Biology Department, University of Pennsylvania, Philadelphia, Pennsylvania 19104; and Marine Biological Laboratory, Woods Hole, Massachusetts 02543
Submitted 15 January 2003 ; accepted in final form 22 May 2003
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ABSTRACT |
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muscle energetics; skinned muscle fibers; sarcoplasmic reticulum calcium ion pumps; cross bridges
Up to now, there was not an analogous specific and potent cross-bridge
blocker that could be used to knock out cross-bridge ATP utilization so that
ATP utilization by SR Ca2+ pumps could be directly measured.
2,3-Butanedione monoximine (BDM) has been used to knock out cross-bridge ATP
utilization, but its effect is incomplete (i.e., at 40 mM, BDM knocks out only
80% of the cross-bridge ATP utilization)
(22). Thus the rate of ATP
utilization of the SR Ca2+ pumps has generally been measured
indirectly as the difference between total ATP utilization and cross-bridge
ATP utilization (measured in the presence of Ca2+ pump blockers)
(20). However, because this
measurement can represent a relatively small difference between two large
numbers, it can be prone to error.
In the special case of the toadfish swimbladder muscle, the far right shift of the force-Ca2+ concentration ([Ca2+]) relationship has permitted direct measurement of ATP utilization by the SR Ca2+ pumps up to the [Ca2+] values at which the cross bridges start generating force and utilizing ATP. Consequently, this measurement could be performed only over a limited range of [Ca2+] values (i.e., up to pCa 5.8) before the confounding effects of cross-bridge ATP utilization were encountered (16).
Recently, by use of combinatorial chemistry and high-throughput screening, Cheung and colleagues (3) found that N-benzyl-p-toluene sulfonamide (BTS) blocked ATPase of myosin II in biochemical assays. In addition, it was found that at micromolar concentrations, BTS markedly reduced force generation in fast-twitch fibers while having little effect on the Ca2+ transient (3). It seemed likely that BTS could be used as a specific cross-bridge blocker that would permit the direct measurement of SR Ca2+ pumps in muscle fibers over a broad range of [Ca2+].
Here we show that BTS can knock out cross-bridge ATP utilization as well as
force at low concentrations (25 µM). Furthermore, by utilizing the
special properties of the swimbladder muscle and working below the threshold
[Ca2+] for cross-bridge activation, we show that 25 µM BTS has
no effect on SR Ca2+ pump ATP utilization rate. After establishing
the efficacy of BTS as a specific cross-bridge blocker, we used it to make
some of the first direct measurements of SR Ca2+ pump ATP
utilization in skinned fibers over a full physiological range of
[Ca2+] (9). Our data
show that SR Ca2+ pumping has a low apparent level of cooperativity
[i.e., the Hill coefficient (nH) is 1.49] compared with
that of cross-bridge force generation (nH =
5 in
swimbladder). Finally, preliminary results show that BTS not only allows one
to measure the rate of ATP utilization of intact SR in saponin-skinned fibers
but can block cross-bridge function in intact fibers as well. Hence, we
surmise that BTS will be a valuable tool for studying integrated SR function
and Ca2+ cycling in muscle during normal motor behavior.
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METHODS |
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Toadfish were kept at 15°Cin flow-through seawater tanks at the Marine
Biological Labs (MBL) or in filtered seawater at the University of
Pennsylvania and fed ad libitum during their captivity. They were sedated in
ice-cooled water until unresponsive and then killed by cervical sectioning and
double-pithing according to guidelines set out by the Institutional Animal Use
and Care Committees of the University of Pennsylvania and the MBL. Swimbladder
muscle was isolated from the toadfish and dissected as described previously
(19). The swimbladder was
quickly removed and placed in chilled Ringer solution (composition in mM: 132
NaCl, 2.6 KCl, 1 MgCl2, 2.7 CaCl2, 10 imidazole, 10
sodium pyruvate, pH 7.7 at 15°C), and bundles of this pure fiber type were
dissected out and checked for strong twitches by electrical stimulation.
Responsive bundles of 100 fibers were then depolarized in a
high-potassium solution (composition in mM: 7.8 MgCl2 ·
6H2O, 50 K2EGTA, 1 KH2PO4, 6.2
sodium ATP, 58.2 TES, pH 7.1 at 15°C). Fibers were dissected down to small
bundles of two to four fibers (fiber diameter
40-50 µm) and
"skinned" with 50 µg/ml saponin (20 min at 4°C), which
permeabilizes the cell membrane
(10,
17,
20) but does not affect the
Ca2+ pumps or SR membrane
(10). The fiber bundle was
secured with foil clips between a force transducer (400 series, Aurora
Scientific) and a fixed hook. Sarcomere length, determined by microscopy
(18), was set at 2.2-2.3
µm.
ATP Utilization Measurements
ATP utilization was measured with a fluorescent coupled assay (20) in a temperature-controlled (15 ± 0.1°C), vigorously stirred 5.5-µl chamber (16, 21). The Ca2+-EGTA solutions were the same as those used previously (16). To block SR Ca2+ pumping a cocktail of 20 µM TBQ and 20 µM CPA was used (as in Ref. 16). Stock solutions of TBQ (7.5 mM), CPA (7.5 mM), and BTS (20 mM) were made in DMSO. We carried out preliminary experiments to confirm that DMSO alone, in the final concentrations used in this study (0.13% for BTS alone and 0.4% with BTS, TBQ, and CPA) had no effect on the rate of either cross-bridge or SR Ca2+ pump ATP utilization. No effect was observed even up to DMSO concentrations of 1%.
All solutions contained 5 mM caffeine to prevent buildup of Ca2+ in the SR and consequent back-inhibition of SR Ca2+ pumping. Previous direct tests of the effect of caffeine concentration on fiber ATP utilization showed that it was constant over caffeine concentrations ranging from 2 to 20 mM (20).
We performed three sets of experiments. The first two sets of experiments were designed to test the effect of BTS on cross-bridge and SR Ca2+ pump ATP utilization, respectively. Having shown that BTS blocks cross-bridge ATP utilization without affecting SR Ca2+ pump ATP utilization, in the third set of experiments we measured the effect of [Ca2+] on SR Ca2+ pumping. These different protocols are described in RESULTS. At the end of each experiment, the fiber bundle was dried, the clips were removed, and the weight of the bundle was determined with a Cahn microbalance (model C-35). Its wet weight was calculated with a conversion factor of 8 (16), and intact fiber volume was calculated by assuming a fiber density of 1.05 kg/l. All data are reported as means ± SE. Statistical significance was set at the P = 0.05 level.
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RESULTS |
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In this set of experiments a dose-response relationship was determined for
ATP utilization and force generation of the cross bridges. To accomplish this
the SR Ca2+ pump ATP utilization was first completely blocked by a
combination of two Ca2+ pump inhibitors (20 µM TBQ and 20 µM
CPA) (16).
Figure 1 shows that 85% of
the control cross-bridge ATP utilization and force are lost with only 5 µM
BTS (note that even at 40 mM, BDM does not produce as large a decline; Ref.
22). The decrease in
cross-bridge ATP utilization and force appeared similar up to 10 µM. As BTS
concentration was increased above 20 µM, however, the force became
indistinguishable from zero while the ATP utilization rate fell to a constant
low level (
4%).
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It is unclear whether this small difference between cross-bridge force
generation and cross-bridge ATP utilization represents a differential response
to high BTS concentrations. The swimbladder muscle generates only about
one-tenth the stress of other skeletal muscles
(15). Because our technique
requires vigorous mixing and small bundle sizes for accurate measurement of
the swimbladder's rapid ATP utilization, it is difficult to resolve small
forces representing only a small percentage of a bundle's low maximum force.
Several additional experiments were performed with larger preparations to
improve force resolution. Although these preparations were too large for
accurate measurements of ATP utilization, we found that a force of 2% of
control remained at 25 µM BTS. This is close to the remaining percentage of
ATP utilization, so it appears that BTS has a similar effect on both
cross-bridge ATP utilization and force generation. These results are
consistent with, and thus do not differentiate between, two possible
mechanisms of action: BTS reduces the number of attached cross bridges without
affecting the kinetics and/or BTS slows the attachment rate constant
f (3).
BTS Does Not Affect SR Ca2+ Pump ATP Utilization
Previous studies have shown that in swimbladder muscle the ATP utilization of the Ca2+ pumps can be obtained without interference of the cross bridges up to a [Ca2+] of pCa 5.8 (16). Hence, to determine the effect of BTS on SR Ca2+ pumping, we measured ATP utilization at pCa 6.2, pCa 6, and pCa 5.8 without BTS (Fig. 2) and then remeasured the values after addition of 25 µM BTS. Each run was performed in duplicate, and the values were averaged. Figure 2 shows that although SR Ca2+ pump ATP utilization increased with increasing [Ca2+], there was no difference in the average values between control and 25 µM BTS. The average ratios of the control values to the BTS values from individual muscle bundles (n = 9) were 0.98 ± 0.05, 0.98 ± 0.04, and 0.99 ± 0.08 for pCa 6.2, pCa 6, and pCa 5.8, respectively. These values were found to be not significantly different from 1.0.
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SR Ca2+ Pumping as a Function of [Ca2+]
Having demonstrated that BTS does not affect SR Ca2+ pumping at
concentrations that effectively block cross-bridge ATP utilization, we were
able to measure SR Ca2+ pump ATP utilization over a wide range of
[Ca2+] values. In what we believe are among the first direct
measurements of their kind in a skinned fiber
(9), we found that the rate of
SR Ca2+ pump ATP utilization increased with increasing
[Ca2+] up to pCa 5.2 (Fig.
3). Furthermore, this relationship could be described by the Hill
equation with a relatively low nH (1.49 ± 0.04;
n = 9), and the 50% maximum pumping rate occurs at pCa 6.08 ±
0.015 (n = 9) or at a [Ca2+] of
0.83 µM. To prove
that this ATP utilization is a measure of the SR Ca2+ pumps rather
than cross bridges, 20 µM TBQ and 20 µM CPA were added at the end of
some of the experiments, and on average the ATP utilization rate fell to
3% (2.7 ± 0.8%; n = 6) of the original value obtained at
pCa 4.4. Interestingly, the rate of SR Ca2+ pump ATP utilization
appeared to decline slightly (12.3 ± 4.2%; n = 6) at very high
[Ca2+] (pCa 4.2-4.4) and was significantly smaller than at pCa 5.2
(P = 0.03; n = 6).
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DISCUSSION |
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For approximately a decade, several potent SR pump inhibitors (e.g., TBQ, CPA) have been used to permit direct measurements of the rate of cross-bridge ATP utilization. Here we demonstrate that the potent and specific cross-bridge inhibitor BTS can be used to knock out cross-bridge ATP utilization in saponin-skinned fibers (Fig. 1) without affecting SR Ca2+ pump ATP utilization (Fig. 2). This permits the direct measurement of SR Ca2+ pump ATP utilization as a function of [Ca2+] (Fig. 3). The impact and future uses of BTS are discussed below.
We found that the Ca2+ pumping rate vs. [Ca2+]
relationship was quite shallow (i.e., nH was small: 1.49)
compared with the value for cross bridges (nH = 5;
Ref. 19). Values of
nH ranging from 1 to 2 have been found previously for SR
Ca2+ pumping with different techniques, muscle preparations, and
temperatures. For instance, using skinned Xenopus fibers at
4.3°C, Stienen and colleagues
(20) found an
nH of 2. Without the benefit of a specific cross-bridge
blocker, rather than making direct measurements, Stienen and colleagues
(20) had to determine the rate
of SR Ca2+ pump ATP utilization by calculating the difference
between the total ATP utilization and the ATP utilization in fibers treated
with Triton (which removes the SR Ca2+ pump ATP utilization). In
addition, they did not measure the rate of ATP utilization between pCa 5.8 and
pCa 4.4 and hence may have missed the peak rate for SR Ca2+ pump
ATP utilization (see Fig. 3),
which in turn may have caused an overestimation of nH.
Using an entirely different technique, Kurebayashi and Ogawa
(9) extracted troponin C (TnC)
from skinned guinea pig fast-twitch muscle fibers, thereby preventing the
activation of cross bridges and hence cross-bridge ATP utilization. They found
that, although ATP utilization due to SR Ca2+ pumping had a
pCa50 (6.15 at 20°C) similar to what we observed, there was a
very low level of cooperativity (nH = 1). A
difference between this study and our study is that Kurebayashi and Ogawa used
an unstirred ATP utilization assay with relatively low enzyme activities, both
of which may have reduced the value obtained for maximum flux and thereby
affected the estimation of nH.
Because the swimbladder is one of only a few muscles in which the number of
SR Ca2+ pumps has been determined by ultrastructural morphometrics
(1), an accurate determination
of intrinsic Ca2+ pump function is possible. The maximum value we
obtained was 1 mmol ATP · s-1 · kg
muscle-1. From this value and the density of pumps
(1), the pump turnover rate for
the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)1 pumps in swim
bladder is 2.5 s-1, very similar to values found in
biochemical vesicle studies for SERCA1 pumps in mammals (Ref.
8; adjusted to 15°C
according to Ref. 7). This
similarity between the maximum pumping rates found for SERCA1 pumps in these
divergent animals reinforces the notion that SERCA1 is highly conserved and
thus the maximum Ca2+ pumping rate of fast muscles is increased
predominantly by adding more pumps, not by changing the kinetics of the pump
(16). This stands in contrast
to cross-bridge function, which is altered almost exclusively by changing
kinetics because the number of myosin heads in a sarcomere varies little.
One further interesting feature of the SR Ca2+ pump ATP utilization is that at [Ca2+] >pCa 5, we found that there was a small but consistent reduction in the rate of ATP utilization. Slower SR Ca2+ pump ATP utilization (and consequently Ca2+ uptake by the SR) at high [Ca2+] values has been observed previously (9), and this reduction generally has been considerably larger than that observed here. It has been proposed that the mechanism for the decline at very high [Ca2+] may be the increase in intra-SR [Ca2+] (6), which has been shown to decrease pump rate due to back-inhibition. Although this was not tested explicitly (as we did not have the means of determining the free [Ca2+] within the lumen of the SR of our saponin-skinned fibers), it appears unlikely under our experimental conditions. The 5 mM caffeine that we used was shown previously to keep the intra-SR [Ca2+] sufficiently low to prevent slowing of SR pumping (20). Furthermore, we did not observe any time-dependent slowing of SR ATP utilization, which would be expected if back-inhibition was occurring.
BTS as a Tool and Swimbladder as a Model for Studying Integrated SR Function and Ca2+ Handling During Normal Motor Behavior
BTS permits study of intact SR in skinned fibers. What advantages
are offered by measurements on skinned fibers over measurements on more
reduced SR preparations? Saponin skinning permeabilizes the sarcolemma but is
not thought to affect SR Ca2+ pumping or other SR function except
for increasing the open time of the Ca2+-release channels
(10). Thus these experiments,
along with Ca2+ accumulation studies on mechanically skinned fibers
(2,
11), provide some of the first
functional measurements of an intact SR
(9,
20). There are several
important differences between a structurally intact SR and reduced SR
preparations (i.e., vesicles). Hence, to obtain quantitative understanding of
Ca2+ cycling in normally functioning muscles, it is necessary to
integrate results from reduced preparations with those from intact SR
preparations. One important advantage in studying intact SR preparations (vs.
vesicles) is that the measurement of SR Ca2+ pump function will
better approximate the value in intact muscle. There are several reasons for
this. First, during homogenization and purification to produce vesicles, some
SR Ca2+ pumps may be damaged and hence the measured rate of SR
Ca2+ pumping (or ATP utilization) may be reduced compared with
intact SR. This could explain the lower values previously obtained in toadfish
swimbladder vesicle studies [0.5 mmol
Ca2+·s-1·kg muscle-1
(4); original measurements at
23°C corrected for 15°C by using a Q10 of 3] than we
observed here (
2 mmol Ca2+ · s-1 · kg
muscle-1; a stoichiometry of 2 Ca2+ per ATP is
assumed).
In addition, most vesicles are made from either "heavy" SR (taken from the terminal cisterna) or "light" SR (taken from the longitudinal segment sitting between the terminal cisternae at either end of the sarcomere) (12-14). The distributions of Ca2+-handling proteins differ in the two types of vesicles. In the heavy SR, the Ca2+-release channels (ryanodine receptors) and the intra-SR Ca2+ binding protein calsequestrin are in high concentration. In contrast, light SR is devoid of Ca2+-release channels and has little calsequestrin but contains a higher concentration of SR Ca2+ pumps (6). The differing ratios of SR Ca2+ pumps (by which Ca2+ enters SR lumen) to Ca2+-release channels and calsequestrin (by which Ca2+ either leaves the lumen or is taken out of solution by binding) confer differing physiological properties. Vesicles of light SR have only a slow leakage of Ca2+ that is inhibited by high external [Ca2+], whereas vesicles of heavy SR have an order of magnitude higher leakage rate that is stimulated by external [Ca2+] (12). Hence, one would anticipate finding differing function in vesicles made from different types of SR (e.g., more back-inhibition in the light SR). Thus, by using an intact SR (as in our saponin-skinned fibers), we study both types of SR at once and hence we are observing their combined function in a quantitatively (and spatially) appropriate manner.
It is interesting to note that in swimbladder, force generation does not
start until much higher [Ca2+] values than SR Ca2+
pumping (Fig. 3). However,
because the force-pCa relationship is much steeper than the pumping-pCa
relationship, both cross bridge and SR Ca2+ pump ATP utilization
reach their maximal value at about the same [Ca2+] (pCa 5;
Fig. 3). This lack of overlap
is unusual. In normal fast-twitch muscles, force is generated at lower
[Ca2+] values (i.e., the force-[Ca2+] relationship is
shifted leftward to higher affinity) and hence overlaps with SR
Ca2+ pumping more fully. However, it is necessary to emphasize that
it is not the unusual properties of the swimbladder muscle that make BTS work;
on the contrary, we have simply taken advantage of these properties to
demonstrate the efficacy of BTS. This evidence that BTS can be used to block
cross-bridge ATP utilization without affecting SR Ca2+ pump ATP
utilization (Figs. 1 and
2) also suggests that BTS will
be highly effective in permitting measurements of SR Ca2+ pumping
in more typical fast-twitch fibers in which the SR Ca2+ pumping
overlaps with cross-bridge force generation. Indeed, this is confirmed by our
own preliminary experiments (unpublished data) in fast-twitch mammalian
fibers.
Comparing Ca2+ pumping in skinned fibers
with in vivo Ca2+ cycling in swimbladder
muscle. Although swimbladder muscle was chosen because its unique
properties permitted us to test the effect of BTS on SR Ca2+ pump
ATP utilization without interference from the cross bridges, the toadfish
swimbladder muscle also represents a near-ideal system to explore the
principles of Ca2+ handling during normal motor behavior
(16). The data obtained in
this study partially explain an interesting discrepancy between our previous
measurement of the rate of SR Ca2+ pump ATP utilization and that
which might be expected given the tremendous speed at which swimbladder muscle
functions. If one assumes that during each twitch sufficient Ca2+
is released (and taken back up) to saturate and desaturate TnC [35 µM;
following Refs. 24 and
25 we assume that [TnC] is
equal to one-half the myosin heavy chain concentration, which is 67 µM
(Ref. 15)], then at 100 Hz,
the Ca2+ pumping rate would need to be
7 mmol ·
s-1 · kg muscle-1 (i.e., 2 Ca2+ per
TnC x 100 Hz) to keep up
(16). We previously reported
(16) that SR Ca2+
pumps use ATP at a rate of 0.45 mmol · s-1 · kg
muscle-1 at pCa 5.8, which is equivalent to a Ca2+
pumping rate of 0.9 mmol · s-1 · kg
muscle-1 (or nearly 8-fold less than that required). It was assumed
that the rate of pumping had reached a maximum level at pCa 5.8 because a
similar value was obtained at pCa 4.4, where the difference of total fiber ATP
utilization (cross bridge + SR Ca2+ pumps) and cross-bridge ATP
utilization was determined. Although this was a reasonable conclusion, we now
demonstrate that the SR Ca2+ pump ATP utilization continues to
increase up to pCa 5.2 and then declines again at pCa 4.4
(Fig. 3). This behavior may
have contributed to an underestimate of the maximum rate of SR Ca2+
pump ATP utilization of swimbladder muscle
(16).
With BTS we were able to study a full range of [Ca2+] values and
thus determine the maximum SR Ca2+ ATP utilization rate. The
maximum value was obtained at pCa 5.2 and was about twofold higher (1
mmol · s-1 · kg muscle-1) than reported
previously. Assuming a stoichiometry of 2 Ca2+ pumped per ATP
utilized, this is equivalent to a Ca2+ pumping rate of
2 mmol
· s-1 · kg muscle-1. This faster
Ca2+ pumping rate helps to explain part of the discrepancy between
the hypothesized Ca2+ cycling rate at 100 Hz and the relatively low
SR Ca2+ pumping rate. However, even after accounting for this
approximately twofold increase, there remains a three- to fourfold difference.
This is likely explained by either (or a combination) of two additional
mechanisms (16): 1)
some of the Ca2+ binds to parvalbumin during the 300-ms call and
then is pumped back at a slow rate during the long 5- to 10-s intercall
interval; and/or 2) the force oscillations of the swimbladder muscle
during calling involve only small changes in troponin occupancy and thus only
a small amount of Ca2+ (i.e., <<70 µmol/kg muscle) is
released and taken back up during each stimulus. The reduced Ca2+
release per twitch is regulated by SR Ca2+ channel inactivation
(5): a result of the high
frequency of muscle stimulation.
Future use of BTS in intact fibers will permit measurement of in vivo
Ca2+ cycling. Although fundamental to normal
Ca2+ handling, these proposed mechanisms cannot be tested without
having the ability to measure SR Ca2+ pump function during
contraction in intact fibers. Up to now, partitioning the ATP utilization rate
has been very difficult in intact muscle. Preliminary experiments, however,
show that BTS can knock out cross-bridge force generation (and thus
cross-bridge ATP utilization) nearly as effectively (reduces to <5%) in
large (150 mg) swimbladder bundles suitable for intact energetics
experiments as in skinned toadfish fibers. Hence, measuring the SR
Ca2+ pump ATP utilization during contraction in intact fibers, and
combining this with a stoichiometry of 2 Ca2+ pumped per ATP, would
enable one to determine the rate, time course, and overall magnitude of
Ca2+ release and reuptake during normal contractions. This may
prove to be the most important use of BTS, and as such, BTS will have a large
impact on the field of muscle energetics and on helping us to understand
Ca2+ cycling and the role of parvalbumin in living skeletal
muscle.
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ACKNOWLEDGMENTS |
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Present address of I. S. Young and C. L. Harwood: Dept. of Veterinary Preclinical Sciences, University of Liverpool, Liverpool L69 7ZJ, UK (E-mail: isyoung{at}liv.ac.uk, charwood{at}liv.ac.uk).
This work was supported by National Institute of Arthritis and Musculoskeletal and Skin Diseases Grants AR-38404 and AR-46125 (L. C. Rome), a Univ. of Pennsylvania Research Foundation grant (L. C. Rome and I. S. Young), a Wellcome Trust Short Term Travel Grant and a MBL Summer Fellowship (I. S. Young), and a Wellcome Trust Prize Traveling Fellowship (C. L. Harwood).
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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