Effects of Reduced Muscle Ca2+ Release and Contractile Protein Function Intact Skeletal Muscle
Effects of Reduced Muscle Ca2+ Release and Contractile Protein Function Intact Skeletal Muscle
Effects of Reduced Muscle Ca2+ Release and Contractile Protein Function Intact Skeletal Muscle
17-29 17
During repeated tetanic contractions of skeletal muscle balance between rates of ATP utilization (intensity and
there is a reduction in force output. This decline in function, duration of contractile activity) and rates of ATP supply
referred to as muscle fatigue, has a complex aetiology which (metabolic pathway, substrate supply and muscle fibre type).
can involve various metabolic and ionic factors (see Fitts, During repeated contractions at high intensities (i.e. 100%
1994; Allen, Liinnergren & Westerblad, 1995) for recent maximum force for -3 min), ATP is resynthesized predomin-
reviews). Metabolic impairment plays a major role in muscle antly by PCr degradation and anaerobic glycolysis (Spriet,
fatigue with the extent of impairment dependent on the 1992). Under these conditions there is an accumulation of
inorganic phosphate (Pi), H+ and lactate (V0llestad, result in an earlier reduction in [Ca2+]i and force and a
Sejersted, Bahr, Woods & Bigland-Ritchie, 1988; Nagesser, shorter time to fatigue. This reduction in force can be
van der Laarse & Elzinga, 1992) and fatigue is thought to explained by decreased Ca2+ release from the SR and reduced
result from some effect of these metabolites either on Ca2+ sensitivity and maximum force-generating capacity of
sarcoplasmic reticulum (SR) Ca2P release (Westerblad & the myofibrillar proteins. Our data also indicate that while
Allen, 1991; Fryer, Owen, Lamb & Stephenson, 1995) or on impaired myofibrillar protein function is always associated
the contractile proteins (Cooke, Franks, Luciano & Pate, with changes in muscle glycogen content, Ca2+ release failure
1988; Godt & Nosek, 1989). During repeated contractions at is due to both glycogen-dependent and glycogen-independent
moderate intensities (i.e. 65-80% maximum, > 30 min), mechanisms.
oxidation of glycogen and triglyceride stores will provide
most of the energy for ATP resynthesis (Spriet,
Heigenhauser & Jones, 1986). Under these conditions there METHODS
is little accumulation of H+ throughout most of the exercise Mice were killed by cervical dislocation and single fibres or fibre
although PCr and glycogen depletion and Pi and lactate bundles were dissected from the flexor brevis muscle. Methods for
accumulation are evident at the point of exhaustion fibre dissection and mounting were similar to those previously used
in this laboratory (Westerblad & Allen, 1991). The mean diameter
(Vollestad et al. 1988). Under conditions of prolonged of single fibres was 40 + 3-5 /sm and the fibre bundles consisted of
moderate-intensity exercise, the cellular mechanisms under- 20-40 fibres. Both single fibres and fibre bundles were clamped at
lying the reduction in force are not well understood. each tendon by platinum foil micro-clips, which were attached to a
The importance of glycogen to muscle function was first force transducer at one end and to an adjustable holder at the other
end. The adjustable holder allowed the single fibres and bundles to
shown by Bergstrom and co-workers (Bergstr6m, Hermansen, be set to the length which gave maximum tetanic tension. All
Hultman & Saltin, 1967) who observed a direct correlation preparations were superfused at 22 °C with a solution containing
between muscle glycogen concentration and time to fatigue. (mM): NaCl, 121; KCl, 5 0; CaCl2, 1-8; MgCl2, 0.5; NaH2PO4, 0 4;
This correlation has been verified by several others (Ahlborg, NaHCO3, 24; and glucose, 5 5. The solution was bubbled with 95%
Bergstrom, Ekelund & Hultman, 1967; Galbo, Holst & 02 and 5% CO2 to give a pH of 7-3. Approximately 0-2% fetal calf
Christensen, 1979) and it is generally accepted that during serum was added.
moderate intensity exercise, muscle glycogen is an obligatory Single fibres were used for studies measuring force and Ca2+ during
substrate and that its depletion induces muscle fatigue. fatigue, and fibre bundles were used for studies measuring changes
While the mechanism by which glycogen depletion disrupts in glycogen concentration. Both the single fibres and fibre bundles
cellular function is not known, its tight association with the were stimulated with platinum electrodes using pulses of 0 5 ms
SR (Entman, Keslensky, Chu & van Winkle, 1980; Friden, duration at an intensity of approximately 1'2 x threshold. All
tetanic contractions were 0 35 s in duration. Force produced during
Seger & Eklblom, 1989) suggests that glycogen depletion unfused tetani was assessed by the mean output over the final
may lead to alterations in excitation-contraction coupling
m00ms.
(E-C coupling). Studies using isolated SR vesicles prepared
from muscles fatigued by prolonged exercise to exhaustion Recording of fluorescence signals
have associated reductions in SR Ca2+-ATPase activity [Ca2+]i was measured with the fluorescent dye indo-1 which was
(Belcastro, Rossiter, Low & Sopper, 1981; Byrd, Bode & micro-injected into the fibres. The experimental set-up was similar
to that described by Lee, Westerblad & Allen (1991) but with a
Klug, 1989) and reductions in SR Ca2+ release (Favero, single illumination wavelength of 360 nm and dual emission
Pessag & Klug, 1993) with fatigue. Byrd et al. (1989) noted wavelengths at 400 and 510 nm. Emitted light was measured by
that the extent of SR Ca2+-ATPase depression correlated two photomultiplier tubes and passed on to an analogue divide
with the extent of muscle glycogen utilization. However, a circuit which allowed continuous monitoring of the 400/510 ratio
reduction in SR Ca2+ pump activity increases tetanic signal. The background fluorescence was small, and was subtracted
intracellular free calcium concentration ([Ca2+]1) and force, from all measurements. The indo-1 ratio was converted to [Ca2+]i
and is not the cause of the reduction in force during fatigue using the in vivo calibration previously described (Westerblad &
(Westerblad & Allen, 1994). In contrast, SR Ca2P release Allen, 1993). The tetanic [Ca2+], was defined by the mean output
over the last 100 ms.
failure would contribute to the loss in force observed during
fatigue (Allen, Lee & Westerblad, 1989; Westerblad & Experimental protocol
Allen, 1991). The aim of the study was to measure muscle glycogen during
fatigue and to assess the extent to which reduced glycogen
The purpose of this study was to examine the effects of concentration may be responsible for the reductions in force and
reduced glycogen concentration on force and Ca2P release [Ca2+], that have previously been observed (Westerblad & Allen,
during fatigue in single skeletal muscle fibres. We also 1991; Westerblad, Duty & Allen, 1993). We used a protocol shown
examined the changes in Ca2+ sensitivity and maximum to fatigue single mouse muscle fibres (Westerblad & Allen, 1991)
CPa2-activated force to assess the effects of altered muscle and extended this regime to include several repeated fatigue bouts.
glycogen content on contractile protein function. Our Various conditions were designed to manipulate muscle glycogen
levels between fatigue bouts. The standard fatigue protocol
findings indicate that reduced levels of muscle glycogen consisted of repeated maximal tetani continued until muscle force
J Physiol. 498.1 Glycogen depletion and calcium release 19
was reduced to less than 30 %. Fibres were stimulated at 100 Hz for Ca50 before and during fatigue were compared in the control (Con),
0 35 s with tetani repeated at an interval of 4 s for the first 2 min, no glucose (-Gluc) and 50 Hz-stimulated (Stim) groups to assess the
and the interval reduced every 2 min to 3 s, 2-5 s, 2-0 s, and so on. effects of glycogen depletion on Ca2+ sensitivity and maximum
Under control conditions (Con), this protocol was followed by Ca2+-activated force.
60 min of recovery during which the fibres were continuously
Muscle glycogen determination
perfused with a standard solution containing 5-5 mm glucose. After To quantify the changes in muscle glycogen during fatigue and
60 min, fibres were fatigued a second time using the same 100 Hz
stimulation protocol described above. The repeated fatigue bouts recovery, these experiments were repeated in small bundles of
will be referred to as fatigue 1 and fatigue 2 for the first and second approximately 20-40 fibres. Separate experiments were required to
fatigue bout, respectively. Fatigue resistance, defined as the time assess the muscle glycogen levels at the end of fatigue 1 (n = 4) and
required to reach 30% of initial force, was compared between after recovery for control (n = 5), no glucose (n = 4) and 50 Hz
fatigue 1 and fatigue 2. The same absolute level of force (i.e. 30 % of stimulation (n = 4) conditions. For each of these samples a control
initial force during fatigue 1) was used to assess fatigue resistance bundle from the same muscle was also perfused with glucose, but
for each fatigue run. In some fibres a third and fourth fatigue bout not stimulated, for the same duration that the fatigued bundles
were also given, each separated by 60 min of recovery. were perfused. The level of glycogen in these bundles represents the
pre-fatigue level of muscle glycogen. Values for stimulated muscle
To examine the effects of reduced muscle glycogen concentration on are expressed as a percentage of control values relative to the
the changes in force and [Ca!+]1 during fatigue, two other conditions unstimulated bundles. Stimulated and unstimulated muscles were
were examined. These conditions were designed to manipulate the quick-frozen in liquid nitrogen and stored at -80 °C until analysed.
glycogen content of the single fibres at the start of the second Glycogen was assessed in the fibre bundles using a fluorometric
fatigue bout. In the first condition, a solution with no glucose was method (Lowry & Passonneau, 1972). Samples were first hydrolysed
used to perfuse the fibres during the 60 min recovery period between in 2 N HCl at 100 °C for 2 h and then glucose concentration
fatigue 1 and fatigue 2. It was expected that this intervention measured in the extract. Total protein of the extract was
would prevent resynthesis of any glycogen used during the first determined using a bicinchoninic acid (BCA) method with serum
fatigue bout. In some fibres (n = 3), glucose was removed during, as albumin as a standard. In a subset of larger samples from the same
well as between, both fatigue bouts (-Gluc). In the second muscle, weight and protein content were measured and the protein
condition, fibres were stimulated (Stim) using a pattern of 50 Hz yield determined (136 + 14 mg g-'). This value was used to express
intermittent tetani between fatigue 1 and fatigue 2 in a solution glycogen concentrations in micromoles of glucosyl units per gram
containing glucose. The Stim protocol consisted of 50 Hz tetani wet weight of tissue (,smol (g wet wt)f).
repeated at a rate of 1 every 10 s for 2 min, then 1 every 9 s for Statistical analyses
2 min and so on, with the tetanus rate increased every 2 min until
50 Hz force reached 30% of its initial value. On average, fibres were All values are expressed as means + S.E.M. One-way analysis of
stimulated 22-6 + 4 min. There was a brief period of recovery variance (ANOVA) for repeated measures was used to test for
(9 min) between fatigue 1 and the 50 Hz tetani and again between statistical differences within each group (pre- vs. post-fatigue). One-
the 50 Hz tetani and fatigue 2, during which force and [CaP+]1 were way random block ANOVAs were used to test for differences
assessed at a range of frequencies (see details below). This protocol between groups (Con vs. -Gluc vs. Stim). Significance was accepted
was designed to gradually reduce muscle glycogen stores below the at P < 0 05.
levels obtained at the end of fatigue 1 as a way of examining changes
during fatigue at moderate work intensities. In preliminary studies RESULTS
we also attempted this stimulation protocol in a glucose-free solution
but no fibres survived the protocol. Force output during repeated fatigue bouts
Contractile protein function assessment Using our standard fatigue protocol, force was reduced to
In order to determine the CP sensitivity and maximum Ca2+- 30% of initial values in 222 + 44 s. Under control
activated force, we used a protocol previously described (Westerblad conditions where the fibres recovered between fatigue bouts
& Allen, 1991). Briefly, before each fatigue bout, fibres were for 60 min in the presence of glucose, fatigue resistance was
activated at a range of frequencies (30, 50, 70, 100 Hz and 100 Hz not significantly different in the second fatigue bout (mean
in the presence of 5 mm caffeine) at 1 min intervals. Force and time to fatigue, 243 + 54 s; n = 6). Figure 1 shows the force
[Ca2+]i at each frequency were used to construct force-Ca2+ curves recorded from a representative fibre in which four successive
and to determine the resting Car+ sensitivity and maximum Ca2+-
activated force. Force-Ca2+ curves were fitted using a Hill equation: fatigue bouts were given. With glucose present during
recovery, there was minimal change in fatigue resistance
F= Fmax[Ca2+]in/(Cason + [Ca2+]in), even after four fatigue bouts.
where F is force, Fmax is the force at saturating [Ca2+]i, n is a
constant describing the steepness of the relation and Ca50 To assess the effects of reduced muscle glycogen concentration
represents the [Ca2+]i required for 50% of maximum force. To on fatigue resistance, time to fatigue was measured after
assess the changes in CaP+ sensitivity during fatigue, force and fatigued fibres recovered for 60 min in the absence of glucose
[Ca2+]1 from the final phase of fatigue, when both are declining, (-Gluc rec). Under these conditions, fatigue resistance was
were used (see Westerblad & Allen, 1991). Force and [Ca2+]i from a significantly reduced (from 273 + 50 to 38 + 17 s; n = 3).
100 Hz tetanus in the presence of caffeine, representing maximum Figure 2 shows the force recorded from a representative
Ca2+-activated force, were measured at the end of the fatigue fibre that recovered without glucose between fatigue 1 and
protocol and added to the force-Ca2P plot which could then be fatigue 2. The time to fatigue in the second fatigue run was
fitted by a Hill equation as described above. Values of Fmax and
abbreviated, with force declining rapidly immediately after
20 E. R. Chin atndI D. G. Allen J Physiol. 498.1
the first tetanus. This reduction in fatigue resistance was resistance was not significantly reduced during fatigue 1
readily reversed when the fibre was allowed to recover for (125 + 28 s, P = 0 113) but was reduced during fatigue 2
60 min in the presence of glucose after fatigue 2. Note the compared with fatigue 1 (30 + 2 s). Since the relative
similar time to fatigue during fatigue 3 and fatigue 1. The reduction in fatigue resistance during fatigue 2 was similar
improved fatigue resistance with an additional period of for -Gluc rec (16%) and -Gluc (18%) and subsequent
recovery with glucose indicates that the reductions in analyses revealed that changes in force and [Ca2+]i were also
fatigue resistance were not due to permanent impairment of similar for these conditions, data for these two groups were
the fibre but were related to the changes in substrate supply. pooled (-Gluc; n = 6). In four other fibres that were
We were also interested in whether fatigue resistance would fatigued once with glucose and once without glucose (glucose
be altered by the presence or absence of glucose during present during recovery; data not shown), fatigue resistance
was not altered (202 + 16 vs. 192 + 27 s). This indicates
fatigue. In fibres where glucose was removed during fatigue 1
and fatigue 2 as well as during recovery (-Gluc), fatigue that glucose removal during recovery was more important
100- Fatigue 1
c
c
50-
0
U-
0-
100 Fatigue 2
C
'm 50-
a
20
0
U-
0-I
100 Fatigue 3
50 -
0
U-
100-i Fatigue 4
.-
L..
50-
a)
0
LL
04
0 100 200 300 400 500
Time (s)
than glucose removal during fatigue in altering the fatigue The reduced fatigue resistance following low-frequency
resistance of the fibres. It also indicates that in these stimulation is therefore not due to permanent damage of the
predominantly type II fibres (Allen, Duty & Westerblad, fibre but probably related to metabolic or ionic alterations at
1993), glucose uptake from the perfusate does not contribute the start of fatigue 2.
much to the energy required for contractions and suggests
that intracellular stores of PCr and glycogen provide most of Force recovery between fatigue runs
the fuel for ATP resynthesis. The changes in force in response to fatigue under the
different conditions is shown in Fig. 4. The mean 100 Hz
Fatigue resistance was also assessed in a group of fibres tetanic force produced by the single fibres was 44 1 +
where a period of 50 Hz tetani was given after the first 1-7 mg or 350 + 13 kPa when normalized for cross-sectional
fatigue bout. Under these conditions, time to fatigue was area, similar to previously reported values (Westerblad &
reduced from 333 + 78 to 187 + 77 s (n = 7). Figure 3 Allen, 1991). Under control conditions, force at the start of
shows the force output of a fibre where 50 Hz stimulation in fatigue 2 was 100 + 2% of initial values, indicating that
the presence of glucose was given between the first and force had been fully restored during 60 min of recovery
second fatigue bout. In this fibre, fatigue resistance was with glucose. Under conditions where no glucose was present
reduced after 50 Hz stimulation (fatigue 2) but increased during recovery, force at the beginning of fatigue 2 was
again after 60 min of recovery with glucose (fatigue 3). reduced to 64 + 8% of force at the beginning of fatigue 1.
100- Fatigue 1
li I I JIIIIIIIUIbILI
I._c
50-
0
U-
0-
100 - Fatigue 2
co
C. 50 -
LL
O -
100 7
Fatigue 3
(a
L-
50-
o
U-
Il. lw
0-
-T..
ll3
R
0 50 100 150 200 250
Time (s)
Figure 2. Force output from a single fibre after recovery without glucose between fatigue 1 and
fatigue 2
The fibre was fatigued using the same protocol described for Fig. 1. Between fatigue 1 and fatigue 2, the
fibre recovered unstimulated for 60 min in the absence of glucose; 5-5 mm glucose was present during each
of the fatigue bouts. The period of glucose-free recovery resulted in a reduction in fatigue resistance during
fatigue 2 which could be reversed following subsequent recovery with glucose (fatigue 3). Tetanic force was
also reduced at the beginning of fatigue 2 and recovered before fatigue 3.
22 E. R. Chin and D. a. Allen J Physiol. 498.1
100 -
ir
U-
.10
0
0) Figure 3. Force output from a single
O- fibre after 50 Hz stimulation between
fatigue 1 and fatigue 2
The fibre was fatigued using the protocol
* 50- described for Fig. 1. Between fatigue 1 and
fatigue 2, the fibre was stimulated with
0 -
100 Fatigue 2 intermittent 50 Hz tetani at a rate of 1 every
t--
I0 s for 2 min, then 1 every 9 s for 2 min, etc.
c
._ with the train rate increased every 2 min
until force reached 30% of the initial 50 Hz
o- 50 - force. For this fibre, 50 IHz stimulation was
a)
stopped after 11 min. Fatigue 2 followed
after a brief rest (9 min) during which force
and [Ca2+]i were measured at various
O-
frequencies. After fatigue 2, the fibre
recovered unstimulated for 60 min. Glucose
(5S5 mM) was present in the perfusing
solution throughout the entire experiment.
100 -1 Fatigue 3 The period of 50 Hz stimulation resulted in a
decrease in fatigue resistance during fatigue 2
which was reversed with unstimulated
recovery before fatigue 3. Tetanic force was
501 reduced at the start of fatigue 2 but also
0
recovered by the start of fatigue 3.
U-
O-I -IXzW1 I
0 30 60 90 120 150 180
Time (s)
In fibres stimulated at 50 Hz between the first and second Intracellular Ca2+ recovery between fatigue runs
fatigue bouts there was also a significant depression in In order to determine whether reductions in Ca2+ release
tetanic force at the start of fatigue 2 (76 + 9% of initial contributed to the reductions in force under the different
levels). Comparing the differences between groups, 100 Hz conditions, changes in [Ca2+]i were also assessed. Examples
force at the beginning of fatigue 2 was significantly reduced of the alterations in [Ca2P]1 associated with the changes in
in the -Gluc and Stim groups compared with the Con group. force during fatigue 1 and fatigue 2 under control and
t
100 -
Figure 4. Mean changes in force during fatigue 1
*t+ and fatigue 2 with different recovery conditions
80- Force at the beginning of fatigue 1 (Fat 1) and fatigue 2
* t.
(Fat 2) (0) and at the end of each fatigue bout (O) is
shown for the different conditions: control (Con) where
60- fibres recovered in the presence of 5f5 mm glucose;
.5 glycogen depleted (-Gluc) where fibres recovered without
a,
0
glucose; and stimulation (Stim) where fibres received
40- 50 Hz tetani intermittently over 23 + 4 min in the
LL
presence of 5-5 mM glucose. Force for each fibre was
T 4
expressed as a percentage of its initial 100 Hz force.
20 - * P < 0 05 vs. first tetanus for fatigue 1. t P < 0 05 vs.
last tetanus of fatigue 1. $ P < 0 05 vs. same time point
for Con.
0-
Fat 1 Fat 2 Fat 1 Fat 2 Fat 1 Fat 2
Con -Gluc Stim
J: Phy-siol. 498.1 Glycogen depletion and calcium release 23
glycogen-reduced conditions are shown in Figs 5 and 6, tetanic [Ca2+]1 at the start of fatigue 1 was 887 + 57 nM,
respectively. Figure 5 shows that under control conditions, which was reduced to 47 + 3% at the end of fatigue 1.
fatigue 1 resulted in a decrease in [Ca2+], from 1144 to Under control conditions, the recovery of [Ca2+]i to 82 +
590 nm when force fell to 30% of initial (Fig. 5a vs. b). 8% of initial levels by the start of fatigue 2 is consistent
Following 60 min of recovery with glucose, [Ca2+]i had with previous observations of prolonged Ca2+ release failure
recovered to 791 nm when force had recovered to its following this type of fatigue protocol (Westerblad et al.
starting level (Fig. 5c). During fatigue 2, force declined with 1993). At the end of fatigue 2, [Ca2+], was again reduced to
a similar time course to that in fatigue 1 and [Ca2P], fell to 48 + 5% of the initial level, a level not different from that
635 nm, a level not different from that at the end of fatigue 1 at the end of fatigue 1. In fibres where glucose was removed
(Fig. 5b vs. d). These results can be compared with Fig. 6, during recovery, [Ca2+], during the first tetanus of fatigue 2
which shows the diminished recovery of [Ca2+]i and force was 57 + 7 % of the initial level. Intracellular [Ca2+] at the
when glucose was not present during recovery. Fatigue 1 end of fatigue 2 was reduced to 38 + 4%, a level not
resulted in a reduction of [Ca2+], from 944 to 519 nm when different from that at the end of fatigue 1. In fibres
force was reduced to 30% (Fig. 6a vs. b). After 60 min of stimulated at 50 Hz between fatigue 1 and fatigue 2, the
recovery without glucose, [Ca2+], had recovered to only changes in [Ca2+]i followed a similar pattern. In seven fibres,
626 nM and force to only 82% of initial (Fig. 6c). During the mean [Ca2+]i during the first tetanus of fatigue 2 was
fatigue 2, [Ca2+], fell to 460 nm when force was reduced to 55 + 6% of initial. At the end of fatigue 2, [Ca2+], was
30%, which was similar to [Ca2+]i at the end of fatigue 1. reduced to 41 + 3% of initial and was not different from
A summary of the changes in [Ca2+]i in response to fatigue [Ca2+], at the end of fatigue 1. Comparing the differences
under the different conditions is shown in Fig. 7. The mean between groups, [Ca2+]1 at the start of fatigue 1 was similar
Fatigue 1 Fatigue 2
100 - a c
I
50-
0
IL
5 mm glucose, 60 min
0
,-M 1-o-
2 05-
0 o- 1-i
100 -
C
I-Ro 50-
0
LL
0-
Figure 5. Data traces representative of the changes in force, [Ca'1I and time to fatigue for
fatigue 1 and fatigue 2 under control conditions
Tetanic (100 Hz) force and [Ca2+]i were reduced at the end of fatigue 1 (a v8. b). After 60 min recovery in
the presence of 55 mm glucose, tetanic force had fully recovered but [Ca2+], remained depressed (b v8. c).
During fatigue 2, tetanic force and [Ca2+]i were reduced (c v8. d) over a similar time course as fatigue 1.
Intracellular [Ca2+] reached the same level at the end of both fatigue bouts (b vs. d) despite starting at lower
levels at the beginning of fatigue 2.
24 E. R. Chin and D. G. Allen J Physiol. 498.1
for all conditions. However, the [Ca2+], at the start of fatigue fatigue 2 there was no recovery of Ca50, but there was a
2 was significantly reduced in the -Gluc and Stim groups partial recovery of Fmax. During the abbreviated second
compared with the Con group. fatigue bout there was no further reduction in Ca2+
sensitivity but Fmax decreased to levels similar to fatigue 1.
Ca2O sensitivity changes between fatigue runs In fibres stimulated at 50 Hz after fatigue 1, Ca50 and F...
The reductions in force observed during fatigue can be had returned to pre-fatigue levels by the start of fatigue 2.
attributed not only to reductions in [Ca2+]i but also to The reversible decrease in Ca50 and Fmax indicates that the
reductions in Ca2P sensitivity of the myofibrils and alterations in Ca2+ sensitivity and maximum Ca2+-activated
maximum Ca2P-activated force. Table 1 shows the changes force induced during fatigue from 100 Hz tetani had
in Ca2P sensitivity (Ca50) and in maximum Ca2P-activated recovered during the period of 50 Hz stimulation. During
force (Fmax) obtained from Hill plots of the force-Ca2P fatigue 2, Ca,O and Fmax decreased again to levels similar to
relationships under the different conditions. Fatigue 1 fatigue 1. Comparing the differences between groups, Ca50
resulted in an increase in Ca50 and a decrease in Fmax in all and Fmax were altered to a similar extent at all times except
three groups, which is consistent with previous reports at the beginning of fatigue 2, when Ca50 was increased and
(Westerblad & Allen, 1991). After 60 min of recovery with Fmax decreased in the -Gluc compared with the Con and
glucose, Ca50 and Fmax had returned to pre-fatigue levels Stim groups.
but during fatigue 2 were again altered to levels not
different from fatigue 1. In fibres that recovered without Muscle glycogen concentrations
glucose, alterations in Ca50 and Fmax were not completely Muscle glycogen concentration was measured in fibre
reversed by 60 min, indicating that recovery of contractile bundles that were fatigued using protocols identical to those
protein inhibition was incomplete. Between fatigue 1 and described for the single-fibre studies. In the bundles, the
a Fatigue 1 Fatigue 2
100 - 1,
C
co
cJ 50 -
I-o
l)20 d
LL
0 glucose, 60 min
01 3I 4 C////ZZ!Zz/
0 100 200 300 400 0 100 200 300 400
Time (s) Time (s)
1-
a b c d
.o-i-
ci
co
>0-5
100
Cf
"O50-
U-
Figure 6. Data traces showing the changes in force, [Ca2+]i and time to fatigue for fatigue 1 and
fatigue 2 for a fibre that recovered without glucose
Tetanic (100 Hz) force and [Ca2P]i were reduced at the end of fatigue 1 (a vs. b). After 60 min recovery
without glucose, tetanic force only partially recovered and [Ca2+]i showed only minor improvement (b vs. c).
During fatigue 2, tetanic force and [Ca2+]i were reduced (c vs. d) more rapidly compared with fatigue 1.
Intracellular [Ca2P] reached the same level at the end of both fatigue bouts (b vs. d) despite starting at lower
levels and declining more rapidly during fatigue 2.
J Physiol.498.1 Glycogen depletion and calcium release 25
prevent glycogen resynthesis but did impair the recovery of results we have assumed that muscle glycogen levels in the
force and Ca2P release. We therefore postulate that changes single fibres were similar to those in the fibre bundles
in force and Ca2P release involve both glycogen-dependent treated with the same protocol.
and glycogen-independent mechanisms. Alterations in Ca2P
We have shown that fatigue resistance within a given
sensitivity and maximum Ca2+-activated force of the muscle fibre, is associated with the availability of glycogen.
myofibrillar proteins were correlated with changes in muscle These data are consistent with the ideas proposed by
glycogen and are probably due to the alterations in Pi or Bergstrom et al. (1967) that muscle glycogen concentration is
other metabolites associated with glycogen breakdown and
resynthesis. important in determining muscle performance during
prolonged exercise. We have also extended this idea by
Muscle glycogen depletion and fatigue resistance showing that both force and Ca2+ release are associated with
In the present study, glycogen concentration measured in glycogen concentration. At the cellular level, glycogen
small bundles of the flexor brevis muscle of mice averaged concentration may alter fatigue resistance by limiting the
26 ,umol (g wet wt)-', values within the range reported for duration for which E-C coupling mechanisms can function
mouse fast twitch muscle (Bonen & Homonko, 1994). normally before Ca2+ release fails and [Ca2+], and force start
Intermittent tetanic stimulation resulted in reductions in to fall. A surprising observation was that fatigue resistance
glycogen concentration to -25%. This is consistent with was not impaired by the small but prolonged reduction in
studies in rat skeletal muscle where a reduction of muscle Ca2+ release induced by a single fatigue bout. This suggests
glycogen to 27 % was observed with a comparable stim- that the duration of a bout of activity depends primarily on
ulation protocol, and a decrease to 40 % was observed with the available energy supply, in this case glycogen, and small
exhaustive swimming (Lindinger, Heigenhauser & Spriet, reductions in Ca2+ release do not affect the endurance
1987). Although we were unable to measure glycogen in capacity of a fibre. However, when the prolonged reductions
single fibres, we would expect the level of glycogen of Ca2+ release are greater than 20 %, such as observed when
utilization to be similar since the stimulation protocol was 50 Hz stimulation followed a single fatigue bout, then the
the same. Based on the reduction in time to fatigue endurance of a fibre is compromised despite sufficient
following recovery without glucose (to 17 % of initial values glycogen availability. Thus, prolonged reductions in E-C
in single fibres vs. 55% in fibre bundles), glycogen in the coupling do not reduce fatigue resistance until the impair-
single fibres may have been reduced to even less than the ments are greater than the extent observed after a single
25% estimated from the fibre bundles. In interpreting our fatigue bout.
200 1
t
175 -
150 4
0
125
C-
100
c
0)
0
0
75
50
25
0
Pre-fat Fat 1 +Gluc -Gluc Stim
Figure 8. Mean muscle glycogen values obtained in fibre bundles after fatigue 1 and following
recovery under the different conditions
Glycogen concentrations are expressed as a percentage of the values in a control bundle from the same
animal that was perfused but unstimulated for 60 min in the presence of 5.5 mM glucose. In the control
bundles, glycogen concentration averaged 26 ± 3 umol (g wet wt)-1, which is shown as the pre-fatigue
glycogen level (Pre-fat). Glycogen levels are also shown for bundles after fatigue 1 (Fat 1), after recovery
from fatigue 1 for 60 min with 5-5 mm glucose (+Gluc), after recovery from fatigue 1 for 60 min without
glucose (-Gluc) and after intermittent 50 Hz tetani following fatigue 1 (Stim). * P < 0 05 vs. Pre-fat.
t P < 0 05 vs. Fat 1. t P < 0 05 vs. +Gluc.
J Physiol. 498.1 Glycogen depletion and calcium release 27
Muscle glycogen and Ca2+ release failure during study was that the changes in maximum Ca2+-activated
fatigue force and Ca2+ sensitivity (and therefore Pi) during a second
During the intermittent stimulation protocol used there are fatigue bout were of a similar magnitude to those of the first
several phases of fatigue, differentiated by the changes in fatigue bout. Also, the reductions in maximum force and
force and [Ca2+]i, which help to identify different underlying Ca2+ sensitivity at the end of the fatigue bout were similar
mechanisms. These have previously been shown in single regardless of the starting levels of muscle glycogen. Thus,
muscle fibres from both mouse (Liinnergren & Westerblad, differences in starting levels of glycogen appear to alter the
1991) and frog (Nagesser et al. 1992) and include an early time course and not the magnitude of contractile protein
reduction in force (phase 1), a plateau phase (phase 2) and a inhibition and recovery. This would be expected if the
final linear fall in force (phase 3). During phase 1, the 15% starting level of muscle glycogen determines how long ATP
reduction in force over the first eight to ten tetani is due to can be supplied by glycolysis before the remaining PCr is
an inhibition of the contractile proteins (Linnergren & hydrolysed and P1 concentration increased.
Westerblad, 1991) caused by an accumulation of metabolites While there were no differences in the extent of contractile
following rapid activation of PCr hydrolysis and anaerobic protein inhibition during fatigue with different starting
glycolysis (Nagesser et al. 1992). The start of phase 2 is glycogen levels, the degree of recovery over 60 min was
thought to coincide with a metabolic steady state where dependent on glycogen availability. Under conditions where
oxidative metabolism can meet the ATP demands of the glycogen was resynthesized (Con, Stim) there was a full
repetitive contractions (Liinnergren & Westerblad, 1991). recovery of myofibrillar protein function. However, when
During this metabolic steady state, muscle glycogen appears glycogen did not recover (-Gluc) there was no recovery of
to be the predominant substrate for ATP resynthesis. This is Ca2P sensitivity and only partial recovery of maximum
consistent with the observation that phases 1 and 2 were Ca2+-activated force. The partial recovery of maximum
abbreviated or completely absent when muscle glycogen force in the absence of any change in Ca2+ sensitivity would
concentration was reduced (-Gluc) and that repletion of not be expected if both effects are mediated only by the
glycogen between the second and third fatigue bouts allowed increases in Pi. Other metabolic changes, including increases
this plateau phase to be re-established. In phase 3, force and in ADP or increases in AMP from the resynthesis of ATP
[Ca2+]i fall rapidly due to failure of E-C coupling via the myokinase reaction, may also contribute to changes
mechanisms (Westerblad & Allen, 1991). When glycogen in contractile protein function when glycogen does not
was reduced, fibres went into phase 3 immediately or much recover. An increase in AMP has been shown to increase
sooner, suggesting that with low levels of starting glycogen Fmax without altering Ca50 (Godt & Nosek, 1989) and could
there is no metabolic steady state. The prevailing metabolic therefore explain the partial recovery of maximum Ca2+-
conditions under these circumstances caused an immediate activated force without any change in Ca2P sensitivity
failure of Ca2+ release and force loss. An important obser- during recovery without glucose. Although we cannot
vation from this study is that the mechanism responsible for precisely determine the metabolites involved, it seems clear
the decrease in force and [Ca2+]i (i.e. E-C coupling failure) that myofibrillar protein function remains depressed when
observed during phase 3 in fatigue is not different when energy is not available for muscle glycogen resynthesis.
muscle glycogen concentration is reduced. Instead, the time
course is altered, with muscle glycogen being utilized earlier Glycogen-dependent and glycogen-independent
and E-C coupling failure occurring much sooner. Thus, the mechanisms of Ca2O release failure
fall in force during phase 3 under control conditions would Recovery of force and [Ca2+]i after fatigue follows a complex
also appear to be due to a glycogen-dependent failure of time course. One component of force and [Ca2+]i recovers over
E-C coupling, albeit much later in the fatigue protocol. 15-30 min and then there is a slow-recovering component
which requires more than 60 min (Westerblad et al. 1993).
Muscle glycogen and myofibrillar protein function This complex recovery pattern and its alteration when
during fatigue glycogen resynthesis was impaired suggests that there are at
The decline in force observed during muscle fatigue has been least two types of mechanisms contributing to the failure of
attributed to an inhibition of the myofibrillar proteins as Ca2P release during fatigue: glycogen-dependent and
well as to impaired Ca2P release from the SR (Fitts, 1994; glycogen-independent mechanisms. The glycogen-dependent
Allen et al. 1995). Inhibition of the myofibrillar proteins is component of Ca2P release failure would account for the
due primarily to increases in Pi and H+ which have been partial recovery of [Ca2+]i observed during the first hour
shown to depress maximum Ca2P-activated force (Cooke after fatigue. In the present study, when fibres recovered in
et al. 1988; Godt & Nosek, 1989) and Ca2P sensitivity (Godt the presence of glucose, glycogen had recovered and [Ca2+]i
& Nosek, 1989) of skinned single fibres. It has previously increased from 47 to 82 % of initial levels. In contrast, when
been shown, however, that there are no large increases in fibres recovered without glucose, glycogen was still depleted
[H+] in intact single mouse fibres with this fatigue protocol and [Ca2+], only recovered to 57% of initial levels. Thus
(Westerblad & Allen, 1992). Therefore the changes in con- - 25 % (82 - 57 %) of the recovery of Ca2P release following
tractile protein function in this preparation are presumably fatigue appears to be glycogen dependent. It seems
due to an accumulation of P1. A novel observation in this reasonable to infer that a similar proportion of the failure of
E. R. Chin and D. G. Allen J Phy8iol. 498.1
Ca2+ release during fatigue can be attributed to glycogen- due to the fact that whole-cell ATP concentrations rarely fall
dependent mechanisms. A postulated glycogen-dependent by more than 30-50% (V0llestad et al. 1988) and muscle
mechanism to explain E-C coupling failure during fatigue is glycogen is never entirely depleted, the concentration of
outlined in a subsequent section. glycogen and ATP within the narrow restricted space of the
triadic gap may fall well below that in the bulk space. The
Glycogen-independent mechanisms compartmentalized supply and utilization of ATP in skeletal
In the present study there were several conditions where the muscle triads is supported by observations that: (i) glycogen
reductions in [Ca2+]i and force were not associated with is bound in distinct regions within mammalian muscle fibres
decreases in muscle glycogen concentration. The prolonged including the lateral ends of the I-band, a region corres-
reduction of [Ca2+]i 60 min after fatigue 1 under control ponding to the terminal cisternae region of the SR (Friden
conditions as well as the decrease in fatigue resistance et al. 1989); (ii) glycogen in the lateral I-band region is
following 50 Hz stimulation were associated with the preferentially depleted following intense exercise in humans
resynthesis of muscle glycogen and appear to be due to (Friden et al. 1989); and (iii) ATP supplied by glycolytic
glycogen-independent mechanisms. We have recently shown enzymes is utilized to phosphorylate proteins in the triadic
that the prolonged impairment of Ca2+ release after a single gap that may be required for E-C coupling (Han et al. 1992).
fatigue bout is due to some Ca2+-activated process (Chin & The role of muscle glycogen may therefore be to provide a
Allen, 1996). This Ca2P-activated mechanism may have rapid and local supply of ATP in the triadic gap for
contributed to the reduction in Ca2+ release and the reduced important phosphorylation-dephosphorylation steps (Han
fatigue resistance during the second fatigue bout following et al. 1992) or direct binding to the Ca2P release channel
50 Hz stimulation. The additional period of high [Ca2+]i (Smith et al. 1985). When localized glycogen stores are
during 50 Hz stimulation may have activated this process. depleted, ATP supply in the SR triadic region falls and a
The reduced fatigue resistance after 50 Hz stimulation could decrease in Ca2+ release and force results. This type of
also be due to elevations in myoplasmic Pi. It has been glycogen-dependent mechanism might play an important
shown in skinned muscle fibres that elevations in P1 to levels feedback role in inactivating muscle E-C coupling under
observed during fatigue (25-50 mM) can decrease both the conditions of metabolic stress such as during muscle fatigue.
rate and magnitude of Ca2+ release from the SR (Fryer et al.
1995). Fryer et al. (1995) proposed that elevated levels of In summary, we have shown that decreases in muscle
myoplasmic Pi may lead to Pi transport into the SR, calcium glycogen concentration contribute to the reduction in force
phosphate precipitation and a reduction in the releasable SR and [Ca2+]i observed during fatigue. These reductions can be
Ca2+ pool. Thus, elevated myoplasmic [Ca2+]i or P1 are two explained by an impairment in Ca2P release from the SR and
possible glycogen-independent mechanisms that alter force, an inhibition of the myofibrillar proteins. Our observations
[Ca2+]i and fatigue resistance. confirm, at a cellular level, the correlation between muscle
glycogen concentration and fatigue and suggest a mechanistic
Glycogen-dependent mechanisms - a hypothesis for link between muscle glycogen and proteins involved in
E-C coupling failure in skeletal muscle fatigue excitation-contraction coupling and force production.
We have shown that, in general, changes in muscle glycogen
are associated with changes in fatigue resistance, force and
[Ca2+]i in single muscle fibres. Based on these associations,
an important question to consider is how the decrease in AHLBORG, B., BERGSTROM, J., EKELUND, L.-G. & HULTMAN, E.
muscle glycogen might contribute to E-C coupling failure. (1967). Muscle glycogen and muscle electrolytes during prolonged
As a working hypothesis, we suggest that there is a physical activity. Acta Physiologica Scandinavica 70, 129-142.
functional coupling between ATP supplied by glycolysis and ALLEN, D. G., DUTY, S. & WESTERBLAD, H. (1993). Metabolic changes
ATP utilized within the SR-t-tubule triadic gap (Han, in muscle during exercise; their effects on muscle function.
Thieleczek, Varsainyi & Heilmeyer, 1992; Xu, Zweier & Proceedings of the Australian Physiological and Pharmacological
Society 24, 65-75.
Becker, 1995). During repeated tetani, when glucose uptake
is not rapid enough to supply the energy required for muscle ALLEN, D. G., LXNNERGREN, J. & WESTERBLAD, H. (1995). Muscle
cell function during prolonged activity: cellular mechanisms of
contraction, intracellular PCr and glycogen stores are utilized. fatigue. Experimental Physiology 80, 497-527.
When PCr and glycogen are reduced below some critical ALLEN, D. G., LANNERGREN, J. & WESTERBLAD, H. (1997). Role of
level, ATP levels may transiently fall before [Ca2+]1 and ATP in the regulation of Ca2P release in normal and fatigued mouse
force output drop. Under these conditions, the net reduction skeletal muscle. Journal of Physiology (in the Press).
in ATP may inhibit excitation-contraction coupling processes ALLEN, D. G., LEE, J. A. & WESTERBLAD, H. (1989). Intracellular
or impair optimal Ca2P release channel function (Smith, calcium and tension during fatigue in isolated single muscle fibres
Coronado & Meissner, 1985; Allen, Liinnergren & Westerblad, from Xenopus laevis. Journal of Physiology 415, 433-458.
1997). Evidence from intact fibres indicates that the release BELCASTRO, A. N., RossITER, M., Low, M. P. & SOPPER, M. M. (1981).
of caged ATP during fatigue can temporarily reverse the Calcium activation of the sarcoplasmic reticulum ATPase following
decrease in force and [Ca2+]1i and overcome the impairment strenuous activity. Canadian Journal of Physiology and
Pharmacology 59, 1214-1218.
in E-C coupling (Allen et al. 1997). Although the energy
limitation hypothesis of muscle fatigue has been criticized
J Physiol.498.1 Glycogen depletion and calcium release 29
BERGUSTROM, J., HERMANSEN, L., HULTMAN, E. & SALTIN, B. (1967). NAGESSER, A. S., VAN DER LAARSE, W. J. & ELZINGA, C. (1992).
Diet, muscle glycogen and phylsical performance. Acta Physiologica Metabolic changes with fatigue in different types of single muscle
Scantdinavica 71, 140-150. fibres of Xenopus laevis. Journal of Physiology 448, 511-523.
BONEN, A. & HowIONKO, 1). A. (1994). Effects of exercise and glycogen SuMITH, J. S., CORONADO, R. & AIEISSNER, G. (1985). Sarcoplasmic
depletion on gliconeogenesis in muscle. Journial of Applied reticulum contains adenine nucleotide-activated calcium channels.
Physiology 76, 175:3-1758. Nature 316, 446-449.
BYRD, S. K., BODE, A. K. & KLUG, G. A. (1989). Effects of exercise of SPRIET, L. L. (1992). Anaerobic metabolism in human skeletal muscle
varying duration on sarcoplasmic reticulum function. Journial of during short-term, intense activity. Canadiani Journal of Physiology
Applied Physiology 66, 1383-1389. anid Pharmacology 70, 157-165.
CHIN, E. R. & ALLEN, 1). G. (1996). The role of elevations in intra- SPRIET, L. L., HEIGENHAUSER, G. J. F. & JONES, N. L. (1986).
cellular [Ca2+] in the development of low frequency fatigue in mouse Endogenous triacylglycerol utilization by rat skeletal muscle during
single muscle fibres. Journial of Physiology 491, 813-824. tetanic stimulation. Journal of Applied Physiology 60, 410-415.
COOKE, R., FRANKS, C., LUCIANO, G. & PATE, E. (1988). The VJ0LLESTAD, N. K., SEJERSTED, 0. M., BAHR, R., WOODS, J. J. &
inhibition of rat skeletal muscle contraction byr hydrogen ions and BIGLAND-RITCHIE, B. (1988). 1lotor drive and metabolic responses
phosphate. Journal of Physiology 395, 77-97. during repeated submaximal contractions in humans. Journal of
DOUEN, A. G., RAMLAL, T., RASTOGI, S., BILAN, P. J., CARTEE, G. D., Applied Physiology 64, 1421-1427.
VRANIC, M., HOLLOSZY, J. 0. & KLIP, A. (1990). Exercise induces XXVESTERBLAD, H. & ALLEN, D. C. (1991). Changes in myoplasmic
recruitment of the 'insulin responsive glucose transporter'. calcium concentration during fatigue in single mouse muscle fibres.
Evidence for distinct intracellular insulin- and exercise-recruitable Journal of Geeneral Physiology 98, 615-635.
transporter pools in skeletal muscles. Journal of Biological WVESTERBLAD, H. & ALLEN, D. G. (1992). Changes of intracellular pH
Chemistry 265, 13427-13430. due to repetitive stimulation of single fibres from mouse skeletal
ENT-MAN, AM. L., KESLENSKY, 8. S., CHU, A. & VTAN WINKLE, WV. B. muscle. Journtal of Physiology 449, 49-71.
(1980). The sarcoplasmnic reticulum-glvcogenolytic complex in XVESTERBLAD, H. & ALLEN, D. G. (1993). The influence of intra-
mammalian fast twitch skeletal muscle. Proposed in1 vitro cellular pH on contraction, relaxation and [CPa2+] in intact single
counterpart of the contraction-activated glycogenolytic pool. fibres from mouse muscle. Journal of Physiology 466, 611-628.
Journial of Biological Chenmistry 255, 6245-6252.
WVESTERBLAD, H. & ALLEN, D. G. (1994). The role of sarcoplasmic
FAVIERO, T. C., PESSAH, 1. N. & KLUG, G. A. (1993). Prolonged exercise
reticulum in relaxation of mouse muscle; effects of 2,5-di(tert-butyl)-
reduces Ca2P release in rat skeletal muscle sarcoplasmic reticulum. 1,4-benzohydroquinone. Journal of Physiology 474, 291-301.
Pfluigers Archiv 422, 472-475.
XVESTERBLAD, H., DUTY, S. & ALLEN, D. C. (1993). Intracellular
FITTS, R. H. (1994). Cellular mechanisms of muscle fatigue (Review). calcium concentration during low-frequency fatigue in isolated
Physiological Reviews 74, 49-94. single fibers of mouse skeletal muscle. Journal of Applied
FRIDEN, J., SECGER, J. & EKBLOM, B. (1989). Topographical Physiology 75, 382-388.
localization of muscle glycogen: An ultrahistochemical study in Xu, K. Y., ZWEIER, J. L. & BECKER, L. C. (1 995). Functional coupling
human vastus lateralis. Acta Physiologica Scandinavica 135,
between glycolysis and sarcoplasmic reticulum Ca2P transport.
381-391. Circulation Research 77, 88-97.
FRYER, AM. WV., OWEN, V. J., LAMIB, G. D. & STEPHENSON, D. (1995).
Effects of creatine phosphate and Pi on Ca2P movements and tension
development in rat skinned skeletal muscle fibres. Journal of Acknowledgements
Physiology 482, 123-140. This study was supported by the National Health and Medical
CALBO, H., HOLST, J. .. & CHRISTENSEN, H. J. (1979). The effect of Research Council of Australia. We thank Patricia Ruell and
different diets and of insulin on the lhoirmonal response to prolonged Dr Martin Thompson at the Faculty of Health Sciences, University
exercise. Acta Physiologica Scandinavica 107, 19-32. of Sydney Cumberland Campus, for their assistance with muscle
GODT, R. E. & NOSEK, T. Ml. (1989). Changes in the intracellular glycogen measurements.
milieu with fatigue or hypoxia depress contraction of skinned rabbit Author's email address
skeletal and cardiac muscle. Journal of Physiology 412, 155-180. D. G. Allen: davida@physiol.su.oz.au
HAN, J., THIELECZEK, R., VARSANYI, AM. & HEILMEYER, L. Ml. G.
(1992). Compartmentalised ATP sy-nthesis in skeletal muscle triads.
Biochemistry 31, 377-384. Received 22 Mlarch 1996; accepted 13 Septemiber 1996.
LANNERGREN, J. & WVESTERBLAD, H. (1991). Force decline due to
fatigue and intracellular acidification in isolated fibers from mouse
skeletal muscle. Journial of Physiology 434, 307-322.
LEE, J. A., WVESTERBLAD, H. & ALLEN, D. G. (1991). Changes in
tetanic and resting [Ca2+]i during fatigue and recovery of single
muscle fibres from Xeniopus laevis. Journal of Physiology 433,
307-326.
LINDINGER, AM. I., HEIGIENHAUSER, C. J. F. & SPRIET, L. L. (1987).
Effects of intense swimming and tetanic electrical stimulation on
skeletal muscle ions and mnetabolites. Joutrntal of Applied Physiology
63, 2331-2339.
LOWRY, 0. H. & PASSONNEAU, J. V. (1972). A Flexible Systemti of
Enzymatic An2alysis. Academic Press, Newv York.