451
J Physiol 560.2 (2004) pp 451–468
Effect of low cytoplasmic [ATP] on excitation–contraction
coupling in fast-twitch muscle fibres of the rat
Travis L. Dutka and Graham D. Lamb
Department of Zoology, La Trobe University, Melbourne, Victoria, 3086, Australia
In this study we investigated the roles of cytoplasmic ATP as both an energy source and a
regulatory molecule in various steps of the excitation–contraction (E–C) coupling process
in fast-twitch skeletal muscle fibres of the rat. Using mechanically skinned fibres with
functional E–C coupling, it was possible to independently alter cytoplasmic [ATP] and free
[Mg2 + ]. Electrical field stimulation was used to elicit action potentials (APs) within the sealed
transverse tubular (T-) system, producing either twitch or tetanic (50 Hz) force responses.
Measurements were also made of the amount of Ca2 + released by an AP in different
cytoplasmic conditions. The rate of force development and relaxation of the contractile
apparatus was measured using rapid step changes in [Ca2 + ]. Twitch force decreased
substantially (∼30%) at 2 mM ATP compared to the level at 8 mM ATP, whereas peak tetanic
force only declined by ∼10% at 0.5 mM ATP. The rate of force development of the twitch
and tetanus was slowed only slightly at [ATP] ≥ 0.5 mM, but was slowed greatly (> 6-fold)
at 0.1 mM ATP, the latter being due primarily to slowing of force development by the
contractile apparatus. AP-induced Ca2 + release was decreased by ∼10 and 20% at 1 and
0.5 mM ATP, respectively, and by ∼40% by raising the [Mg2 + ] to 3 mM. Adenosine inhibited
Ca2 + release and twitch responses in a manner consistent with its action as a competitive
weak agonist for the ATP regulatory site on the ryanodine receptor (RyR). These findings
show that (a) ATP is a limiting factor for normal voltage-sensor activation of the RyRs, and
(b) large reductions in cytoplasmic [ATP], and concomitant elevation of [Mg2 + ], substantially
inhibit E–C coupling and possibly contribute to muscle fatigue in fast-twitch fibres in some
circumstances.
(Received 28 May 2004; accepted after revision 10 August 2004; first published online 12 August 2004)
Corresponding author G. D. Lamb: Department of Zoology, La Trobe University, Victoria, 3086, Australia.
Email: g.lamb@latrobe.edu.au
The cytoplasmic [ATP] is very important in all cells
because it is ATP hydrolysis that provides the energy for
vital cellular processes. ATP may also act as a regulatory
molecule. In resting skeletal muscle fibres the cytoplasmic
[ATP] is ∼7–8 mm (expressed per litre cytoplasmic water),
and it is maintained near this level in most circumstances
by aerobic and anaerobic glycolysis, and in the short
term, by the creatine kinase reaction, which utilizes the
high concentration of creatine phosphate (PCr) normally
present (∼40 mm) (Fitts, 1994; Allen et al. 1995). However,
it has been recently shown that when human subjects
perform a fatiguing 25-s maximal cycling bout, the [ATP]
in fibres containing type IIX myosin heavy chain drops
to as low as 0.7–1.7 mm (2–5 µmol (g dry weight)−1 )
(Karatzaferi et al. 2001). This immediately raises the
question of what effect such low cytoplasmic [ATP] has
on the various steps in the excitation–contraction (E–C)
coupling process in skeletal muscle fibres and in particular
C The Physiological Society 2004
whether a low [ATP] and the associated changes could
contribute to muscle fatigue.
Muscle fatigue is the term used to describe the decrease
in force and power output that occurs with activity,
and it is clear that muscle fatigue has many forms
and many causes (Fitts, 1994; Allen et al. 1995). In
certain circumstances, in particular with high frequency
stimulation, muscle force can decline at least in part
owing to failure of excitability, that is, failure in the
generation and propagation of action potentials (APs)
on the sarcolemma and in the transverse tubular (T-)
system, caused by changes in the electrochemical gradients
for K+ and Na+ (Sejersted & Sjøgaard, 2000; Nielsen
et al. 2004). With repeated or strenuous activity, force can
also decline because of changes in the ‘metabolic state’
of the cytoplasm. This is due to the high usage of ATP
which, depending on the relative contributions of aerobic
and anaerobic pathways of ATP re-synthesis, can cause
DOI: 10.1113/jphysiol.2004.069112
452
T. L. Dutka and G. D. Lamb
various changes including decreased levels of PCr, ATP
and glycogen, and increased levels of inorganic phosphate
(Pi ), H+ , free Mg2+ , ADP, AMP, inosine monophosphate
(IMP) and lactate (Edwards et al. 1975; Nagesser et al.
1992, 1993; Karatzaferi et al. 2001). With repeated tetani,
force shows an initial moderate decline to a plateau level,
followed some time later by a large and steep decline
(Allen et al. 1995). The initial decline is evidently due
to reductions in the maximum force production and
Ca2+ sensitivity of the contractile apparatus, possibly
caused mostly by the increased [Pi ] (Allen et al. 1995;
though see Debold et al. 2004). The later steep decline in
force, on the other hand, is due to a decline in Ca2+ release
from the sarcoplasmic reticulum (SR), though the cause(s)
of this has not been clearly established, and quite possibly
it differs in different circumstances and muscle fibre types.
The decrease in Ca2+ release is evidently not caused by the
rise in concentrations of H+ (Lamb et al. 1992; Lamb &
Stephenson, 1994; Bruton et al. 1998; Chin & Allen, 1998),
lactate (Westerblad & Allen, 1992a; Posterino et al. 2001)
or IMP (Blazev & Lamb, 1999b; Laver et al. 2001). Instead,
it may be the result of cytoplasmic Pi entering the SR and
precipitating with Ca2+ , thereby reducing the amount of
Ca2+ available for rapid release (Fryer et al. 1995; Allen &
Westerblad, 2001; but see Steele & Duke, 2003).
Of importance to the present study, in some
circumstances reduced Ca2+ release might also be caused
by low cytoplasmic [ATP] and related changes, such as
the concomitant rise in free [Mg2+ ] (Westerblad & Allen,
1992b; Owen et al. 1996; Westerblad et al. 1998; Steele &
Duke, 2003). As mentioned, measurements made in single
fibres show that cytoplasmic [ATP] can drop to low levels
with intense exercise in certain fibre types (Nagesser et al.
1992, 1993; Karatzaferi et al. 2001). Furthermore, as ATP
usage and re-synthesis is compartmentalized, the [ATP] in
local regions of high usage probably drops lower than the
average level present in the cytoplasm (Korge & Campbell,
1995). In apparent support of the proposal that low
cytoplasmic [ATP] limits Ca2+ release, Allen et al. (1997)
found that photolysis of caged ATP produced an increase
in Ca2+ release and force in fatigued fibres of the
mouse. However, these authors subsequently showed that
photolysis of caged ADP and caged phosphate had a similar
effect, leading them to conclude that the increase in Ca2+
release resulted from the reduction in the amount of caged
compound rather than the release of biologically active
molecules (Allen et al. 1999).
We have previously shown, using ionic substitution
to depolarize the T-system in skinned muscle fibres,
that depolarization-induced Ca2+ release is inhibited
substantially by either raising the free [Mg2+ ] in the
cytoplasm from 1 mm (approximately the normal resting
level, Westerblad & Allen, 1992b,c) to 3 mm (Lamb &
Stephenson, 1991, 1994), or by decreasing [ATP] to 0.5 mm
(Owen et al. 1996), although in mammalian muscle fibres
J Physiol 560.2
no reduction in Ca2+ release could be detected by lowering
[ATP] unless it was also accompanied by an increase
in [Mg2+ ] (Blazev & Lamb, 1999a). However, it is not
certain that these findings can be extrapolated to infer
what happens in normal E–C coupling when Ca2+ release
is triggered by APs. This is because (a) the voltage-sensors
were activated about two orders of magnitude more
slowly than with an AP (∼200 ms versus < 1 ms), and (b)
the depolarization was applied for much longer (∼2–3 s
versus ∼2–3 ms). Thus, the ionic substitution experiments,
which assessed the total amount of Ca2+ release in response
to comparatively slow, prolonged stimulation, may not
have detected even large differences in the rate of Ca2+
release in the various conditions. Furthermore, it is quite
possible that depolarization by APs, where the whole
array of voltage sensors in the T-system are activated in
a rapid, co-ordinated fashion, might be a particularly
potent stimulus to the apposing array of ryanodine
receptor/Ca2+ release channels (RyRs), activating them
irrespective of the prevailing cytoplasmic conditions.
In the present study we used electrical stimulation to
trigger AP-induced Ca2+ release in mechanically skinned
fibres (Posterino et al. 2000), and report the effects
of low [ATP] and related changes on individual steps
and the overall behaviour of E–C coupling. The study
provides evidence that ATP must be bound to a cytoplasmic regulatory site on the RyR for the RyR to be
properly activated by the voltage sensors in the T-system
during physiological activation by APs. This is important
for understanding the coupling mechanism between the
voltage sensors and RyRs and also for identifying a possible
cause of muscle fatigue in fast-twitch fibres in certain
circumstances. Finally, the beneficial side of such muscle
fatigue could be that it would help prevent complete
exhaustion of ATP levels and the damage that otherwise
may ensue.
Methods
Skinned fibre preparation and force recording
Male Long-Evans hooded rats (∼5 months old) were
killed with an overdose of halothane as approved by La
Trobe University Animal Ethics Committee. An extensor
digitorum longus (EDL) muscle was excised and pinned
at resting length under paraffin oil in a dish kept on
an icepack. Individual fibres were mechanically skinned
with jeweller’s forceps and a segment (length, ∼3 mm;
diameter, 30–50 µm) was mounted on a force transducer
(AME801, Horten, Norway) with a resonance frequency
> 2 kHz at 120% of resting length. The paraffin oil was
then replaced with a Perspex bath containing 2 ml of
the appropriate potassium-based solution according to
whether AP-induced or Ca2+ -activated force responses
were being examined. Force responses were amplified
with a Bioamp pod (ADInstruments, Sydney, Australia)
C The Physiological Society 2004
J Physiol 560.2
Effect of low [ATP] on E–C coupling
attached to a 400 series Powerlab and simultaneously
recorded on both chart recorder and computer using Chart
software (version 4.2). All experiments were carried out at
23 ± 2◦ C.
Solutions
Unless otherwise stated all chemicals used were
purchased from Sigma Chemicals (St Louis, MO,
USA). The standard K-HDTA solution contained (mm):
hexamethylene-diamine-tetraacetate (HDTA2− , Fluka,
Buchs, Switzerland) 50, total ATP 8, Na+ 37, K+ 126,
total Mg2+ 8.5 (giving 1 mm free [Mg2+ ]), PCr, 10;
EGTA 0.05, Hepes 90 and N3 − 1; pH 7.1 and pCa
(− log10 [Ca2+ ]) 7.0, except where stated. Low [ATP]
solutions (i.e. 0.1, 0.5, 1 or 2 mm) were made by replacing
appropriate amounts of ATP by PCr, with all other
parameters maintained constant. This was achieved by
mixing appropriate amounts of the standard solution (e.g.
8 mm ATP and 10 mm PCr) and a matching solution with
no ATP and 18 mm PCr. The latter solution contained
only 1.66 mm total Mg2+ so as to keep the free [Mg2+ ]
constant at 1 mm. This amount of Mg2+ was calculated
based on apparent affinity constants for Mg2+ binding to
PCr, ATP and HDTA of 15 m−1 , 6.9 × 10−3 m−1 and 8 m−1 ,
respectively (Stephenson & Williams, 1981). K-HDTA
solutions with 3 mm free Mg2+ , and various [ATP] were
made in a similar way.
For examination of the properties of the contractile
apparatus, solutions similar to the standard HDTA
solution were made in which the 50 mm HDTA was
replaced by 50 mm EGTA for strong Ca2+ buffering.
The standard ‘relaxing solution’ had no added Ca2+
(pCa > 10) and the ‘maximum activation’ solution (max)
had 49.5 mm added Ca2+ (pCa 4.7), with total magnesium
of 10.3 and 8.1 mm, respectively, to maintain the free
[Mg2+ ] at 1 mm (see Stephenson & Williams, 1981).
These solutions were mixed in various proportions to
produce solutions at intermediate pCa values. Similar
EGTA solutions were also made with low [ATP] in the same
manner as for the HDTA-based solutions. Free [Ca2+ ]
of solutions in the range pCa 3–7.3 was measured with
a Ca2+ -sensitive electrode (Orion Research Inc, Boston,
MA, USA).
Other additions to solutions
Glybenclamide (GLYB) was prepared as a 100 mm stock
solution in dimethyl sulfoxide (DMSO) and then diluted
2000-fold to give a concentration of 50 µm in the final
solution, with the same volume of DMSO added to
the matching control solution. In experiments where
exogenous creatine phosphokinase (CPK) was added, it
was made up as a stock in the relevant solution at 10 times
the required level and then diluted to give a CPK
activity of ∼30 or 150 units ml−1 in the final solution.
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453
Adenosine stock (8 mm) solutions were prepared simply by
dissolving the adenosine in either the K-HDTA standard
solution or in the zero ATP-containing K-HDTA solution.
This was possible because adenosine negligibly binds
Mg2+ . Solutions of various [adenosine] could then be
made without changing the concentration of the other
constituents (e.g. Mg2+ , PCr, HDTA, Na+ , K+ , etc.) in the
final test solution. There was only a very slight increase
in osmolality (and no change in the ionic strength), and
this would have had negligible effects on the properties
of either the contractile apparatus or Ca2+ release (Lamb
et al. 1993).
The amount of Ca2+ released by an AP was assayed
in the appropriate K-HDTA solutions (control and low
[ATP]) with the addition of 50 µm 2,5-di-tert-butyl-1,
4-hydroquinone (TBQ), a blocker of the SR Ca2+ -ATPase,
and set amounts of the fast Ca2+ -buffer BAPTA
(120–200 µm) (and with all EGTA omitted). A 50 mm TBQ
stock solution (dissolved in DMSO) was diluted 1000-fold
in the final solution. BAPTA (Molecular Probes, OR, USA)
was added from a 50 mm stock that was similar to the
standard K-HDTA solution except that all 50 mm HDTA
was replaced by BAPTA.
AP-induced force responses triggered by transverse
electric field stimulation
When examining twitch and tetanic force responses, the
skinned fibre segment was bathed in the standard (weakly
Ca2+ -buffered) K-HDTA solution (8 mm ATP, 1 mm free
Mg2+ , 50 µm EGTA, pCa 7.0). The segment was positioned
midway between two platinum stimulating electrodes
and electric field pulses (duration, 2 ms; 70 V cm−1 ) were
applied by an in-house stimulator in order to generate APs
in the sealed T-system (see Posterino et al. 2000). Twitch
and tetanic force responses were elicited with single pulse
and 50 Hz stimulation, respectively. When measuring
the effect of a given treatment (e.g. lowered [ATP]
or raised [Mg2+ ]), the fibre segment was equilibrated
in the appropriate solution for > 30 s and bracketing
measurements were obtained in the same fibre segment
under the control conditions (i.e. in the standard solution
with 8 mm ATP and 1 mm free Mg2+ ). Where appropriate,
the magnitude of the applied voltage was varied in order
to ascertain the threshold for AP generation, or pairs of
supra-maximal pulses were applied with the interpulse
interval varied from 2 to 20 ms in order to study the
refractory behaviour of AP generation.
TBQ-BAPTA assay of amount of Ca2 + released
by an AP
The amount of Ca2+ released from the SR in response
to an AP was determined using the technique described
by Posterino & Lamb (2003). The twitch response was
454
T. L. Dutka and G. D. Lamb
first measured in the standard conditions as described
in the previous section. Then the fibre segment was
transferred into a K-HDTA solution with 50 µm TBQ
and a known [BAPTA] for 15 s before being stimulated
electrically. The TBQ completely blocks uptake by the SR
Ca2+ -ATPase and the Ca2+ released by the AP rapidly binds
to the BAPTA and the troponin C (TnC) sites on the
contractile apparatus. The size of the resulting force
response can then be used to quantify the total amount
of Ca2+ released by the AP, as described by Posterino &
Lamb (2003), with two small adjustments being made
to take into account: (a) the greatly reduced amount of
Ca2+ binding to ATP in the solutions with only 0.5 mm
ATP (i.e. Ca2+ binding to ATP reduced by a factor of 16);
and (b) the +0.2 pCa unit shift in the Ca2+ sensitivity
(i.e. pCa50 ) of the contractile apparatus in solutions with
3 mm Mg2+ (Blazev & Lamb, 1999a). In some experiments,
the SR was loaded with additional Ca2+ by bathing the
fibre segment for a set period (30–60 s) in a loading
solution (standard K-HDTA solution, with 1 mm total
EGTA at pCa 6.7) prior to being exposed to the
TBQ-BAPTA solution.
Rapid activation and relaxation of force
by the contractile apparatus
2+
When examining the steady-state Ca -dependence of the
contractile apparatus, the freshly skinned fibre segment
was activated in a set of solutions in which the [Ca2+ ]
was heavily buffered with 50 mm EGTA at progressively
higher [Ca2+ ] (pCa 6.7–4.7), first under control conditions
J Physiol 560.2
(8 mm ATP and 1 mm Mg2+ ), then under the given test
conditions (e.g. 0.5 mm ATP), and then again under
control conditions, as done by Lamb & Posterino (2003).
In other experiments the contractile apparatus was rapidly
activated by changing the bathing solution from one
with weak Ca2+ buffering (100 µm EGTA) at pCa 7.0 to
one with heavy Ca2+ buffering (all HDTA replaced by
50 mm CaEGTA) at pCa 4.7. This method greatly reduces
the diffusional delays limiting the rise in [Ca2+ ] within
the skinned fibre (for details see Moisescu, 1976). The
two bathing solutions (weakly and heavily Ca2+ -buffered)
contained the same concentration of all other constituents
(e.g. PCr, ATP and Mg2+ ). In such experiments, the
fibre segment was first treated with Triton X-100 to
remove the SR and other membranous compartments
(5 min exposure to 2% (v/v) Triton X-100 in relaxing
solution, followed by two washes without detergent).
Similar experiments were also performed in which the fibre
was relaxed by rapidly lowering the [Ca2+ ] by exchanging a
solution weakly Ca2+ -buffered at pCa 5 (1 mm total EGTA)
for one very strongly buffered at pCa > 10 with 50 mm free
EGTA. In such experiments, all other constituents were
maintained constant (e.g. at 8 mm ATP or at 0.5 mm ATP).
Statistical analysis
Values are presented as means ± s.e.m. with n indicating
the number of fibres examined. Statistical significance
was determined with Student’s t test (1-tailed, paired or
unpaired, as appropriate), with mean values considered
significantly different if P < 0.05.
Figure 1. Low cytoplasmic [ATP]
decreases and slows the twitch
response
A–D, representative examples of twitch
responses at various low [ATP]; each
panel shows data from a different
skinned fibre. Unless indicated otherwise,
the skinned fibre was bathed in the
control conditions (8 mM ATP and 1 mM
free Mg2+ ). Several twitches were
elicited under each condition with similar
results. Timing of stimulus (2 ms pulse,
70 V cm−1 ) is indicated by a vertical bar
below each force recording. See Table 1
for mean data.
C The Physiological Society 2004
Effect of low [ATP] on E–C coupling
J Physiol 560.2
Results
Effect of low [ATP] on twitch
and tetanic force responses
Twitch and tetanic force responses could be elicited in
the skinned EDL fibres by triggering APs in the T-system
with brief electric field pulses, as previously described
(Posterino et al. 2000; Posterino & Lamb, 2003). When
the cytoplasm contained 8 mm ATP and 1 mm free Mg2+ ,
the tetanic response to 50 Hz stimulation typically reached
close to the maximum Ca2+ -activated force level measured
in the same fibre (94 ± 5%, n = 43). Tetanic stimulation
was always applied for a sufficiently long enough period
for the force to reach its peak level under the given
conditions. With the free [Mg2+ ] maintained at 1 mm,
reducing the cytoplasmic [ATP] to very low levels caused
a marked reduction in the peak size of both the twitch
(Fig. 1) and tetanic (Fig. 2) responses. These responses
were also slower to develop and to relax (Figs 1 and 2,
summarized in Table 1). The peak of the twitch force
declined progressively as [ATP] was decreased. At 2 mm
ATP the peak twitch force was only ∼70% of the control
level measured with 8 mm ATP, and with 100 µm ATP it
was only ∼30% of the control level (Table 1). The peak
tetanic force was less sensitive to reductions in [ATP],
the response with 0.5 mm ATP still being ∼90% of the
control level. However, when the [ATP] was decreased
to 100 µm, the peak tetanic force only reached ∼47%
of the control level. Both the rate of force development
and the rate of relaxation of the tetanic response became
substantially slower even at 2 mm ATP (∼1.4-fold and
1.7-fold, respectively) (Table 1), showing that such a
reduction in [ATP] had a major effect on the response
of the muscle fibres.
It should be noted that the [ATP] within the skinned
fibres here is likely to be better controlled than would
be the case in metabolically exhausted intact fibres at
similar low [ATP]. This is because the [ATP] in the
skinned fibre is continuously replenished by diffusion of
the bathing solution into the fibre and, more importantly,
because the low [ATP] solutions contained 16–18 mm PCr
(and no creatine) which would have greatly stimulated
resynthesis of ATP from ADP. Mechanically skinned
fibres normally retain appreciable amounts of CPK for
prolonged periods (Stephenson et al. 1999), and only
∼5% of the endogenous CPK is evidently required to
support normal activity in CPK-knockout mice (Dahlstedt
et al. 2003). In accord, we found that addition of
30 units ml−1 (Table 1) or even 150 units ml−1 (data not
shown) of CPK had no significant effect on the reduction
in twitch and tetanic force responses occurring in low
[ATP].
Figure 2. Effect of low cytoplasmic [ATP] on tetanic responses
A–D, representative examples of tetanic responses at various low [ATP]; each panel shows data from a different
skinned fibre. A period of 2 min was allowed between successive tetani. Period of stimulation (50 Hz) is indicated
by the rectangle below each force recording; the duration of the stimulation was longer in low [ATP] to ensure
that peak force was obtained. The rates of rise and decay of the tetani were slowed as [ATP] was decreased, and
peak tetanic force was reduced at [ATP] ≤ 1 mM (see Table 1 for mean data).
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456
T. L. Dutka and G. D. Lamb
J Physiol 560.2
Table 1. Summarized data on the effect of low [ATP] on AP-induced force responses
Treatment
n
Peak
(%)
RT10–90
(%)
RFD10–90
(mN s−1 )
FT90–10
(%)
RFR90–10
(mN s−1 )
Twitch
8 mM ATP
2 mM ATP
2 mM ATP + GLYB
1 mM ATP
0.5 mM ATP
0.5 mM ATP + CPK
0.5 mM ATP + GLYB
0.1 mM ATP + GLYB + CPK
82
7
3
24
51
11
3
8
100
71 ± 4%∗
74 ± 16%
66 ± 3%∗
56 ± 3%∗
54 ± 7%∗
61 ± 4%∗
28 ± 4%∗
100
107 ± 6%
106 ± 7%
121 ± 9%∗
120 ± 4%∗
128 ± 6%∗
123 ± 7%∗
182 ± 18%∗
8.3 ± 0.9
5.5 ± 4.7
5.9 ± 4.5
4.5 ± 1.9∗
3.9 ± 0.8∗
3.5 ± 0.8∗
4.1 ± 1.3∗
1.3 ± 0.3∗
100
136 ± 11%∗
131 ± 12%∗
163 ± 18%∗
176 ± 10%∗
172 ± 17%∗
188 ± 14%∗
502 ± 18%∗
3.2 ± 0.4
1.7 ± 0.5∗
1.8 ± 0.7∗
1.3 ± 0.5∗
1.0 ± 0.1∗
1.0 ± 0.2∗
1.0 ± 0.2∗
0.2 ± 0.0∗
Tetani
8 mM ATP
2 mM ATP
1 mM ATP
0.5 mM ATP
0.5 mM ATP + CPK
0.1 mM ATP + CPK + GLYB
43
6
10
27
23
7
100
102 ± 2%
92 ± 3%∗
90 ± 2%∗
87 ± 5%∗
47 ± 7%∗
100
141 ± 10%∗
127 ± 10%∗
158 ± 12%∗
151 ± 17%∗
340 ± 55%∗
4.3 ± 0.9
3.2 ± 0.8∗
3.2 ± 1.2∗
2.5 ± 0.5∗
2.5 ± 0.8∗
0.6 ± 0.1∗
100
174 ± 22%∗
245 ± 36%∗
255 ± 25%∗
249 ± 36%∗
456 ± 104%∗
1.4 ± 0.4
0.8 ± 0.3∗
0.5 ± 0.1∗
0.5 ± 0.1∗
0.5 ± 0.1∗
0.2 ± 0.0∗
Twitch and tetanic force responses elicited as in Figs 1 and 2. Values are the mean ± S.E.M. in n fibres for the given parameter,
as a percentage of that in 8 mM ATP. The mean twitch peak in 8 mM ATP was 58 ± 5% of maximum Ca2+ -activated force, the
mean rise time from 10 to 90% of peak force (RT10–90 ) was 29 ± 3 ms, and the mean fall time from 90 to 10% of peak force
(FT90–10 ) was 76 ± 10 ms. The mean RT10–90 for the tetani in 8 mM ATP was 92 ± 9 ms and the mean FT90–10 was 282 ± 25 ms.
The average rate of force development from 10 to 90% maximum (RFD10–90 ), and rate of force relaxation from 90 to 10%
of maximum (RFR90–10 ) (both in absolute units, mN s−1 ) are also given. Such values, which are influenced by the changes in
maximum force Ca2+ -activated force occurring at the different [ATP], can be directly compared with the force responses to
applied Ca2+ (Table 2). GLYB, glybenclamide (50 µM); CPK, exogenous CPK (30 units ml−1 ). ∗ Significantly different from that in
8 mM ATP (paired t test). With glybencamide and CPK, data were obtained with and without treatment in the same fibre.
The reduction in the AP-induced force responses at
low [ATP] was evidently not caused by alterations in
T-system membrane potential or AP generation. Firstly,
the threshold electric field required for AP generation
(detected by the sudden transition in twitch force from 0
to > 70% of maximum twitch force) was not significantly
altered at low [ATP] (36.0 ± 0.8 V cm−1 with 8 mm ATP,
34.3 ± 1.3 V cm−1 with 0.5 mm ATP, n = 8 fibres; paired
observations). Secondly, the refractory behaviour of the
T-system AP was not detectably altered in low [ATP]. This
was assessed by applying a pair of closely spaced, supramaximal pulses and determining the minimum interpulse interval required to elicit a quantal increase in force
(typically ∼30%), which was indicative that the second
pulse occurred sufficiently long enough after the first pulse
to elicit another AP (see Fig. 3 in Posterino et al. 2003).
This interval was not significantly different at 8 mm and
0.5 mm ATP (5.1 ± 0.4 ms and 5.3 ± 0.4 ms, respectively,
n = 7 fibres; paired observations). This behaviour was not
assessed at 100 µm ATP because the twitch response even to
single pulses did not stay constant enough over the number
of repetitions required. Finally, because it is known that
reducing [ATP] to very low levels can open ATP-sensitive
K+ channels (Fink & Lüttgau, 1976; Spruce et al. 1985),
which might alter the resting membrane potential or AP, we
examined the effect of glybenclamide, a potent and specific
blocker of ATP-sensitive K+ channels (Light & French,
1994; Barrett-Jolley & Davies, 1997). It was found that the
presence of 50 µm glybencamide did not significantly alter
the effect of low [ATP] on the twitch response (see data
for 2 mm and 0.5 mm ATP in Table 1; paired responses),
indicating that the reduction in twitch force was not due
to opening of ATP-sensitive K+ channels.
Effect of low [ATP] on Ca2 + -activated force
development by the contractile apparatus
The properties of the contractile apparatus were examined
in order to ascertain whether the reduction in twitch and
tetanic peak force occurring at low [ATP] was due to effects
on the contractile apparatus or due to a reduction in
SR Ca2+ release. First, the steady-state properties of the
contractile apparatus were examined in freshly skinned
fibres that had not been treated with Triton X-100, and
which were thus fully comparable to the fibres used in
the AP-stimulation experiments. Each fibre was exposed
to sequences of heavily Ca2+ -buffered solutions (50 mm
total EGTA) with 8 mm ATP (control) or with 0.5 mm
ATP, containing progressively higher free [Ca2+ ] until
maximum Ca2+ -activated force was achieved (at pCa 4.7)
(data not shown). Force was plotted against pCa, and a Hill
curve fitted individually for each condition in each fibre
C The Physiological Society 2004
J Physiol 560.2
Effect of low [ATP] on E–C coupling
(as by Lamb & Posterino, 2003). In the four fibres
examined, 0.5 mm ATP caused a significant potentiation
of maximum Ca2+ -activated force (113 ± 3%, compared
to bracketing controls), but the [Ca2+ ] eliciting 50% of
maximum Ca2+ -activated force (pCa50 ) was unchanged
(difference in pCa50 , −0.004 ± 0.036 pCa units). This
potentiation of maximum force without change in
Ca2+ sensitivity is very similar to that found previously
when decreasing [ATP] to 1 mm (Godt & Nosek, 1989).
In contrast, in six other fibres that were treated with
Triton X-100 to destroy all membranes (leaving only the
contractile elements), 0.5 mm ATP caused a substantial
increase in Ca2+ sensitivity (change in pCa50 , +0.149 ±
0.012 pCa units). The increase in maximum Ca2+ activated force in 0.5 mm ATP was no different between
fibres that were treated or not treated with Triton
X-100 (114 ± 5% and 113 ± 3% of that with 8 mm
ATP, respectively). Of importance, when 30 units ml−1 of
exogenous CPK was present in the solutions, the increase
in Ca2+ sensitivity seen with 0.5 mm ATP was no longer
manifested, and the pCa50 value was not appreciably
different from that in 8 mm ATP (final difference in
pCa50 , +0.007 and +0.006 pCa units, examined in two
of the six Triton X-100-treated fibres above). This strongly
suggests that the endogenous CPK normally present in
mechanically skinned fibres was lost during the Triton
X-100-treatment and washing procedure, and that the shift
in Ca2+ sensitivity was due to accumulation of ADP within
the myofibrils (Godt & Nosek, 1989). It also demonstrates
that the exogenously added CPK was active and sufficient
to replace the endogenous CPK normally present in the
skinned fibres.
The ability of the contractile apparatus to develop force
in response to rapid rises in [Ca2+ ] was then examined,
in order to make comparisons with the time course of the
twitch and tetanic force responses. This was achieved by
transferring the mechanically skinned fibre from a weakly
Ca2+ -buffered (100 µm EGTA) solution at pCa 7.0 to a
heavily Ca2+ -buffered (50 mm CaEGTA) solution at high
[Ca2+ ] (pCa 4.7) (see Moisescu, 1976). The fibre segment
was pre-treated with Triton X-100 to eliminate any effect
of the SR on Ca2+ movements and to improve diffusional
access to the contractile apparatus. With this procedure,
the free [Ca2+ ] within the skinned fibre rises to the
micromolar level within tens of milliseconds (Moisescu
& Thieleczek, 1978). As seen in Fig. 3, this resulted in
rapid contractile activation, with the force reaching
more than 50% of maximum in less than 100 ms in the
presence of 8 mm ATP. As in the measurements of steadystate activation, the level of maximum Ca2+ -activated
force was potentiated in low [ATP] conditions
(maximum force: 115 ± 1%, n = 4; 117 ± 2%, n = 27;
127 ± 4%, n = 4; and 124 ± 8%, n = 4, compared to the
control level (8 mm ATP) for 2 mm, 1 mm, 0.5 mm and
100 µm ATP, respectively). In the presence of very
C The Physiological Society 2004
457
low [ATP] (i.e. 100 µm), the rate of isometric
force development (RFD) was dramatically slowed
compared to control conditions (Fig. 3 and Table 2)
and appeared to exhibit at least two distinct ‘phases’
(initially the force developed rapidly but then slowed
markedly). In contrast, at [ATP] ≥ 0.5 mm, the rate of
force development from 10 to 50% of maximum force
development (RFD10–50 ) was not detectably different
from that under the control conditions (8 mm ATP)
(Table 2). Thus, from these experiments it seems that the
slowing of force development by the contractile apparatus
observed at 100 µm ATP was a major factor contributing
to the slow rise and reduced peak of the tetanic response
under such conditions. With 0.5–2 mm ATP, it appeared
that the rate of force development by the contractile
apparatus was not greatly slowed; however, it is not
possible to conclude that slowing of force development
by the contractile apparatus played no part at all in
the reduction in the twitch response at 0.5 mm ATP.
This is because the highest rate of force development
measured in the Ca2+ -activation experiments with
Figure 3. Force development in response to a rapid rise in
[Ca2 + ] at various [ATP]
A, the [Ca2+ ] within a skinned fibre was rapidly raised by transferring
the fibre from a weakly Ca2+ -buffered solution (100 µM EGTA) at
pCa 7.0 to a strongly buffered solution (∼50 mM CaEGTA) at pCa 4.7.
The procedure was repeated with [ATP] set at each of the levels
indicated. The SR and other membranous compartments were
removed beforehand by treating the fibre with TritonX-100. All
solutions had 30 units ml−1 exogenous CPK added. B, superimposed
and expanded records of the force responses from A. The unevenness
in the trace for 100 µM ATP was caused by movement during the
solution exchange procedure. See Table 2 for mean data.
458
T. L. Dutka and G. D. Lamb
Table 2.
J Physiol 560.2
Force development by the contractile apparatus when rapidly raising [Ca2+ ]
RT10–50
(%)
RFD10–50
(mN s−1 )
RT10–90
(%)
RFD10–90
(mN s−1 )
8 mM ATP (n = 4)
2 mM ATP
0.5 mM ATP
0.1 mM ATP
100
119 ± 6∗
125 ± 6∗
1641 ± 548∗
3.0 ± 0.1
2.9 ± 0.2
2.8 ± 0.2
0.4 ± 0.2∗
100
113 ± 4∗
189 ± 36
3203 ± 810∗
1.6 ± 0.1
1.6 ± 0.1
1.1 ± 0.2∗
0.1 ± 0.0∗
8 mM ATP (n = 27)
1 mM ATP
100
117 ± 3∗
3.1 ± 0.2
2.9 ± 0.3
100
153 ± 14∗
1.4 ± 0.1
1.0 ± 0.1∗
The contractile apparatus was directly activated by rapidly raising the [Ca2+ ] as shown in
Fig. 3. The rise time between 10 and 50% (RT10–50 ) and between 10 and 90% (RT10–90 ) of
peak force in low [ATP] were expressed as a percentage of that in control conditions
(8 mM ATP) in the same fibre. The mean rate of force development (RFD) over the
respective ranges is shown in units of mN s−1 . Data were obtained at each of the indicated
concentrations in the same fibres. ∗ Significantly different from value in 8 mM ATP (paired
t test). Exogenous CPK (30 units ml−1 ) was present in all solutions.
0.5–8 mm ATP (RFD10–50 , 2.8–3.0 mN s−1 ; Table 2) was
evidently limited by the speed of Ca2+ diffusion into and
throughout the fibre; even though the force development
was quite fast, it was not as fast as the highest rate of
force development measured over a comparable force
range during a twitch (rate of force development from
10 to 90% maximum (RFD10–90 ), 8.3 mN s−1 ; Table 1).
The rate of force development during a twitch and when
applying exogenous Ca2+ would both be similarly affected
by the kinetic properties of contractile apparatus events
subsequent to Ca2+ binding to TnC. The faster rate
of force development during a twitch stems from the
fact that an AP induces within several milliseconds the
release of a large bolus of Ca2+ (∼230 µmoles Ca2+ per
litre fibre volume; Posterino & Lamb, 2003) at release
zones less than 1–2 µm from the TnC binding sites. This
pulse of Ca2+ delivered simultaneously throughout the
fibre effectively ‘supercharges’ Ca2+ binding to TnC and
consequently force development is even faster than when
applying Ca2+ exogenously with the heavy-buffering
technique. In view of this, the question of what caused the
reduced twitch and tetanic responses at 0.5 mm ATP was
instead addressed by determining whether there was any
reduction in Ca2+ release from the SR (see later).
Effect of low [ATP] on force relaxation
Noting the very slow decline in the tetanic responses
at low [ATP] (Fig. 2), it was also relevant to examine
the rate at which the contractile apparatus relaxed when
[Ca2+ ] was rapidly decreased. This was achieved in a
manner analogous to that used to rapidly raise [Ca2+ ].
After treating a fibre with Triton X-100, it was activated
maximally in a high [Ca2+ ] solution (pCa 4.7) buffered
with 1 mm CaEGTA and then plunged into a zero Ca2+
solution (pCa > 10) buffered with 50 mm EGTA, with the
[ATP] kept the same in both solutions. The fall time
from 90 to 10% peak force (FT90–10 ) was examined at
various [ATP] in each of four fibres with exogenous CPK
present (e.g. Fig. 4) and in every case the fall time became
progressively longer as [ATP] was reduced (Table 3, fibre
subset 3). This direct effect of low [ATP] on the contractile
apparatus suggests that a reduction in [ATP] in this range
in muscle fibres would slow relaxation even if there were
no reduction in the rate of Ca2+ uptake by the SR Ca2+
pump. It was also found that this slowing of relaxation
was very greatly enhanced in Triton X-100-treated fibres
when no exogenous CPK was added and the [ATP] was low
(≤ 0.5 mm) (e.g. FT90–10 > 3 s in 100 µm ATP; see Table 3).
This demonstrates the great importance of the creatine
kinase reaction in keeping [ATP] from decreasing to very
low levels in regions of high usage (see Discussion).
Table 3 also presents data on the effect of low [ATP] on
the rate of relaxation after a tetanus. As mentioned earlier,
the rate of relaxation slowed considerably when the [ATP]
was decreased to ≤ 0.5 mm. The decline of tetanic force
was not affected by addition of exogenous CPK, indicating
that increasing the total amount of CPK above the amount
already present in the skinned fibres conferred no benefit.
It is also very apparent that the force declined more slowly
after a tetanus than it did when the [Ca2+ ] was rapidly
decreased by the solution change method. This is evident in
Table 3 both from the mean data from all fibres examined
and from the subset of five fibres (subset 1) in which tetanic
responses were first recorded in each fibre and then the
fibre was treated with Triton X-100 (and CPK added) and
its response to lowering [Ca2+ ] recorded. The latter data
show that in 8 mm ATP it took ∼200 ms longer to relax
after a tetanus than it did when the [Ca2+ ] was rapidly
decreased, and in 0.5 mm ATP this difference was ∼760 ms.
At least some of the difference in the time course of force
decline must be accounted for by the time taken for the SR
to remove Ca2+ from the cytoplasmic space at the end of
a tetanus.
C The Physiological Society 2004
Effect of low [ATP] on E–C coupling
J Physiol 560.2
Measurement of Ca2 + release with AP stimulation
at different [ATP]
We next measured how much Ca2+ was released from
the SR by AP stimulation at different [ATP]. This was
done using the method described recently by Posterino
& Lamb (2003) in which all Ca2+ uptake by the SR is
blocked with TBQ and the amount of Ca2+ released by
an AP is ascertained from the size of the force response
produced in the presence of a known amount of the fast
Ca2+ buffer, BAPTA. When all Ca2+ uptake is blocked, the
[Ca2+ ] within the fibre declines only very slowly (time
constant, > 600 ms), limited primarily by diffusion of
Ca-BAPTA out of the fibre. This means that even if reduced
[ATP] slows the rate of force development of the contractile
apparatus to some extent, the peak of the force response
should still be indicative of the total amount of Ca2+
released by an AP. It is apparent from Fig. 3B that the
rate of force development in response to a rapid rise in
[Ca2+ ] is little if at all slower in 0.5 mm ATP compared
to that at 8 mm ATP, which means that the TBQ-BAPTA
method should allow reliable estimation of the amount of
Ca2+ released by an AP in 0.5 mm ATP. Figure 5 shows the
protocol used. The amount of Ca2+ released by an AP in
0.5 mm ATP was compared to bracketing measurements
of release in 8 mm ATP. Measurements were made (a) in
fibres with the SR Ca2+ content close to its normal endogenous level and with 140 or 160 µm BAPTA present to
chelate the released Ca2+ , and (b) in fibres with the SR
loaded ∼50% above the endogenous level and with 200 µm
BAPTA present. The results with 8 mm ATP were similar
to those obtained by Posterino & Lamb (2003): the peak
amplitude of the first response in 140, 160 and 200 µm
BAPTA was 53 ± 8 (n = 5), 44 ± 4 (n = 7) and 36 ± 4%
(n = 10) of maximum force, respectively, corresponding to
release of 210 ± 14, 218 ± 11 and 237 ± 5 µm Ca2+ (values
expressed as Ca2+ per unit fibre volume; see Posterino
& Lamb, 2003). When the [ATP] was reduced to 0.5 mm
ATP, the amount of Ca2+ released by an AP decreased by
∼20%, irrespective of loading and buffering conditions
used (release relative to bracketing measurements in 8 mm
ATP: 140 µm BAPTA, 81 ± 2%, n = 5; 160 µm BAPTA,
80 ± 2%, n = 7; 200 µm BAPTA, 81 ± 3%; n = 8). This
demonstrates that the ability of the voltage sensor to
activate the Ca2+ release channels is adversely affected
at low cytoplasmic [ATP]. This reduction in AP-induced
Ca2+ release at 0.5 mm ATP accounts for much if not all
of the reduction in twitch force (Fig. 1 and Table 1) (see
below and Discussion).
Ca2+ release in response to pairs of APs was also
measured. In intact mammalian fibres, the second and
subsequent APs in a train (all 10 ms apart) only release
∼25% as much Ca2+ as the first AP, apparently owing
to Ca2+ -dependent inactivation of the release channels
(Hollingworth et al. 1996). In accord, when a skinned
EDL fibre with 8 mm ATP present in the cytoplasm is
stimulated by two APs (10 ms apart) rather than by a single
AP, the total Ca2+ release is only increased by 25 ± 2%
(n = 30) (Posterino & Lamb, 2003). When this double
pulse stimulation was applied to the fibres here with
0.5 mm ATP present, the total amount of Ca2+ release was
77 ± 3% (n = 7, 200 µm BAPTA) of that released by the
same double pulse stimulus in bracketing measurements
in 8 mm ATP. This indicates that the amount of Ca2+
released in the low [ATP] by the second AP in a pair is
reduced to a similar (or perhaps slightly greater) extent as
occurred with the first AP (i.e. reduced to ≤ 80% of that
at 8 mm ATP).
The above data are fully consistent with the responses
observed with single and double pulse stimuli in the
absence of TBQ-BAPTA. As seen in the lefthand side of
Fig. 5, the peak of the force response in 0.5 mm ATP to a
pair of APs 10 ms apart (D10 response) was only slightly
smaller than the response to a single AP in 8 mm ATP
Figure 4. Relaxation upon rapidly lowering [Ca2 + ] is slowed at low [ATP]
Maximum force (at various [ATP]) was elicited by exposing the Triton X-100-treated fibre to a solution with moderate
Ca2+ buffering (1 mM CaEGTA) at pCa 4.7. Force was then rapidly abolished by transferring the fibre (at vertical
dotted line) to a very strongly buffered relaxing solution (50 mM free EGTA) at pCa > 10, with the [ATP] unchanged.
The horizontal bar indicates the maximum Ca2+ -activated force level in each condition. The unevenness in force
traces was caused by the solution exchange procedure. The time for the response to fall from 90 to 10% of
maximum (FT90–10 ) is shown under each trace. All solutions contained 30 units ml−1 exogenous CPK. See Table 3
for mean data.
C The Physiological Society 2004
459
460
T. L. Dutka and G. D. Lamb
Table 3.
J Physiol 560.2
Comparison of FT90–10 at the end of a tetanus and when rapidly lowering [Ca2+ ]
Decline in force to lowered [Ca2 ± ]
Decline of tetanic force
No added CPK
8 mM ATP
2 mM ATP
0.5 mM ATP
0.1 mM ATP
282 ± 25 (43)
491 ± 146 (6)
718 ± 108 (27)
—
With CPK
No added CPK
With CPK
357 ± 46 (20)
—
771 ± 99 (23)
983 ± 200 (7)
170 ± 14 (15)
—
347 ± 45 (8)
3186 ± 1832 (7)
172 ± 14 (13)
154 ± 14 (4)
216 ± 15 (12)
327 ± 43 (8)
Subset 1
8 mM ATP
0.5 mM ATP
—
—
421 ± 20 (5)
990 ± 191 (5)
—
—
215 ± 10 (5)
227 ± 9 (5)
Subset 2
8 mM ATP
0.5 mM ATP
276 ± 22 (7)
1090 ± 169 (7)
278 ± 22 (7)
877 ± 177 (7)
—
—
—
—
Subset 3
8 mM ATP
2 mM ATP
0.5 mM ATP
0.1 mM ATP
—
—
—
—
—
—
—
—
—
—
—
—
134 ± 16 (4)
154 ± 14 (4)
181 ± 18 (4)
254 ± 12 (4)
Values are means ± S.E.M. (n fibres) for the force decline for the tetani (as in Fig. 2) and when
lowering [Ca2+ ] to nanomolar levels by solution change (as in Fig. 4), both with and without
exogenous CPK (30 units ml−1 ). Top section shows the grand mean for all fibres examined. Lower
sets of data show three subsets of fibres where the indicated parameters were all compared in
each fibre. The decline in force when lowering the [Ca2+ ] was considerably faster than the decline
of tetanic force (subset 1). Addition of exogenous CPK did not significantly affect the decline of
tetanic force (subset 2, P > 0.05). The rate of force decline when lowering [Ca2+ ] slowed as [ATP]
was lowered (subset 3), and was much slower at low [ATP] in the absence of CPK (also confirmed
by pairwise comparison in some fibres, not shown).
(mean data, 95 ± 5% in 10 fibres). This fits with Ca2+
release in 0.5 mm ATP being only 80% of that in 8 mm
ATP and with a pair of APs eliciting ∼25% more Ca2+
release than a single AP, as this means that the total Ca2+
released by a pair of APs in 0.5 mm ATP would be almost
exactly the same as that released by a single AP in 8 mm
ATP.
Effect of adenosine on twitch responses
The reduction in AP-stimulated Ca2+ release at low cytoplasmic [ATP] is most likely due to there being insufficient
ATP present to saturate the stimulatory ATP-binding sites
on the RyR (K a , ∼0.4 mm ATP; Meissner et al. 1986;
Laver et al. 2001). Further evidence supporting this was
obtained by examining the effect of adenosine, which
is a competitive weak agonist for the ATP binding site
of the RyR (Duke & Steele, 1998; Laver et al. 2001). As
force was used as an indicator of Ca2+ release, it was
first necessary to determine whether adenosine altered
the ability of the contractile apparatus to develop force.
Blazev & Lamb (1999b) previously showed that 3 mm
adenosine had little effect on the steady-state properties
of the contractile apparatus compared to the control
level without adenosine (maximum Ca2+ -activated force
decreased < 2%, without altering either the Hill coefficient
or the pCa50 ). Using the rapid [Ca2+ ] procedure shown in
Fig. 3, it was found here that addition of 2 mm adenosine
to solutions with 1 mm ATP had no significant effect on
either the level of maximum Ca2+ -activated force or the
rate of force development (maximum force, 101.8 ± 1.6%;
RFD10–90 , 99.8 ± 7.4%, n = 13, with adenosine compared
to bracketing measurements without adenosine). This
indicates that any changes in twitch force occurring in
the presence of adenosine are not due to effects on the
contractile apparatus, and instead must be due to changes
in Ca2+ release. Twitch responses were elicited in the
presence of various ratios of ATP:adenosine. As seen in
the examples in Fig. 6, the presence of 2 mm adenosine
had little or no effect when the [ATP] was high (8 mm)
but caused progressively greater depression of the twitch
response as the [ATP] was decreased. The mean peak size
of the twitch response (as a percentage of the response in
8 mm ATP) for various ratios of ATP:adenosine (in mm)
was 95.5 ± 2.0% (n = 3) for the 8:2 ratio, 53.6 ± 5.6%
(n = 4) for the 2:2 ratio, 30.9 ± 3.5% (n = 17) for the 1:2
ratio, and 19.1 ± 2.9% (n = 7) for the 1:4 ratio. These data
are fully consistent with adenosine competitively interfering with ATP binding to its stimulatory site on the RyR,
thereby making the voltage sensor less effective at releasing
Ca2+ . The reduction in Ca2+ release in the presence of
adenosine was further confirmed using the TBQ-BAPTA
C The Physiological Society 2004
J Physiol 560.2
Effect of low [ATP] on E–C coupling
assay described above (with the initial Ca2+ content of the
SR increased ∼50% above the endogenous level in order to
make all the measurements in the sequence required before
the SR became depleted of Ca2+ ). When 4 mm adenosine
was present with 1 mm ATP the mean amount of Ca2+
released by an AP in the six fibres examined was only
131 ± 12 µm, whereas in the bracketing measurements in
1 mm ATP without adenosine in the same fibre it was
204 ± 10 µm (note the [BAPTA] had to be decreased to
120 µm in the solution with adenosine in order to observe
any force response at all upon AP stimulation). Thus,
decreasing the [ATP] from 8 to 1 mm reduced the amount
of Ca2+ release by ∼10%, and the presence of 4 mm
adenosine reduced it by approximately a further 36% (see
Fig. 7).
461
maximum Ca2+ -activated force increased (to 125 ± 1%
of that with 1 mm Mg2+ −8 mm ATP, n = 5) and the
rate of force development was slightly slowed (mean
RFD10–90 , 139 ± 17% of control, n = 5), just as seen
in 1 mm Mg2+ (see earlier; Table 2). Thus, maximum
Ca2+ -activated force was unchanged or potentiated in
3 mm Mg2+ , whereas tetanic force was markedly reduced
(Fig. 8), with the disparity being greater at 0.5 mm ATP.
This difference cannot be accounted for by the decrease
in Ca2+ sensitivity of the contractile apparatus occurring
in 3 mm Mg2+ , implying that Ca2+ release was inhibited.
This was confirmed by measuring the amount of Ca2+
release in response to single AP stimulation with the
TBQ-BAPTA assay procedure shown in Fig. 5. In the five
fibres examined, it was found that Ca2+ release in 3 mm
Mg2+ (with 8 mm ATP) was substantially reduced, being
only 62 ± 4% of that in 1 mm Mg2+ .
Effect of elevated free [Mg2 + ] on twitch
and tetanic force responses
Finally, we investigated the effect of increased cytoplasmic
[Mg2+ ] on twitch and tetanic responses, because if [ATP]
in a fibre decreases to ∼1 mm the free [Mg2+ ] must rise
very considerably (theoretically by up to ∼6 or 7 mm,
depending on its buffering). As seen in the example in
Fig. 8, when the free [Mg2+ ] was raised from the control
level of 1 mm to 3 mm, with the total [ATP] kept constant
at 8 mm, the peak of the twitch response was reduced about
6-fold and the tetanus reduced about 2-fold; in the 10 fibres
examined, the mean twitch peak and mean tetanus peak
in 3 mm Mg2+ were 15 ± 5% and 65 ± 5%, respectively,
of their values in 1 mm Mg2+ . When the [Mg2+ ] was
raised to 3 mm and the [ATP] reduced to 0.5 mm, the
twitch and tetanic responses were even further reduced
(P < 0.05; mean twitch peak, 3 ± 2%, n = 5; mean tetanus
peak, 40 ± 5%, n = 5, relative to response with 1 mm free
Mg2+ and 8 mm ATP). There was also a significant slowing
in the rate of force development of the tetanus in the
presence of 3 mm free Mg2+ −8 mm ATP and 3 mm free
Mg2+ −0.5 mm ATP compared to the bracketing control
responses (mean relative mean rise time from 10 to
90% of peak force (RT10–90 ), 117 ± 6% and 149 ± 13%,
respectively).
The reduction in tetanic force was due largely or
entirely to a reduction in Ca2+ release and not to effects
on the contractile apparatus. Maximum Ca2+ -activated
force was not altered in 3 mm Mg2+ (mean, 100 ± 2%;
n = 5) and there was no detectable change in the rate
of force development in response to a fast [Ca2+ ] step
(applied as in Fig. 3; mean RFD10–90 , 98 ± 4% of bracketing
control in 1 mm Mg2+ , n = 5). Previous measurements
of steady-state properties showed that 3 mm Mg2+ only
caused a moderate reduction in Ca2+ sensitivity (pCa50
decreased ∼0.21 pCa units, Blazev & Lamb, 1999a). When
the [ATP] was decreased to 0.5 mm at 3 mm Mg2+ ,
C The Physiological Society 2004
Figure 5. Assay of amount of Ca2 + released by AP-stimulation
at different [ATP]
Twitch and tetanic (50 Hz) force responses were first elicited under
control conditions (8 mM ATP) as in Figs 1 and 2. In the presence of
0.5 mM ATP, the twitch response (to a single pulse) reached only
∼40% of the control twitch, and a double pulse stimulus (D10; two
pulses 10 ms apart to elicit two successive APs) produced a peak force
that was ∼95% of that produced by a single pulse in 8 mM ATP. The
same fibre was then transferred to corresponding solutions with
50 µM TBQ and 160 µM BAPTA added, and stimulated by single pulses
at 15 s intervals. In TBQ-BAPTA the force response declined very slowly
(∼1 s) because all Ca2+ re-uptake by the SR was blocked. The peak of
the force response was indicative of the amount of Ca2+ released by
the stimulus; this amount could be calculated from the size of the
force response and the known amount and Ca2+ -binding properties
of the BAPTA and TnC (see Methods). The single AP stimuli elicited the
release of ∼206 µM Ca2+ on each of the first two trials in 8 mM ATP,
only ∼141 µM Ca2+ in the two trials in 0.5 mM ATP, and then
∼174–183 µM Ca2+ after returning to 8 mM ATP. Note that the
presence of BAPTA meant that force was detected only when the
Ca2+ release exceeded a certain threshold level, and also that the SR
became progressively depleted of Ca2+ during the successive stimuli in
TBQ-BAPTA. The 50 Hz tetanic response reached the maximum
Ca2+ -activated force level (latter not shown).
462
T. L. Dutka and G. D. Lamb
Discussion
This study documents the effects of low cytoplasmic [ATP]
on E–C coupling in rat fast-twitch fibres, examining both
the overall effects on twitch and tetanic responses, as
well as the effects on individual steps in the process,
in particular on AP-induced Ca2+ release and on force
development and relaxation by the contractile apparatus.
This was achieved using a mechanically skinned fibre preparation that retains functional E–C coupling, and in
which it was possible to manipulate cytoplasmic [ATP]
and other factors such as [Mg2+ ] independently and
thus characterize their individual effects on the various
processes. This enabled a parametric examination of the
effects of low [ATP] on E–C coupling steps that could not
be performed with intact fibres.
The force response, rather than an exogenous Ca2+
indicator, is used here to assay Ca2+ release because, even
though it is a comparatively slow sensor of Ca2+ release,
its dependence on cytoplasmic [Ca2+ ] can be accurately
determined in situ under all the relevant conditions.
Fluorescent and light absorbing Ca2+ indicators are the
only way to gain information about the time course of
rapid changes in free [Ca2+ ], but there can be considerable
difficulties with Ca2+ saturation and with calibrating the
indicators in situ to take account of their binding within
the fibre and consequent changes in their properties.
For example, there is currently more than a 10-fold
disparity between the estimates of peak tetanic free [Ca2+ ]
in intact fast-twitch fibres of the mouse obtained using
different Ca2+ indicators (∼20 µm with furaptra, Baylor
J Physiol 560.2
& Hollingworth, 2003; ∼1–2 µm with indo-1, Westerblad
et al. 1998; Allen et al. 1999). Furthermore, such indicators
show only the free [Ca2+ ], not the total amount of Ca2+
released, which is 10–100 times higher than the free [Ca2+ ]
and has to be calculated based on many assumptions
about the amount and properties of Ca2+ binding to
various known and unknown sites within the fibre. Here,
we instead assay the amount of Ca2+ release using our
recently developed TBQ-BAPTA method (Posterino &
Lamb, 2003) where most of the released Ca2+ binds to
a known amount of exogenously applied buffer (BAPTA)
and force can be used to determine the free [Ca2+ ] (and
hence the total amount of Ca2+ on the BAPTA) because
all re-uptake and Ca2+ binding by the SR Ca2+ pump is
blocked with TBQ.
Effect of low [ATP] on the rise and peak
of the tetanic response
It was found here that reducing the [ATP] to 0.5 mm or
1 mm, with no change in [Mg2+ ], caused a significant
reduction in peak tetanic force (by ∼10%; Fig. 2 and
Table 1). This was evidently not due to effects on the
contractile apparatus; maximum Ca2+ -activated force
actually increased ∼15% or more and Ca2+ sensitivity was
not changed. The reduction in tetanic force was instead
primarily due to a reduction in AP-induced Ca2+ release
(by ∼20% and 10% at 0.5 and 1 mm ATP, respectively;
Fig. 7). The rate of development of tetanic force was also
significantly slowed in 0.5 and 1 mm ATP (by ∼60% and
30%, respectively; Table 1). This was probably due not
Figure 6. Effect of adenosine on the
twitch response at various [ATP]
A–D, twitch responses were elicited by
single 2 ms pulses as in Fig. 1. Different
fibres shown in each panel. Control
responses (8 mM ATP) were always produced
before and after exposure to the different
ratios of ATP:adenosine (ADEN). Several
twitches were elicited under each condition
(not shown). A, ratio 4:1 (i.e. 8 mM
ATP and 2 mM ADEN). B, ratio 1:1
(2 mM ATP and 2 mM ADEN). C, ratio
1:2 (1 mM ATP and 2 mM ADEN). D, ratio 1:4
(1 mM ATP and 4 mM ADEN). Clearly, as the
ratio ATP:ADEN was decreased so too was
the relative peak of the twitch response.
C The Physiological Society 2004
J Physiol 560.2
Effect of low [ATP] on E–C coupling
only to the reduced Ca2+ release, but also to slowing of
the rate of force development by the contractile apparatus,
which was observed in the [Ca2+ ] step experiments to
decrease by ∼35% at such [ATP] (Table 2, RFD10–90 ).
This is consistent with previous findings showing that the
velocity of shortening in chemically skinned mammalian
fast-twitch fibres dropped by ∼25% when the [ATP] was
decreased from 5 mm to 0.5 mm (Cooke & Bialek, 1979).
When the [ATP] was decreased even further, to 100 µm,
tetanic force was reduced very substantially (to ∼28% of
that in 8 mm ATP). This was probably caused not only
by a further reduction in AP-induced Ca2+ , but also by
the marked slowing of force development by the contractile apparatus that occurs at such [ATP], as seen in Fig. 3
and Table 2. The latter is in accord with the substantial
decrease in the velocity of shortening (> 3-fold) observed
at 100 µm ATP (Cooke & Bialek, 1979). Thus, muscle
power production drops precipitously if the [ATP] falls
to such levels.
Effect of low [ATP] on twitch responses
The force response to single APs, while not of direct
physiological relevance, was also examined because it
enabled quantitative comparison of the Ca2+ release and
the force response to a rapid, submaximal stimulus. The
twitch response was attenuated to a greater degree by low
[ATP] than was the tetanic response (Table 1); this is largely
a consequence of the fact that the amount of Ca2+ released
by a single AP under control conditions was sufficient
to only partially activate, rather than fully activate, the
contractile apparatus, and hence any change in the amount
Figure 7. Relative amount of Ca2 + released by AP stimulation in
different conditions
The bars show the relative amount of Ca2+ released by a single AP
(open bars) or a pair of APs (hatched bar) in the indicated conditions,
expressed as a percentage of that released by a single AP in the
control conditions with 8 mM total ATP and 1 mM free Mg2+ . Data
obtained by measurements in TBQ-BAPTA as shown in Fig. 5.
C The Physiological Society 2004
463
of Ca2+ release had a more severe effect on the peak force
reached. It was found that the twitch response to a single
AP in 0.5 mm ATP decreased to ∼50% of that in 8 mm ATP
(e.g. Figs 1 and 5, and Table 1), even though the amount of
Ca2+ released was only reduced by ∼20% (Fig. 7). It was
established that the reduced force response was indeed
due to the reduction in Ca2+ release and not to changes
in the response of the contractile apparatus, because
stimulating the fibre with two APs in close succession in
0.5 mm ATP elicited approximately the same total Ca2+
release as a single AP in 8 mm ATP (Fig. 7) and also
approximately the same peak force (Fig. 5). Thus, the
data obtained with single AP stimulation in 0.5 mm ATP
demonstrated that a relatively small reduction in Ca2+
release causes a much larger reduction in twitch force.
This could result from a relatively large proportion of the
Ca2+ released by a single AP (perhaps ∼50% or ∼110 µm)
rapidly binding to sites not directly involved in force
generation. Possibly many of these sites are on the SR
Ca2+ pumps, because such binding appears to be lost when
these sites are occluded with TBQ (Posterino & Lamb,
2003). It is also apparent that these Ca2+ -binding sites are
close to fully saturated by the Ca2+ released by a single AP,
because most if not all of the additional Ca2+ released by
the second AP in a pair (∼25% more) appears to contribute
directly to force development (Fig. 5). Thus, the first AP in
a train elicits considerably more Ca2+ release (∼4-fold)
than the subsequent APs (Hollingworth et al. 1996;
Posterino & Lamb, 2003) and this seems well designed
Figure 8. Effect of 3 mm Mg2 + on twitch and tetanic responses
at high and low [ATP]
Twitch and tetanic force responses recorded in the presence of 3 mM
Mg2+ and 8 mM ATP or 0.5 mM ATP, with bracketing responses under
control conditions (8 mM ATP and 1 mM Mg2+ ). Raising the [Mg2+ ] to
3 mM at constant 8 mM ATP reduced the twitch response by ∼6-fold
and slowed and reduced the tetanic response by ∼2-fold. There was a
greater reduction in both the twitch and tetanic response when the
rise in [Mg2+ ] was accompanied by a decrease in [ATP] from 8 mM to
0.5 mM (see text for mean data in five fibres). Twitches elicited by
single stimuli and tetani by 50 Hz stimulation (respectively indicated by
ticks and rectangles under traces).
464
T. L. Dutka and G. D. Lamb
to rapidly fill the non-force generating Ca2+ -binding sites
in the fibre, hence ensuring that there is adequate Ca2+
available for activating force production. In intact fibres
some of this initial Ca2+ released could also bind to any
free parvalbumin (i.e. that not occupied by Mg2+ ), but this
would not have occurred in the skinned fibres here, because
the parvalbumin was rapidly lost when the fibre was
transferred from oil to the aqueous solution (Stephenson
et al. 1999).
Effect of raised [Mg2 + ]
It was also found here that when the free [Mg2+ ] was
raised from 1 to 3 mm the tetanic force response was
reduced to ∼65% of its control level (e.g. Fig. 8). This is
similar to the ∼50% reduction in tetanic force occurring
in intact murine fast-twitch fibres when the free [Mg2+ ]
in the cytoplasm was raised to 3 mm by injection of Mg2+
(Westerblad & Allen, 1992b). That study did not ascertain
how much of the reduction in force was due to effects on the
contractile apparatus and how much to a reduction in
cytoplasmic [Ca2+ ], and even if the cytoplasmic [Ca2+ ]
had been measured it would not have been straightforward
to determine whether or to what degree the total Ca2+
release was altered. The results of the present study
show that AP-induced Ca2+ release is indeed substantially
reduced (by ∼40%) when the free [Mg2+ ] is raised to 3 mm
with the total [ATP] kept unchanged. The reduction in
twitch force (to ∼15%) was much larger than the reduction
in the amount of Ca2+ release, which is consistent with
the non-linear relationship between twitch force and Ca2+
release in 0.5 mm ATP discussed above.
When the [Mg2+ ] was raised to 3 mm and the [ATP]
lowered to 0.5 mm, tetanic force was reduced considerably
more (to ∼40% of that with 1 mm Mg2+ and 8 mm
ATP), despite the fact that maximum Ca2+ -activated
force was substantially increased and Ca2+ sensitivity was
unchanged. This indicates that Ca2+ release under these
conditions was substantially lower than in 3 mm Mg2+ , and
hence that low cytoplasmic [ATP] augments the inhibition
of Ca2+ release occurring in 3 mm Mg2+ . Thus, it is likely to
be the combined effect of both these factors (and possibly
also an associated build-up of ADP and AMP; Blazev &
Lamb, 1999a), not the rise in [Mg2+ ] alone, which causes
the decline in Ca2+ release underlying fatigue in fast-twitch
fibres (Westerblad & Allen, 1992b,c).
Voltage-sensor control of Ca2 + release
through the RyRs
By assaying the amount of Ca2+ released by APstimulation, this study provided clear evidence that the
normal physiological mechanism of releasing Ca2+ from
the SR is inhibited both by lowering [ATP] (to 0.5 mm)
and by raising free [Mg2+ ] (to 3 mm) (Figs 5 and 7).
J Physiol 560.2
Using skinned fibres it had been previously shown that
the amount of Ca2+ released by T-system depolarization
by ionic substitution was inhibited by raised [Mg2+ ],
but no significant change was found when lowering
[ATP] without raising [Mg2+ ] (Blazev & Lamb, 1999a).
Such experiments involved applying relatively slow and
prolonged depolarizations (∼2–3 s, see Introduction),
and consequently they would not have been able to
detect whether there were changes in the rate of Ca2+
release on a physiological timescale under the various
conditions. The present experiments show that even when
the whole array of voltage sensors are activated in a rapid,
co-ordinated fashion by an AP, their ability to open the
apposing RyRs is still modulated by the prevailing cytoplasmic conditions, in particular by the [ATP] and the free
[Mg2+ ]. It is interesting to note that raising the [Mg2+ ]
to 3 mm caused a quantitatively similar decrease in the
amount of Ca2+ released by a single AP (∼40%, Fig. 7)
as occurred with a 2–3 s maximal depolarization elicited
by ion substitution (∼40%; Blazev & Lamb, 1999a).
Given the great differences in speed and duration of the
two methods of stimulation, this similarity may seem
somewhat surprising. However, such a finding would be
expected if raised [Mg2+ ] reduces RyR open probability
(and consequently Ca2+ release) by a similar proportion
regardless of how rapidly or for how long the stimulation
occurs. Moreover, despite its much more rapid time course,
a single AP elicits the release of a substantial fraction
(∼25%) of the total Ca2+ present in the SR (Posterino
& Lamb, 2003), and such substantial release might be
expected to show broadly similar modulation by cytoplasmic factors as occurs when releasing almost all of the
SR Ca2+ by depolarization by ion substitution.
The inhibitory action of raised [Mg2+ ] on AP-induced
Ca2+ release is almost certainly due to increased Mg2+
occupation of both the Ca2+ -activation site and the
low-affinity Ca2+ –Mg2+ inhibitory site on the RyR (Laver
et al. 1997), leading to reduced activation of the RyR by the
voltage sensors (Lamb & Stephenson, 1991, 1994; Lamb,
2002). Similarly, the inhibitory action of low [ATP] is
almost certainly mediated by actions on the RyR. Skeletal
muscle RyRs in synthetic bilayers are activated by ATP
independently of Ca2+ binding to the Ca2+ -activation site,
with half maximal activation at ∼0.36 mm ATP and a
Hill coefficient of ∼1.8 (Laver et al. 2001). The present
results showing a 20% decrease in AP-induced Ca2+ in
0.5 mm ATP is quite consistent with this if ATP must
be bound to its cytoplasmic regulatory site on the RyR
in order for the RyR to be properly activated by the
voltage sensor (dihydropyridine receptor) molecules in the
T-system during an AP. This explanation is also strongly
supported by the effects of cytoplasmic adenosine, which
was found to reduce AP-induced Ca2+ release in a manner
fully consistent with its known action as a weak competitive
agonist for the ATP regulatory site on the RyR (Laver et al.
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J Physiol 560.2
Effect of low [ATP] on E–C coupling
2001). Adenosine had little or no effect on AP-induced
Ca2+ release in the presence of high [ATP] (e.g. Fig. 6A)
but caused progressively more inhibition as the ratio of
adenosine:ATP was increased. It is interesting that the
degree of inhibition of AP-induced Ca2+ release occurring
both (a) when lowering [ATP] alone (20% inhibition
in 0.5 mm ATP), and (b) when adding adenosine (36%
relative inhibition with 4 mm adenosine at 1 mm ATP),
is only about half of that observed with isolated RyRs in
bilayers (Laver et al. 2001). A difference of this magnitude
is perhaps not surprising given the number of differences
in the two types of experimental assays. Nonetheless, it may
well be that this difference mainly reflects the fact that a
‘crystalline’ array of RyRs, such as that in a muscle fibre,
is more easily activated than a single isolated RyR, owing
to co-operative interactions between adjacent RyRs in the
array.
Effect of low [ATP] on force relaxation
A further, very prominent effect of low [ATP] was the
slowing of relaxation, particularly for the tetani (Fig. 2
and Table 1). This effect, which was quite large even at
2 mm ATP, was not due to any change in the steady-state
Ca2+ sensitivity of the contractile apparatus. When rapidly
decreasing the [Ca2+ ] in Triton X-100-treated fibres,
force was observed to decline relatively quickly even
in 100 µm ATP (FT90–10 , 250 ms) (e.g. Fig. 4), provided
that exogenous CPK had been added to the bath to
compensate for the loss of endogenous CPK caused by
the Triton X-100-treatment (Table 3). However, when
exogenous CPK was not added, the contractile apparatus
displayed substantial slowing of relaxation when the
bathing solution contained 0.5 mm ATP, and extreme
slowing of relaxation with 100 µm ATP (FT90–10 , 3000 ms;
Table 3). This indicates that if the cytoplasmic [ATP] is
initially ∼0.5 mm and poorly clamped by the creatine
kinase reaction, as may occur in a severely exhausted fibre
with almost no PCr, local depletion of ATP (and build-up
of ADP) occurring near the contractile apparatus during
activation is great enough to substantially modify the force
response. However, such extreme effects at very low [ATP]
do not readily account for the marked slowing of the
relaxation of the tetanic response observed in 2 mm ATP
when the [ATP] was presumably kept relatively constant
by the creatine kinase reaction with 16 mm PCr present.
(Note that these fibres were not treated with Triton X-100
and retained endogenous CPK (Stephenson et al. 1999)
and that there was no effect of adding exogenous CPK;
Table 3). One possibility is that the slowing of relaxation
is due to effects of low [ATP] on the SR Ca2+ pump.
In 8 mm ATP with CPK present, the tetanic response
declined considerably more slowly than did the contractile apparatus when decreasing [Ca2+ ] directly (Table 3
subset 1: FT90–10 , 421 ms and 215 ms, respectively). This
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465
difference presumably primarily reflects the additional
time taken for the SR Ca2+ pump to resequester the Ca2+
in the cytoplasm at the end of the tetanus. When the [ATP]
was decreased to 0.5 mm, the rate of relaxation measured
in the contractile apparatus experiments only increased
to a small degree (FT90–10 , 227 ms), whereas that of the
tetanus increased greatly (FT90–10 , 990 ms). This seems to
indicate that Ca2+ uptake by the SR Ca2+ pump was substantially slowed at 0.5 mm ATP, and this limited the rate of
relaxation of the tetanus in the fibres here. This is consistent
with studies of pump function showing that the maximum
rate of Ca2+ uptake is modulated by ATP binding at
a regulatory site with an apparent affinity of ∼1 mm
(Dupont et al. 1985). This explanation of the slow decline
of the tetanus in low [ATP] needs to be tested in future
experiments by direct measurement of the time course
of decline of cytoplasmic [Ca2+ ] with a Ca2+ indicator.
Nevertheless, irrespective of its cause, the slowing of tetanic
relaxation seen here in skinned fibres when the [ATP] was
presumably kept close to 2 mm suggests that this effect is
probably of some importance in fatigued muscle fibres.
It should be borne in mind though that the slowing of
the tetanic relaxation observed here at low [ATP] may
well not be observed as such in an intact fibre, because
when an intact fibre has been stimulated sufficiently to
deplete the [ATP] to a low level, other factors, particularly
the raised concentration of inorganic phosphate and
free Mg2+ , will also modify the overall response of the
contractile apparatus and Ca2+ movements. Furthermore,
the presence of parvalbumin in intact fibres may help
mitigate the effects of a decline in the rate of Ca2+ uptake
by the SR.
Muscle fatigue and the possible protective role
of low [ATP]
Karatzaferi et al. (2001) measured PCr and ATP in various
fibre types from the vastus lateralis muscle of human
subjects immediately after a 25 s maximal cycling bout
in which power output decreased to an approximately
steady level of ∼50% of the initial level. In most fibres
containing the fastest myosin heavy chain isoform (type
IIX), the [ATP] dropped to between ∼0.7 and 1.7 mm.
As the [ATP] presumably recovered to some degree while
obtaining the biopsy (it recovered to ∼50% of control
within 90 s), it is likely that the [ATP] was even lower at
the end of exercise than that measured. Moreover, these
values are the average levels in the cytoplasm, so given
that the ATP was poorly buffered by the low PCr levels,
the [ATP] in the triad junction (where there are many
ATPases), and in the vicinity of the myosin heads, may
well have dropped to 0.5 mm or below, with the free [Mg2+ ]
increasing accordingly (i.e. to > 3 mm; see Westerblad &
Allen, 1992b,c). The fact that the exercise regime rapidly
depleted > 90% of the high energy phosphates, clearly
466
T. L. Dutka and G. D. Lamb
shows that the average rate of ATP utilization over the
exercise period greatly exceeded its rate of resynthesis.
As the rate of recovery of ATP and PCr during the
post-exercise period was very much lower than the average
rate of utilization during the exercise, it has to be concluded
that the rate of ATP utilization at the end of the exercise
period was very much lower than the average rate during
the exercise. If this were not the case and the exercise had
continued even slightly longer, all cellular ATP would have
been completely used up, and consequently (a) rigor force
would have developed and (b) the SR Ca2+ pump would
not have operated and Ca2+ would have stayed in the
cytoplasm causing large-scale Ca2+ -dependent damage
(Lamb et al. 1995; Westerblad et al. 1998). Evidently this
does not readily happen in normal exercise. Thus, the rate
of ATP utilization must have been very low near the end
of the exercise period and this implies a low power output.
Of most importance, it also implies that there must have
been very little release of Ca2+ from the SR, because ATP is
consumed at a high rate during Ca2+ re-uptake into the SR
(Szentesi et al. 2001). Even if myosin ATPase activity were
to drop greatly, ATP could only be preserved by decreasing
the rate of Ca2+ re-uptake, which means reducing Ca2+
release. Finally, as the [ATP] drops to very low levels in the
type IIX fibres, but stops before actually reaching zero, it
seems most likely that it is the low [ATP] itself that, directly
or indirectly, causes the reduction in Ca2+ release from the
SR.
The above considerations suggest that in some physiological circumstances (e.g. in strenuous exercise in the
fastest twitch fibres) muscle fatigue may be caused,
at least in part, by a reduction in cytoplasmic [ATP]
and the concomitant rise in free [Mg2+ ] (possibly
also compounded by local increase in ATP metabolites,
ADP and AMP); this also agrees with conclusions
of experiments in intact fibres from mouse and frog
(Westerblad & Allen, 1992b,c). The present results show
that such changes cause a large reduction in AP-induced
Ca2+ release from the SR and force production, apparently
by direct inhibitory effects of these changes on the
responsiveness of the RyRs to activation by the voltage
sensors. However, we note that the results here were
obtained at room temperature, as were those concerning
fatigue in mouse and frog fibres in vitro (Westerblad
& Allen, 1992b,c), and we have not established that
the same phenomenon occurs at mammalian body
temperature, though we do expect this to be the case. In the
skinned fibres here, reducing [ATP] to 0.5 mm inhibited
Ca2+ release without any evident change in membrane
excitability. However in some circumstances, low [ATP]
may cause an additional reduction in Ca2+ release by also
interfering with membrane excitability, as Duty & Allen
(1995) found that in fatigued murine fast-twitch fibres
addition of glybenclamide produced some degree of
recovery of cytoplasmic [Ca2+ ] in three of the six fibres
J Physiol 560.2
examined. Finally, it is important to note that other
factors besides low [ATP] and raised [Mg2+ ] are likely
to play a role in causing muscle fatigue in various
circumstances. In particular, Pi released by PCr breakdown appears to precipitate with Ca2+ in the SR and
thereby reduce Ca2+ release (Fryer et al. 1995; Allen
& Westerblad, 2001). In view of this, it seems likely
that this latter effect also contributes to the overall
reduction in Ca2+ release that evidently occurs when
fast-twitch fibres are stimulated sufficiently to deplete PCr
and ATP levels, though as discussed above, it seems that
it is some mechanism closely linked to cytoplasmic [ATP]
itself, not [Pi ], that must ultimately terminate Ca2+ release
when cellular ATP is nearly completely exhausted.
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Acknowledgements
We are grateful to Professor George Stephenson for helpful
discussions and to the National Health and Medical Research
Council of Australia for financial support (grant no. 280623).
C The Physiological Society 2004