postexercise. At exhaustion,
homogenate Ca2+ uptake and
Ca2+ adenosinetriphosphatase
activity were reduced by 17 ± 4 and 21 ± 5%, respectively, and
remained depressed after 60 min recovery (P 
0.01). Muscle ATP, creatine
phosphate, and glycogen were all depressed at exhaustion
(P 0.01). Peak tension during a maximal voluntary contraction, a twitch, and a 10-Hz stimulation were
reduced after exercise by 28 ± 3, 45 ± 6, 65 ± 5%,
respectively (P 0.01), but no
slowing of half relaxation times were found. Thus fatigue induced by
prolonged exercise reduced muscle
Ca2+ uptake, but this did not
cause a slower relaxation of evoked contractions.
O2) was calculated by
using a metabolic cart (model MMC 2900, Sensor Medics, Anaheim, CA).
The highest O2 during a
1-min interval in the incremental test was termed peak
O2
(O2 peak).
The submaximal O2 and
O2 peak were used to
determine power output for the prolonged exercise test. A warm-up
consisting of 10 min of cycling at 40%
O2 peak was followed
by measurement of contractile properties. Subjects then cycled to
exhaustion at a power output calculated to elicit 70%
O2 peak while
maintaining a pedal cadence between 70 and 80 revolutions/min (rpm).
Cadence became irregular as subjects approached exhaustion, so, to
elicit a clear end point for each subject, exhaustion was defined as an
inability to maintain pedal cadence above 55 rpm, despite verbal
encouragement. At 15-min intervals during prolonged exercise,
O2 was determined over a
3-min period and 100 ml water were ingested. Rectal temperature was
monitored continuously by using a rectal probe (Yellow Springs Instruments, Yellow Springs, OH) inserted 12 cm beyond the anal sphincter. Heart rate was determined continuously during both exercise
tests from an electrocardiograph.
Measurement of muscle contractile function.
All muscle contractile measurements, including both stimulated and
voluntary contractions, were made on the quadriceps muscle group of the
left leg. Muscle contractile properties were measured at rest (after
warm-up), at exhaustion (60-75 s after completion of exercise),
and after 10, 20, and 60 min of recovery. The stimulation protocol
comprised three maximal twitches, followed by 2 and 3 s of electrical
stimulation at 10 and 100 Hz, respectively. Electrically evoked low-
and high-frequency contractions permit muscle contractile function to
be assessed independent of the central nervous system. The protocol
chosen allowed contractile function to be studied in different states
of activation, because the tetanic intracellular [Ca2+] and the number
of active cross bridges would each be greater at high compared with low
stimulation frequencies.
The current used to elicit a maximal twitch was determined in each
individual by increasing the stimulation current until no further
increase in tension was observed, despite further increments in
current. This procedure ensured that each twitch was truly maximal for
each individual, with the current ranging from 55 to 85 mA among
individuals. For the 10- and 100-Hz stimulation procedures, the current
was increased to the highest tolerable level determined by the subject,
which ranged from 20 to 35 mA. Thirty seconds after the stimulation at
100 Hz, subjects performed a 3-s maximal voluntary isometric
contraction (MVC) of the knee extensors. For measurement of all muscle
contractile properties, the subject was seated with the hips flexed at
90° and with the chest and lower abdomen secured by straps to
prevent upper body movement. Knee angle was set at 60° from leg
extension, and the ankle was secured in a precast mould to minimize
lateral movement. The mould was attached to a force transducer (model
2000 N X-Tran, Applied Measurement). Electrical stimulation involved
percutaneous muscle stimulation via two 8 × 13-cm oval pad
electrodes (Medtronic Nortech Division) placed proximally and distally
on the anterolateral thigh. Square-wave stimulation pulses (400 V, 100 µs) were initiated by a stimulator (model DS7, Digitimer,
Hertfordshire, UK), and the frequency was set by a digitimer programmer
(model D4030, Digitimer). The tension output was amplified (model
RD201A, Applied Measurement) and digitized (model DT2801, Data
Translation). Data were sampled at 1 kHz, stored on a computer
(IBM-compatible personal computer), and analyzed for peak tension and
RT1/2 with software written in a
Forth-language derivative (Asyst; Keithley Instruments). Twitch
RT1/2 was defined as the time from
peak tension until half initial tension during the relaxation phase.
For 100-Hz stimulation, RT1/2 was
defined as the time taken for tension to fall from 95 to 50% of steady
plateau tension after the last stimulus in a train of pulses. No
measures of RT1/2 were made after
the 10-Hz contractions because these were found to be unreliable in
pilot testing. The reproducibility of all contractile measurements used in this study were assessed in eight subjects by using two trials conducted on the same day. Subjects performed a 10-min warm-up at 40%
O2 peak, followed by
contractile property measures, and then rested for 60 min before
repeating both the warm-up and contractile measurements. There were no
significant differences (P 0.01) in
the peak tension or RT1/2 of
voluntary or evoked contractions for the two trials. The coefficients
of variation for the contractile measurements were low for MVC (1.2%),
peak twitch tension (2.9%), peak tension at 100 Hz (3.1%), twitch
RT1/2 (4.7%), and 100-Hz RT1/2 (5.7%), with larger
variation for peak tension at 10 Hz (11.1%).
Muscle biopsy and processing procedures.
Needle biopsy samples were taken from incisions made under local
anesthesia (Xylocaine, 1%) in the middle one-third of the right vastus
lateralis muscle, with suction applied to the needle. The resting
biopsy was taken with subjects supine on a laboratory bed before they
commenced the warm-up before the prolonged cycling test. Immediately on
cessation of cycling exercise, the subject reclined on the ergometer
with the trunk supported from behind, toe clips were rapidly loosened,
the right leg was supported, and within 15 s of completion of exercise,
a second biopsy was taken from the same incision. During
recovery, muscle biopsies were taken immediately before the contractile
measurements at 20 and 60 min postexercise from a second incision
located ~1-2 cm from the first incision. Approximately
80-100 mg of muscle tissue were removed, placed on a precooled
petri dish, and rapidly divided into two portions; one was immediately
frozen and stored in liquid N2 for
later analysis of metabolites. The remaining tissue was weighed (49 ± 4 mg), placed in chilled homogenizing buffer composed of 40 mM
tris(hydroxymethyl)aminomethane, 0.3 M sucrose, and 5 mM
dithiothreitol, pH 7.9, and immediately homogenized on ice with a
handheld electric homogenizer by using 3 × 15-s bursts (model
1000, Omni International). The protein concentration of each muscle
homogenate sample was determined by a modified Lowry procedure with
sodium dodecyl sulfate, by using a commercial protein solution as a
standard (Boehringer Mannheim).
Measurement of the maximum rate of
Ca2+uptake.
The methods for the measurement of
Ca2+ uptake are detailed elsewhere
(47). The maximum rate of muscle homogenate
Ca2+ uptake was measured at
37°C, with stirring, by using the fluorescent Ca2+ indicator indo 1 and a
luminescence spectrometer (series 2, Aminco Bowman). The excitation
wavelength was 349 nm, and the emission wavelength alternated between
410 and 485 nm (for Ca2+-bound and
Ca2+-free indo 1, respectively),
with ratiometric data obtained every second. Excitation and emission
band-pass widths were set to 1 and 8 nm, respectively.
Ca2+ uptake was determined in
triplicate after addition of 50 µl of homogenate to the assay medium
(2.2 ml) composed of 20 mM
N-2-hydroxyethylpiperazine-N
-2-ethanesulfonic acid, 150 mM KCl, 10 mM NaN3, 6.8 mM oxalate, 5 µM
N,N,N,N-tetrakis(2-pyridylmethyl)-ethane diamine, 4.5 mM MgATP, and 1 µM indo 1, pH 7.0. The
decrease in [Ca2+] due
to uptake by the SR was determined from the ratio of emission signals
at 410 and 485 nm (47). The dissociation constant for the
Ca2+-indo 1 complex in 150 mM KCl
buffer and 20 mM
N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid, pH 7.0, was found to be 170 nM by using precise mixtures of
Ca2+-ethylene
glycol-bis(
-aminoethyl
ether)-N,N,N,N-tetraacetic acid.
Measurement of the maximum rate of
Ca2+-adenosinetriphosphatase
(ATPase) activity.
Homogenate Ca2+-ATPase activity
was measured in triplicate spectrophotometrically at 37°C with
ionophore A-23187 as previously described (47).
Ca2+-ATPase activity was
calculated by subtracting
Ca2+-independent (basal) ATPase
from total (Ca2+-dependent + Ca2+-independent) ATPase
activities.
Measurement of muscle metabolites.
Muscle samples were freeze-dried, dissected free of blood and
connective tissue, powdered, and extracted according to the methods of
Harris et al. (27). The neutralized extract was assayed enzymatically
for ATP, creatine phosphate (CP), creatine (Cr), and lactate
(Lac
) by fluorometric
analyses. With the exception of muscle
Lac, metabolites were
adjusted to the peak total Cr for each subject to correct for
variability in blood, connective tissue, or other nonmuscle
constituents between biopsies. A further portion of freeze-dried muscle
was homogenized at 0°C in 100 vol of 145 mM KCl, 10 mM NaCl, and 5 mM sodium iodoacetate, and homogenate pH was measured while
stirring by using a pH microelectrode (model AEP341, Activon).
Blood sampling and processing.
A catheter (20 gauge; Jelco) was inserted into a distal superficial
forearm vein, and blood (8 ml) was drawn into a preheparinized syringe
at rest, at 15-min intervals throughout exercise, at exhaustion, and at
20 and 60 min postexercise. Blood samples were arterialized before
sampling; at rest, the forearm and hand were placed in a heated perspex
chamber; and during the early stages of exercise and recovery heating
was by a handheld fan. Periodic infusions of isotonic
saline (1-2 ml) were used to keep the catheter patent. Air bubbles
were expelled from the syringe, the blood was well mixed, and ~1 ml
of blood was portioned into an Eppendorf tube. Whole blood (250 µl)
was deproteinized in 500 µl of 0.6 M cold perchloric acid and
centrifuged, and the supernatant was drawn off and stored at
20°C for later triplicate analysis of
Lac by an enzymatic
technique. Blood glucose was determined by using a glucose-lactate
analyzer (model 2700, Yellow Springs Instruments). All analytic
instruments were calibrated before and during the analyses with
precision standards.
Statistical methods.
For all biochemical assays and muscle contractile measurements, a
multivariate repeated-measures analysis of variance was used.
Significant differences over time (rest, exhaustion, and recovery) were
assessed via Hotteling's T2 statistic.
Pair-wise comparisons between means were confirmed by paired
t-tests
(P 0.01). Linear regression was
performed between the desired dependent and independent variables. All
data are expressed as means ± SE.
RESULTS
O2 peak was
3.57 ± 0.15 l/min. During prolonged exercise,
O2 was 75 ± 2% of
O2 peak at 15 min and
increased slightly (not significant) to reach 83 ± 2% of
O2 peak at 60 min of
exercise. The mean O2 over
the whole exercise bout was equivalent to 79 ± 2% of
O2 peak, with
exhaustion achieved at 72 ± 4 min. Heart rate rose progressively
throughout exercise to reach 96 ± 3% of peak heart rate at
exhaustion (P 0.01). Rectal
temperature increased from 37.7 ± 0.2°C before exercise to 39.5 ± 0.3°C at the end of exercise
(P 0.01) and remained elevated at 38.6 ± 0.3°C (P 0.01) 20 min postexercise.
Hematologic responses
Blood [Lac]
peaked (P 0.01) at 6.43 ± 0.47 mM after 15 min exercise and remained elevated
(P 0.01) at exhaustion (5.39 ± 0.74 mM) and at 20 min postexercise (2.84 ± 0.43 mM). Plasma pH was
unchanged by exercise. Blood glucose declined from rest (5.72 ± 0.25 mM) by 14% after 15 min exercise and at exhaustion (P 0.01) but did not differ
significantly from rest at 20 and 60 min postexercise.
Muscle metabolites.
At exhaustion, muscle glycogen, CP, and ATP contents had fallen by 90, 58, and 20%, respectively (P 0.01;
Table 1). Muscle CP and ATP had completely
recovered by 20 min postexercise, but no significant resynthesis of
glycogen was evident at 60 min postexercise (Table 1). Muscle
Lac increased at exhaustion
(P 0.01) but was not different from rest throughout recovery, whereas muscle pH was unchanged during exercise or recovery (Table 1).
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1 · mg
protein1, respectively. No
differences were found in the repeat resting biopsies for two subjects
for either Ca2+ uptake
(subject 1: 10.76 vs. 9.86 nmol · min1 · mg
protein1 for
rest 1 and rest
2, respectively; subject
2: 6.17 vs. 6.55 nmol · min1 · mg
protein1 for
rest 1 and rest
2, respectively) or
Ca2+-ATPase activity (87.99 vs.
88.98; 52.44 vs. 49.02 nmol · min1 · mg
protein1). After
prolonged exercise, there were significant reductions in the rate of
Ca2+ uptake, paralleled by
reductions in the activity of the
Ca2+-ATPase enzyme (Fig
1). At exhaustion,
Ca2+ uptake and
Ca2+-ATPase activity were reduced
by 17 ± 4 and 21 ± 5% from resting values, respectively
(P 0.01). After 20 min of recovery,
Ca2+ uptake and
Ca2+-ATPase activity remained
depressed by 22 ± 5 and 28 ± 4%
(P 0.01), with no recovery evident
at 60 min postexercise. When measured in one subject after 6 h
recovery, Ca2+ uptake and
Ca2+-ATPase activity were 18 and
5% below rest, respectively. The association between
Ca2+ uptake and
Ca2+-ATPase activity was unaltered
by exercise and for all data was represented by
Ca2+ uptake = 3.94 ± (Ca2+-ATPase activity × 0.67); (r = 0.62, P 0.01).
) and
Ca2+-ATPase activity (
) after
prolonged exercise to exhaustion (Exh; 72 ± 4 min). Values are
means ± SE; n = 10 subjects.
* Significantly different from rest,
P 0.01.
Muscle contractile responses. The responses elicited by percutaneous muscle stimulation at rest and at exhaustion in one subject are shown in Fig. 2. The effects of prolonged exercise on the isometric contractile properties are shown in Table 2. Both voluntary and involuntary tension declined after exercise (Fig. 3). Knee extension MVC declined from 814 ± 37 N at rest to 587 ± 34 N at exhaustion (P
0.01) and remained
depressed at 10 and 20 min postexercise
(P 0.01), with nearly complete
recovery at 60 min postexercise. At rest, 100-Hz stimulation elicited a
peak tension equal to 66 ± 2% of MVC. In contrast to the MVC, peak
tension for an electrically evoked contraction at 100 Hz was unchanged
at the end of exercise and gradually increased throughout recovery to
an elevation of 13 ± 5% above rest at 60 min postexercise
(P 0.01). No potentiation of peak
tension at 100 Hz was evident when measured in one subject after 6 h
recovery. Exhaustive exercise had a pronounced effect on the peak
tension of evoked contractions during low-frequency stimulation. At
rest, 10-Hz stimulation elicited a peak tension of 116 ± 7 N, which
was reduced by 65 ± 5% at exhaustion
(P 0.01) and remained depressed at
10 and 20 min postexercise (P 0.01). After 60 min recovery, peak tension at 10-Hz stimulation had
recovered to 84 ± 4% of the initial value. When measured in one
subject after 6 h recovery, peak tension at 10 Hz remained 24% less
than at rest. The electrically evoked maximal twitch peak tension was
96 ± 7 N at rest and was reduced by 45 ± 6% at the end of
exercise (P 0.01); it remained
depressed after 10 and 20 min (P 0.01) but not at 60 min recovery.
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) and during evoked contractions [twitch (
), 10-Hz tetanus (
), and 100-Hz tetanus
()] in quadriceps muscle group after prolonged exercise to Exh
(72 ± 4 min). Values are means ± SE;
n = 10 subjects. * Significantly
different from rest, P 0.01.
Prolonged exhaustive exercise had a profound effect on the RT1/2 of a maximally evoked twitch but not for a tetanic contraction at 100 Hz (Fig. 4). Twitch RT1/2 was reduced from 86 ± 5 to 51 ± 3 ms at exhaustion and remained 20 ± 3% shorter than at rest at 60 min postexercise (P
0.01). However, no significant changes were found in the
RT1/2 of a tetanic contraction at
100 Hz at exhaustion or during recovery.
0.01.
Interrelationships between SR and contractile function. There was no significant relationship between Ca2+ uptake and muscle contractile function at rest or after exercise. When all data were pooled, no significant associations were found between Ca2+ uptake and peak twitch tension (r = 0.34), peak tension at 10 Hz (r = 0.16), peak tension at 100 Hz (r = 0.06), or MVC (r = 0.36). Similarly, there was a dissociation between Ca2+ uptake and RT1/2 for an evoked twitch or a tetanic contraction at 100 Hz, at rest, and after exercise. Pooled data revealed no significant associations between Ca2+ uptake and RT1/2 for either a maximal twitch (r = 0.48) or 100-Hz contraction (r = 0.22; Fig. 5).
1 · mg
protein1) at rest and
after prolonged exercise to Exh. , Rest; , Exh; , 20 min
postexercise; , 60 min postexercise. There were no significant
linear relationships at any time point or for pooled data for twitch
and 100-Hz comparisons (r = 0.48, r = 0.22, respectively).
DISCUSSION
Prolonged cycling exercise was performed to induce a state of fatigue
that might impair both SR and contractile function. The exercise bout
imposed considerable strain on the subjects, evidenced by increases in
heart rate, blood
[Lac], and
rectal temperature, coupled with a decrease in blood glucose and almost
total depletion of muscle glycogen. Fatigue was substantiated by an
inability to maintain the required power output during dynamic exercise, as well as reduced postexercise voluntary and evoked isometric force production. The major finding from this study was that
the SR Ca2+ uptake rate was
dissociated from the rate of muscle relaxation after fatigue induced by
prolonged exercise; fatigue attenuated SR
Ca2+ uptake rate but did not
prolong muscle relaxation.
O2 peak
(49), consistent with the observed rise in rectal temperature to
39.5°C in this study. It is, therefore, worth considering the
possibility that increased muscle temperature may also contribute to
the depressed SR Ca2+ uptake and
Ca2+-ATPase activity with
prolonged exercise in humans. It is unlikely that an elevated muscle
temperature per se was directly responsible for the in vitro depression
in muscle Ca2+-ATPase activity
observed after prolonged exercise. The direct effect of elevated
temperature is an increased
Ca2+-ATPase activity, up to nearly
50°C (30, 38), beyond which inactivation of
Ca2+-ATPase occurs (30). In
contrast, fatiguing exercise depressed SR
Ca2+-ATPase activity in rat
muscle, and this was evident across a range of temperatures from 15 to
42.5°C (38). Similarly, in the present study,
Ca2+-ATPase activity measured in
vitro at 37°C was depressed with fatigue. At temperatures above
40°C, muscle SR Ca2+ uptake
was uncoupled from Ca2+-ATPase
activity (30), suggesting impairment in vectorial
Ca2+ translocation but unaltered
ATP binding and hydrolysis. Thermal inactivation of the muscle SR
Ca2+-transport system can occur
under normal physiological conditions (temperature 37°C,
[Ca2+] <10 µM),
but this effect is characterized by a depression in the rate of
Ca2+ uptake, with only small
effects on Ca2+-ATPase activity
(12, 41). With prolonged exercise in humans, both
Ca2+ uptake and
Ca2+-ATPase activity were
proportionally depressed, suggesting that thermal inactivation did not
occur. It has been suggested that the temperature-induced impairment of
SR Ca2+ uptake but elevated
Ca2+-ATPase activity is due to a
partial unfolding of the enzyme (12); this would leave cytosolic ATP
bindings sites exposed, but reduce access to
Ca2+ binding sites, therefore
inhibiting vectorial displacement of Ca2+. Such a conformational change
is inconsistent with the finding of a decreased number of ATP binding
sites found on the Ca2+-ATPase
enzyme after prolonged exercise in rats (38). From the above findings,
it seems unlikely that elevated muscle temperature is directly
responsible for the exercise-induced depression in SR
function. However, in contrast, increased temperature in
rat muscle in situ was demonstrated to depress
Ca2+-ATPase to the same extent as
that seen during prolonged exercise (28). The effects of elevated in
vivo temperature on muscle SR function, therefore, require further
investigation.
The SR is known to be a layered structure of lipids throughout which
the Ca2+-ATPase enzyme extends,
with enzyme activity dependent on the presence of lipids (30). It has
been suggested that the
Ca2+-ATPase enzyme activity could
be impaired at temperatures above 37°C by altered protein-lipid
interaction through changes to the fluidity and structure of the lipid
layers (30). However, in contrast to these thermal denaturation
experiments, prolonged exhaustive exercise does not induce gross
changes in lipid composition, as shown in rat gastrocnemius muscle
(38). This finding suggests that gross alterations in lipid structures
are probably not responsible for the decline in
Ca2+-ATPase with prolonged
exercise seen in humans and other species, and this also argues against
free radical-induced lipid peroxidation (see below).
Prolonged exercise has been shown to induce widespread structural
alterations to muscle membranous structures, including disruptions and
dilation of the t-tubular system, the terminal cisternae, and the
longitudinal tubules of the SR (40). Although the exact mechanisms by
which these SR structural changes might impair SR function is not
known, a relationship has been suggested by the parallel changes
observed in these variables during exercise and recovery (7, 40).
Associated with reduced Ca2+
uptake with fatigue is an increased resting intracellular
[Ca2+] (55, 57).
Elevated resting intracellular
[Ca2+] and the
repetitive increases in
[Ca2+] during
contraction could potentially cause muscle protein degradation and
cellular dysfunction by stimulating
Ca2+-sensitive proteases and
phospholipases (33). Consistent with this hypothesis was an increased
activity of the neutral
Ca2+-sensitive protease
calpain in muscle after prolonged exercise in rats (1). In single mouse
fibers, the calpain-inhibitor calpeptin did not prevent the onset of
low-frequency fatigue, although possible direct effects on
Ca2+ uptake were not reported (9).
Whether activation of
Ca2+-sensitive proteases during
exercise in human skeletal muscle is sufficient to degrade SR
Ca2+-ATPase and impair
Ca2+ uptake remains unclear and
worthy of further investigation. However, it is clear that elevated
cytosolic [Ca2+] can
have marked effects on excitation-contraction coupling. Physiological
elevations in intracellular
[Ca2+] for only 10 s
uncoupled excitation-contraction in rat and toad fibers, in association
with distortion or severing of the triads, elongation of the t tubules
and SR vesiculation (34). Furthermore, the calculated
[Ca2+]-time integral
during repetitive stimulation was related to the depression in force
with fatigue in single murine fibers (9). Although
Ca2+ pump function did not appear
to be impaired in these uncoupled fibers (34), the long-term effects of
elevated intracellular [Ca2+] on
Ca2+ pumps may well be more
substantial. To our knowledge there have been no studies investigating
Ca2+ transients in intact muscle
fibers during a prolonged (i.e., >1-h) stimulation period and at
temperatures expected in active muscle, presumably because of
difficulties in fiber survival. Therefore, discussion on the effects of
very prolonged contractions on cytosolic
[Ca2+] during exercise
can only be speculative. However, it seems reasonable to hypothesize
that qualitatively similar changes in cytosolic [Ca2+] will be seen
during prolonged exercise, as observed in intact fibers after shorter
stimulation periods and at room temperature (57).
Exercise results in an increased formation of free radical compounds in
muscle, including the reactive oxygen species superoxide and hydrogen
peroxide (15, 32, 45, 46). Cellular accumulation of free radicals has
been associated with an impaired SR membrane integrity and suggested as
a possible cause of exercise-induced damage to skeletal muscle SR (15).
Consistent with this, cardiac SR
Ca2+-ATPase activity and
Ca2+ accumulation were depressed
by the superoxide radical at pH 7 (29). Oxidative stress induced by
strong oxidizing compounds markedly reduced skeletal muscle SR
Ca2+-ATPase activity and
Ca2+ accumulation, but this was
independent of free radicals, being primarily due to oxidation of
Ca2+-ATPase sulfhydryl groups
(50). However, reactive oxygen species such as singlet oxygen can
directly inhibit skeletal muscle SR function (52). In contrast,
hydrogen peroxide did not affect single muscle fiber relaxation rates,
suggesting no direct inhibition of SR
Ca2+-ATPase (5). Lipid
peroxidation has been suggested as a possible cause of SR damage as a
result of free radical accumulation in muscle (15). However, the
decline in Ca2+-ATPase activity
due to oxidative stress was not due to lipid peroxidation, because the
decline in Ca2+-ATPase was
independent of the degree of lipid oxidation, exposure to peroxylipids
did not induce enzyme inhibition, and removal of lipid peroxidation
products was also without effect (50). In addition, prolonged exercise
did not induce any gross changes in lipid structure in rat muscle (38).
This suggests that lipid peroxidation is not a major factor in the
exercise-induced depression in SR
Ca2+ uptake. Although reactive
oxygen species can damage skeletal muscle SR, whether this occurs with
exercise and, if so, whether it can acccount for the depression in SR
function seen in this study remain to be clarified.
This study investigated the relationship between muscle SR
Ca2+ uptake and muscle relaxation
after prolonged exercise. Ideally, muscle SR
Ca2+ uptake and relaxation would
both be determined in vivo, but this remains impractical in exercising
humans for measurements of Ca2+
uptake. In this study Ca2+ uptake
was measured in vitro, whereas muscle relaxation was determined in
vivo. It is, therefore, important to consider differences in Ca2+ uptake determined in vitro
vs. in vivo. It is likely that the in vivo depression of SR
Ca2+-ATPase activity and
Ca2+ uptake with prolonged
exercise is even more marked than that measured in vitro in this study.
After intense fatiguing contractions lasting 4-6 min in an intact
murine myocyte, the rate of SR
Ca2+ uptake was depressed by 47%
after only 10 tetani, with the maximal reduction being 87% (56). This
was substantially greater than the 55% depression of in vitro
Ca2+ uptake at fatigue in horses
exercised for a comparable time period (7). This discrepancy suggests
structural alterations occurred that impaired SR
Ca2+-ATPase activity measured in
vitro, with additional local reversible effects acting in vivo, most
likely exerted by cytosolic metabolic changes. That a greater
depression of Ca2+-ATPase activity
might be expected in vivo further strengthens our finding of
dissociated rates of muscle Ca2+
uptake and relaxation.
Possible direct inhibitory effects on the
Ca2+-ATPase enzyme may result from
marked metabolic disturbances. Prolonged exercise to exhaustion reduced
total muscle ATP (20%), CP (58%), and glycogen (90%), suggesting
that in vivo Ca2+ pump activity
and Ca2+ uptake may have been
reduced at exhaustion. This impairment may result from an attenuated
ATP production rate or from reduced free energy for ATP hydrolysis as a
result of changes in [ATP], [ADP],
[Pi], and
[Mg2+] (16). The full
recovery in muscle ATP and CP by 20 min postexercise might suggest that
in vivo energetic limitations are unlikely to impair in vivo
Ca2+ uptake. However, this
conclusion is based on our measurements of ATP from the bulk space,
which cannot be assumed to reflect changes occurring in local
compartmentalized areas of the muscle fiber (26). Evidence exists that
Ca2+ uptake is achieved through
the preferential use of compartmental ATP synthesized in the SR triads
(26). The chain of glycolytic enzymes from aldolase onward has been
found to be directly associated with SR membranes, with glycolytic ATP
production capable of directly fueling SR
Ca2+ uptake (58).
Furthermore, compartmental glycogen stores are located in areas
corresponding to the SR triads, and these were also preferentially
depleted during prolonged exercise (20). The 90% reduction in total
muscle glycogen stores during prolonged exhaustive exercise in the
present study would imply depletion of glycogen stores associated with
the SR, which could impede ATP synthesis in the triadic region and thus
directly reduce Ca2+ uptake.
Because no recovery in total muscle glycogen was seen in the 60 min
postexercise, it is conceivable that glycogen stores located in close
association with the SR also did not recover. Therefore, although total
muscle ATP was fully restored during the recovery period, possibly
remaining compartmentalized glycogen depletion may still adversely
affect ATP synthesis in the SR triads and therefore in vivo SR function
during recovery. However, these metabolic changes cannot
be factors accounting for the reduced in vitro SR
Ca2+ pump activity and
Ca2+ uptake found in the present
study, unless local substrate depletion directly and irreversibly
modified SR membrane or
Ca2+-ATPase structure.
Effects of prolonged exercise on muscle tension.
Because electrically evoked contractions are independent of the central
nervous system, the decreases in both peak twitch tension and peak
tension at 10 Hz after exercise indicate that the major contribution to
fatigue was peripheral in origin. During fatigue, the decline in
tension has been associated with a reduction in the number of cycling
cross bridges or with an increase in the number of cross bridges bound
in a weak state (11, 19). Although ATP was reduced by 20% at
exhaustion, there is little evidence to support a decline in ATP of
this magnitude as a limiting factor to cross-bridge cycling (22).
Similarly, although CP was markedly decreased at exhaustion in the
present study, it is unlikely that this reduced the ATP turnover rate
and impaired cross-bridge cycling during isometric contractions,
because reduced CP did not decrease tension in skinned muscle fibers
(23). In the present study, ATP and CP had recovered by 20 min
postexercise, whereas tension remained depressed, further suggesting
that metabolic factors were not a primary mechanism of decreased
tension.
The substantial depression of tension at low-frequency, but the
near-normal tension at high-frequency stimulation, has been termed
low-frequency fatigue and previously demonstrated in human muscle (17)
and in isolated single mouse fibers (9, 57). The most likely cause of
low-frequency fatigue is decreased SR Ca2+ release, because
Ca2+ sensitivity and maximal
Ca2+-activated tension were
unchanged and SR Ca2+ uptake was
depressed (9, 57). In the present study, the dramatic decrease in peak
twitch tension and peak tension at 10 Hz but unchanged peak tension at
100 Hz after prolonged exercise are consistent with decreased SR
Ca2+ release and the subsequent
effects on low- and high-frequency-stimulated tension production. The
greater decline in twitch and low-frequency tension can most likely be
explained by the intracellular
[Ca2+]-tension curve,
as demonstrated by Westerblad et al. (57). At low frequencies of
stimulation, tetanic intracellular
[Ca2+] is on the steep
part of the curve, where a moderate decrease in tetanic intracellular
[Ca2+] during fatigue
results in a large tension decline. At high frequencies of stimulation,
tetanic intracellular
[Ca2+] is on the
horizontal part of the curve, where moderate decreases in tetanic
intracellular [Ca2+]
during fatigue would have little effect on tension. The proposed mechanism for depressed SR Ca2+
release was a structural change to one of the proteins involved in
excitation-contraction coupling (9, 57). After rats performed prolonged
exercise, muscle SR vesicle Ca2+
release was decreased in oxidative muscle (18). Furthermore, reduced
ryanodine binding in fatigued muscle SR was indicative of reduced
number of open Ca2+-release
channels, most likely due to structural alterations to the SR
Ca2+-release channels (18). This
may in part be related to intracellular accumulation of reactive oxygen
species, because small elevations in hydrogen peroxide reduced SR
Ca2+ release (5). A further
possibility is that SR Ca2+
release is decreased after prolonged exhaustive exercise through a
depletion or substantial lowering of SR
Ca2+ stores. Adequate stores of SR
Ca2+ have been demonstrated in
isolated muscle fibers fatigued by several minutes of tetanic
stimulation, with caffeine and potassium contractures essentially
restoring force output (36). However, it is possible that more profound
effects on SR Ca2+ stores may be
observed after 70 min of exhaustive prolonged exercise in vivo. It has
also recently been suggested that reduced SR
Ca2+ release with fatigue may
result from lowered free Ca2+ in
the SR lumen because of the formation of calcium phosphate precipitates
in the SR lumen (21).
Previous work has shown that ATP may activate the SR
Ca2+-release channel (51) and that
the Ca2+-release properties may be
modulated by the local [ATP] (26, 44). If, as suggested
earlier, ATP synthesis in the SR triads is glycogen dependent and both
ATP and glycogen could fall well below that in the bulk space after
prolonged exercise, SR Ca2+
release could be comprised. Consistent with this
hypothesis was the association found between low glycogen and decreased
SR Ca2+ release in fatigued,
glycogen-depleted-skeletal muscle fibers, which was also fully
reversible with addition of glucose to resynthesize glycogen (8).
The peak tension at 100 Hz was potentiated by ~15% at 60 min
postexercise. Potentiation of submaximal 100-Hz contractions (i.e.,
peak tension less than MVC peak tension) has been reported in human
triceps surae muscle 15 min after cessation of 1-2 h of running
(14). Under the same conditions, peak tension for a maximal 100-Hz
contraction (i.e., peak tension approximately equal to an MVC) was
reduced by ~19%. These findings indicate that the peak tension of
submaximally activated muscles during high-frequency stimulation can be
potentiated after exercise. During repetitive stimulation, the
phosphate content of a class of myosin light chain, P light chain, is
increased with increasing stimulation frequency (43). The
effect of P-light chain phosphorylation is to augment tension at
subsaturating [Ca2+]
(54), with little effect at
[Ca2+] found during
maximal muscle activation (53). Such a mechanism might exist in vivo to
oppose fatigue and compensate for the loss of tension at lower
frequencies of stimulation (25). However, the present findings provide
little evidence for potentiation of low-frequency stimulation. The
degree of potentiation after fatiguing exercise will be the net effect
of potentiating and fatiguing factors acting on the contractile
proteins. Thus it is likely that the depressive effects of fatigue on
tension elicited by low-frequency stimulation were much greater than
potentiating factors. Peak tension at 100 Hz was submaximal (~66%
MVC), and intracellular
[Ca2+] would,
therefore, be at subsaturating levels, with the possibility of further
reductions in [Ca2+]
due to fatigue (57). Under these conditions, some potentiation of
tension may have resulted. This and the greater fatigue resistance of
high-frequency compared with low-frequency contractions after prolonged
exercise (17) might have potentiated tension at 100 Hz.
A further contribution to tension at 100 Hz might have been through an
increase in muscle temperature. Evoked submaximal contractions of the
human triceps surae muscle during 100-Hz stimulation were potentiated
by 40% when muscle temperature was raised 3°C (13). During
low-frequency-evoked contractions (10 Hz), tension may decline with an
increase in muscle temperature (13). After prolonged exercise, the
dramatic decline in peak tension during 10-Hz stimulation after
exercise was possibly due to the cumulative effect of fatigue and
increased muscle temperature. Of note was the recovery of peak tension
at 10 Hz between 20 and 60 min postexercise. During this time rectal
temperature (38.6-37.7°C), and most likely muscle temperature,
returned to resting levels.
Effects of prolonged exercise on muscle relaxation.
Fatigue in skeletal muscle is generally accompanied by a decline in
tension and a slowing of relaxation. In this study, after prolonged
exercise, reduced peak tension of evoked and voluntary contractions was
not accompanied by a slowing of
RT1/2; twitch RT1/2 was shortened, with no
change in the RT1/2 of an evoked contraction at 100 Hz.
The prolongation of relaxation is generally associated with
short-duration, intense contractions and is mostly attributed to the
accompanying acidosis that slows both cross-bridge cycling and/or SR Ca2+-uptake
rates. Under these exercise conditions, metabolic perturbations, including increased concentrations of ADP and
Pi and decreased concentrations of
ATP and CP, could also slow cross-bridge cycling and/or
Ca2+ uptake (16). However,
prolonged exercise did not change muscle pH, and the metabolic changes
(excluding glycogen) were less marked than would be expected during
short-term intense exercise. Accordingly, no slowing of relaxation was
evident in the present study after exhaustive prolonged cycling,
consistent with other studies (14, 48). A relationship between reduced
Ca2+ uptake and a prolongation of
relaxation has been suggested in human skeletal muscle fatigued by
intense contractions (24). Because the removal of
Ca2+ from the myoplasm is the
stimulus for Ca2+ dissociation
from the regulatory sites on the troponin complex that precedes
relaxation, any slowing of the rate of
Ca2+ removal should prolong
relaxation. This theory is not supported by the present finding of a
dissociation between Ca2+ uptake
and either twitch or tetanic RT1/2
at rest and after prolonged exercise. One possible reason for this is
that a critical rate of Ca2+
uptake exists below which muscle relaxation may be slowed. Only a 20%
reduction in Ca2+ uptake was found
in this study compared with a 40% reduction after intense exercise,
which was accompanied by the slowing of relaxation (24). Thus the
smaller reduction in Ca2+ uptake
after prolonged rather than intense exercise might have been
insufficient to impact on the contractile rate. However, this seems
unlikely because of the far greater depression in
Ca2+ uptake expected in vivo
compared with in vitro, as discussed earlier. A second possibility is
that contractile properties and Ca2+ uptake were always measured
in different legs. Although we do not have data on interleg variability
for Ca2+ uptake, the possibility
of leg-selection bias seems quite unlikely given the opposing nature of
the responses (i.e., slowed Ca2+
uptake, but faster twitch
RT1/2). In addition, muscle
metabolic changes are unlikely to have differed in the two legs with
exercise (27).
A third possible explanation for the lack of slowing in relaxation with
fatigue is that an increase in muscle temperature during prolonged
exercise impacted on the contractile rate, which shows a strong thermal
dependence (4). A shorter time course of contraction with increasing
muscle temperature has previously been demonstrated in human skeletal
muscle (13). Twitch RT1/2 was
decreased by 16% when human triceps surae muscle temperature was
increased to 38°C by either heating the muscle or running at 70%
O2 max for 15 min (13).
The thermal dependency of the contractile rate may, in part, account
for some of the dramatic slowing of relaxation in fatigued isolated
preparations where temperature was maintained at 22-25°C to
avoid fiber deterioration (19, 56, 57). These temperatures are well
below muscle temperature that can occur in vivo during exercise. In the
present study, the shorter twitch
RT1/2 but not 100-Hz
RT1/2 is consistent with the
greater thermal dependency of twitch
RT1/2 (4). During prolonged
exercise, an increased muscle temperature could be the primary
modulating factor accelerating relaxation in vivo. However, during
short-term intense exercise, the effect of increasing muscle temperature on contractile rate may be secondary to the inhibitory effects of decreased pH and other metabolic changes. It was previously demonstrated in fatigued, intact murine fibers that the slowing of
relaxation was not due to a depression in SR
Ca2+ uptake but rather to a slowed
rate of cross-bridge detachment (56). The unchanged
relaxation in the present study suggests that any fatigue-induced
slowing of cross-bridge detachment was outweighed by an accelerated
contractile rate due to increased muscle temperature.
In conclusion, prolonged exercise to exhaustion in humans resulted in a
long-lasting depression in the rate of muscle homogenate Ca2+ uptake and
Ca2+-ATPase activity. Despite the
reduced tension-producing capacity of the muscle after exhaustive
prolonged exercise, fatigue did not result in a slowing of the
contractile process. On the contrary, a shortening of twitch
RT1/2 was found. After prolonged
exercise, the reduced rate of Ca2+
uptake in vitro was not related to
RT1/2 of a twitch or an evoked contraction at 100 Hz in vivo, nor was it related to any other change
in contractile function. Thus fatigue induced by prolonged exhaustive
exercise in humans reduced Ca2+
uptake, but this did not cause a slowing of relaxation of evoked contractions.
ACKNOWLEDGEMENTS
We acknowledge the inspiration of our great friend and colleague, Professor John R. Sutton, who died on February 7th, 1996. He loved life and lived it to the fullest.
FOOTNOTES
Address for reprint requests: M. McKenna, Victoria Univ. of Technology, Dept. of Human Movement, Recreation and Performance, PO Box 14428, MCMC, Melbourne, Victoria 8001, Australia (E-mail: michaelmckenna{at}vut.edu.au).
Received 16 January 1996; accepted in final form 5 March 1997.
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