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Department of Biochemistry and Biophysics and Cardiovascular Research Institute, University of California, San Francisco, California 94143
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
ACKNOWLEDGEMENTS
FOOTNOTES
REFERENCES
ABSTRACT 
Myburgh, Kathryn H., and Roger Cooke. Response of
compressed skinned skeletal muscle fibers to conditions that simulate fatigue. J. Appl. Physiol. 82(4):
1297-1304, 1997.
During fatigue, muscles become weaker, slower,
and more economical at producing tension. Studies of skinned muscle
fibers can explain some but not all of these effects, and, in
particular, they are less economical in conditions that simulate
fatigue. We investigated three factors that may contribute to the
different behavior of skinned fibers. 1) Skinned fibers have increased
myofilament lattice spacing, which is reversible by osmotic
compression. 2) A myosin subunit becomes phosphorylated during fatigue.
3) Inosine 5
-monophosphate (IMP) accumulates during fatigue. We tested the response of
phosphorylated and unphosphorylated single skinned fibers (isometric
tension, contraction velocity, and adenosinetriphosphatase activity) to changes in lattice spacing (0-5% dextran) and IMP (0-5 mM)
in the presence of altered concentrations of
Pi (3-25 mM),
H+ (pH 7-6.2), and ADP
(0-5 mM). The response of maximally activated skinned fibers to
the direct metabolites of ATP hydrolysis is not altered by osmotic
compression, phosphorylating myosin subunits, or increasing IMP
concentration. These factors, therefore, do not explain the discrepancy
between intact and skinned fibers during fatigue.
pH; phosphate; force; velocity; mechanics; inosine
5 INTRODUCTION PROLONGED PHYSICAL ACTIVITY results in an accumulation
of metabolites in muscle and in a number of changes in the mechanics and energetics of contraction (for review, see Ref. 17). For example,
during a lengthy tetanic contraction, tension, velocity and energy
expenditure all are inhibited, although not all to the same extent.
Energy expenditure is decreased more than tension so that the economy,
or energy cost, of tension production improves (4, 11, 12, 16, 18). The
tension economy of living fibers has been defined as the tension-time
integral divided by the ATP used, and here we define the tension
economy of skinned fibers as the ratio of isometric tension
(Po) divided by the
adenosinetriphosphatase (ATPase) activity of the fiber. Despite
considerable effort, the exact mechanisms by which these changes are
produced remain controversial. Previous work in skinned muscle fibers
has shown that increases in the concentrations of some metabolites,
e.g., phosphate (Pi) and
H+, can apparently account for
some of the decrease in force observed during fatigue and an increase
in H+ concentration inhibits
velocity (for review, see Ref. 26). However, these studies do not
explain a number of other observations.
For example, there is controversy regarding the role of pH during
fatigue, particularly in vivo (1, 2, 6, 27), and a mechanism for
improved economy during fatigue has yet to be found despite several
observations of this effect in intact fast skeletal muscle during
isometric contractions (4, 11, 12, 16) and isotonic tetani (13). This
effect has also been shown in rat diaphragm, which has both
fatigue-sensitive and fatigue-resistant fibers (8) and in human
quadriceps (15). The magnitude of increase in economy is between two-
and fourfold (11, 15). In contrast, the economy of contraction of
skinned fibers is decreased in the presence of either phosphate
(0.7-fold) or H+
(0.7-fold) or both (0.5-fold) (26, 10, 29). In living fibers the
contraction velocity decreases before tension; however, in skinned
fibers the conditions that simulate fatigue inhibit tension more than
velocity. These discrepancies between the responses of intact fibers to
fatigue and of skinned fibers to the increased metabolites of fatigue
suggest that some mechanisms that occur in vivo are lost in the skinned
fiber preparation.
One important difference between intact and skinned fibers is that the
diameter of muscle fibers increases when they are skinned, resulting in
increases in the lattice spacing between adjacent thick filaments in
relaxed fibers of up to 28% (19). However, the lattice spacing can be
returned to physiological spacing by osmotic compression (19), which
increases tension production by 4% but decreases ATPase activity by
26%, thus increasing the economy of contraction by ~40% (22, 35).
We hypothesize that the economy of compressed fibers may undergo
further changes in response to the conditions of fatigue so that their
contractile properties become more akin to that seen in vivo. This
hypothesis has been investigated, in part, in a study by Martyn and
Gordon (25), who showed that osmotic compression of skinned fibers did
not alter the effect of changing pH from 7 to 6. However, this has not
been done in the presence of other metabolites, such as
Pi, ADP, or inosine
5 Myosin P light chains of intact fibers in animal models (31) and in
human muscle (21) become phosphorylated during fatigue, and this occurs
at about the same time as economy increases (11). Phosphorylation is
associated with twitch potentiation (21, 31) but may not be associated
with increased economy of contraction (4). However, myosin
phosphorylation appears to have little effect in maximally activated
skinned fibers (20, 28). Because phosphorylation occurs during
fatigue, it is plausible that its effect may become evident
only in the presence of the metabolites of fatigue. Alternatively, its
effect may be enhanced by simultaneous compression to physiological
lattice spacing. The myosin head is sufficiently long that it more than
spans the interfilament distance at in vivo spacing, and there is some
evidence that an interaction does occur between the myosin
P light chain and the core of the thick filament (30).
Such an interaction could be influenced or eliminated if the
interfilament spacing were to become too great.
During fatigue in intact fibers, a number of additional metabolites
build up that have been given only scant attention in previous
experiments that simulate fatigue in skinned fibers. Among these
metabolites, IMP is a likely candidate to play a role in
modifying muscle function during fatigue (14, 33) because it is a
direct product of nucleotide degradation. Furthermore, in a recent
study of intact muscle in which high levels of IMP were maintained by
inhibiting its resynthesis to AMP, the muscles had an increased economy
of contraction (18).
We hypothesized that either myosin light chain phosphorylation or
increased IMP concentration, or both, might alter the response of fully
activated skinned fibers to the direct metabolites of ATP hydrolysis,
especially under conditions of simultaneous myofilament lattice
compression. In particular, we hypothesized that one or more of these
additional factors would lessen the decrease in force production
previously observed or enhance the decrease in the velocity of
contraction or ATPase activity so that the economy of tension
generation would improve. In contrast to expectation, we found that
none of these factors, either singly or in combination, produced
effects that could explain the apparent discrepancies in mechanics and
energetics of the actomyosin interaction between fatigued intact fibers
and skinned fibers tested under conditions simulating fatigue.
METHODS
-monophosphate; phosphorylation; lattice compression
-monophosphate (IMP), or a combination of several of these.
20°C until used. The extent of
phosphorylation of fibers from each preparation was monitored by
isoelectric focusing gel electrophoresis (IEF gel) before mechanical
testing. The IEF gel method has been previously described in detail
(Refs. 28, 31; see Fig. 1 for a
representative gel). Only fiber preparations that were 90-100% phosphorylated were used for mechanical testing. Control fibers were
between 0 and 10% phosphorylated.
Fig. 1.
Isoelectric focusing gel showing level of phosphorylation of fibers
from representative preparations. Lane
1, purified myosin control; lane
2, control fibers; lane
3, fibers phosphorylated by incubation in a rigor
solution with 50% glycerol and (in mM) 5 potassium fluoride, 5 Mg ATP,
and 20 Pi; lane
4, control fibers after activation. Only region of gel
containing myosin light chain 2 is shown. Bands are identified as
dephosphorylated (LC2) and phosphorylated (LC2~P). An additional
component of myofibril runs just above LC2~P (dashed line and
asterisk). Level of LC2~P appears to be <5% in
lane 2, >90% in
lane 3, and approximately <10% in lane 4.
[View Larger Version of this Image (26K GIF file)]
, Displacement of motor necessary
to maintain tension (P) at a particular fraction of isometric tension
(Po) plotted against time; 
,
fiber length (L) determined from
position of 1st diffraction line. Slope of least squares regression
line fit to data obtained from 10th to 40th ms was calculated and
represents velocity of contraction and could be determined from
displacement of motor
(Vmotor) and 1st diffraction line
(Vdiff). Fiber
was initially activated in relaxing solution and transferred to
activating solution containing activating buffer + 4 mM ATP, 3 mM
Pi at pH 7 with no dextran (A); same buffer with 5% dextran
(B); activating buffer with 4 mM
ATP, 25 mM Pi at pH 6.2 without
dextran (C); or same buffer with 5%
dextran (D). Isotonic tension during
40-ms interval was between 10 and 12%
Po; velocities in muscle
lengths/s and L were Vmotor = 1.26 lengths/s, Vdiff = 1.12 lengths/s, L = 3.4 mm
(A); Vmotor = 0.97 lengths/s, Vdiff = 1.13 lengths/s, L = 3.4 mm
(B); Vmotor = 0.73 lengths/s,
Vdiff = 0.81 lengths/s, L = 2.5 mm
(C); and
Vmotor = 0.66 lengths/s, Vdiff = 0.55 lengths/s, L = 2.5 mm (D). Resting sarcomere length of
each fiber was ~2.3 µm; sarcomere length decreased during 40-ms
clamp by 105, 100, 83, and 51 nm in A,
B, C,
and D, respectively.
Experimental conditions. The basic rigor buffer contained (in mM) 120 KAc, 5 MgCl2, 3 phosphate, 1 ethylene glycol-bis(
-aminoethyl
ether)-N,N,N,N-tetraacetic acid, and 20 3-(N-morpholino)propanesulfonic acid (pH 7) or
40 2-(N-morpholino)ethanesulfonic acid (pH 6.2). To this, 20 mM creatine phosphate, 4 mM ATP, and 1 mg/ml creatine
kinase were added to relax the fiber. In all conditions, the
temperature of the solutions was maintained at 10°C and fibers were
maximally activated with calcium chloride. Initially, several fibers
were titrated with increasing amounts of calcium to establish the
concentration of calcium required for full activation. Because all
fibers were fully activated by addition of 1.1 mM calcium (= 0.1 mM
free Ca2+), this quantity of
calcium was subsequently routinely added.
Compression of the fiber lattice can be achieved by bathing the fiber
in a solution containing a high-molecular-weight polymer, such as
dextran. These large molecules do not penetrate the myofibrillar space
but increase the osmolarity of the solution bathing the fiber. Water
and electrolytes are removed from the myofibrillar space, causing a
decrease in cross-sectional area. Normal lattice spacing is achieved by
the addition of between 3 and 5% Dextran T-500 (Sigma Chemical, St
Louis, MO) (19). There were, therefore, four buffers in which baseline
mechanical measurements were made, namely, pH 7, 3 mM
Pi with and without 5% dextran,
and pH 6.2, 3 mM Pi with and
without dextran. To these baseline buffers, the metabolites ADP,
Pi, or IMP were added. Ionic
strength was maintained at the same level as the basic rigor buffers by
varying KAc concentration. The experiments in which ADP concentration
was varied were carried out in the presence of 2.5 mM ATP and the
absence of creatine phosphate or creatine kinase because
the competition between ATP and ADP is difficult to measure in the
presence of an ATP regeneration system. For experiments in which ADP
was added, the rigor buffer contained 100 µM diadenosine
pentaphosphate, which inhibited the activity of adenylate kinase. The
effect of 5% dextran was determined in two ways: first, by using a
two-well system we alternated between wells containing solutions with
either 0 or 5% dextran, starting in the 0% dextran solution and
switching to the 5% dextran solution or starting in the 5% dextran
solution and washing out the dextran by rapid mixing in a 0% dextran
solution; second, by using a one-well system we determined whether
there was any difference in the effect of addition of metabolites of
fatigue in the presence or absence of dextran.
The effect of phosphorylation on mechanics of contraction was
determined by using two methods: first, by phosphorylating the fibers
during glycerination as described above and, second, by measuring
Po and
V10 in activated
control fibers and subsequently in the same bundle of 2-4 fibers
after incubation in a relaxing solution containing calcium-insensitive
myosin light chain kinase (MLCK). Aliquots of calcium-dependent MLCK
were clipped by 
-chymoptrypsin to a calcium-independent fragment
(32), which was used on the same day for phosphorylation of the myosin
P light chains. The mounted fiber bundles were incubated for 15 min at
10°C before the second set of mechanical measures were made. The
entire fiber or fiber bundle was used in an IEF gel to determine the
extent of phosphorylation. Samples were >80% phosphorylated by
incubation for 15 min. MLCK was a gift of Dr. James Stull.
To determine the effect of a combination of the above fatigue factors
on muscle mechanics, the following two conditions were compared in the
presence of dextran: 1) 4 mM ATP and
3 mM Pi, pH 7, and 2) (in mM) 1 ATP, 30 Pi, 1 ADP, and 4 IMP, pH
6.2. No ATP regeneration system was used for these two experiments. For a more complete picture of the effects of fatigue on force and velocity
of contraction, data were obtained from force clamps ranging between 5 and 40% of Po for construction of
a force-velocity curve.
Determination of myosin ATPase activity.
Bundles containing 3-4 skinned fibers were mounted between two
stainless steel pegs by wrapping the ends around the pegs and gluing
with acetone-diluted nail polish. The fibers and pegs were immersed in
activating and relaxing solutions of different compositions in wells
containing 25 µl of solution at 10°C as described previously (34). In short, after incubation in rigor solution, the fiber bundle
was transferred to a well with rigor solution and 4 mM ATP and 1.1 mM
CaCl2 for a specified time period
of between 1 and 6 min. The rigor solutions used to
determine the ATPase activity were similar to those used during
tensiometer experiments, except that no additional
Pi was added. Once the fiber had
been transferred to a new well, the entire 25 µl were removed from
the completed well and quenched in 1 ml rigor containing 3% perchloric
acid. Pi production was measured
by using a modified Malachite Green assay (34). The results for the
amounts of Pi produced at
different time points (0-6 min) were plotted against time, and the
slope of linear regression fits was taken as the rate of
Pi production. Protein content was
determined for the suspended portion of the fiber bundles by the
standard Bradford assay. From scans of protein gels performed
previously in our laboratory (9), myosin content was assumed to be 45%
of the total fiber protein content. Control experiments were performed
in which similar bundles of fibers were mounted to the pegs and ATPase
rate was determined. The suspended portion of the bundle was removed
for determination of protein content, and the remaining fiber, glued to
the pegs, was tested for ATPase activity. The
Pi production of the remaining
portion of the fibers in these control experiments was 7 ± 4%
(n = 8) of the overall ATPase
activity, indicating that the suspended portion of the fiber bundle
utilized by far the greatest proportion of ATP. The economy of force
production (force produced/ATP hydrolyzed) was calculated as
Po/ATPase
activity from the average values of Po expressed in newtons corrected
for cross-sectional area (N/mm2)
and the average ATPase activity expressed per myosin head per second.
Cross-sectional area was calculated from measurements of fiber width
made at 3-6 positions along the fiber while it was bathed in a
rigor solution. If all width measurements were similar, the fiber was
assumed to be uniformly circular. If not, the fiber was assumed to be
elliptical, and the squares of the different measurements of width were
averaged to give an average area.
Data reduction.
A two-tailed, unpaired Student's
t-test was performed to determine
whether there were differences in the response of fibers to various
conditions.
RESULTS
Lattice compression does not alter the effects of the products of ATP
hydrolysis. The effects of the direct products of ATP hydrolysis on
Po and
V10 were measured
by using two types of experimental protocols. In one, the fibers were
activated in a particular set of conditions and switched between two
solutions, one of which included dextran. In the second, the responses
in two conditions were measured, keeping dextran constant at either 0 or 5%. The effect of dextran on the width of single fibers mounted and
bathed in a relaxing solution was measured. Changing a relaxing bathing solution to a similar solution containing 5% dextran decreased fiber
width by 7.3 ± 1.4% (n = 11;
P < 0.001). Figure
3 shows the effect of increasing
Pi from 3 to 25 mM, at either pH 7 or 6.2, on fiber tension and velocity. Compression of the fiber
increases tension at pH 7. However, compression has much less effect at pH 6.2. In the absence of dextran, the effect of
Pi was similar to previous
results, and compression did not alter this effect.
), pH 7 + 5% dextran (
), pH 6.2 (), and pH 6.2 + 5% dextran (
)
(n = 3-5 fibers at each data
point). Only increase in tension at pH 7 with addition of dextran is
significant (P < 0.05).
To test whether lattice compression alters the competition between ATP and ADP in the fiber, the effect of increasing the ADP concentration was measured. In the absence of dextran, the addition of 5 mM ADP increased Po by 15 ± 2% and decreased V10 by 27 ± 5% (n = 3). The presence of 5% dextran did not change the mechanical response of skinned psoas fibers to increases in ADP concentrations, with Po increased by 14 ± 2% and V10 decreased by 20 ± 7% (n = 9, respectively; changes not significant; P > 0.1). Although the concentrations of ADP utilized in these experiments were greater than those found to occur in vivo, it is evident that the competition between ATP and ADP [competition that depends on both the dissociation constant (Km) for ATP and the inhibition constant (Ki) for ADP] was not changed by fiber compression. The values of Km and Ki measured previously show that the buildup of ADP in vivo would be insufficient to alter muscle properties appreciably (26). Although the Ki for inhibition of fiber velocity or ATPase activity is 200 µM, indicating tight binding of ADP, the binding of ATP is far stronger, Km 15-150 µM, so that physiological levels of ADP do not compete effectively with ATP, even in severe fatigue (26).
ATPase activity. Fiber ATPase activity was measured in a separate series of experiments in the absence of added Pi at pH 7 or 6.2. Figure 4 shows the effects of 5% dextran on Po, ATPase activity, and economy (Po/ATPase activity). Dextran increased Po and decreased ATPase activity at both pH 7 and 6.2. The effect of compression on the ATPase activity was more dramatic at pH 7 than at 6.2, leading to less improvement in the economy of force production at the lower pH (Fig. 4).
Phosphorylated fibers. We addressed the question of whether light chain phosphorylation affected fiber properties in conjunction with a change in a metabolite (increasing Pi and decreasing pH) or with fiber compression, or both. As shown in Fig. 4, phosphorylation did not significantly alter the tension, ATPase activity, or economy, or the changes in these on addition of dextran. The effect of compression on the ATPase activities of phosphorylated fibers was also determined by phosphorylating the fibers via incubation in MLCK, as described in METHODS. Phosphorylation did not alter ATPase activity, as determined by measuring Pi production in an activating solution before and after incubation with MLCK for 15 min. The ATPase activity of control fibers incubated for 15 min in a relaxing solution that did not contain MLCK decreased slightly over that time period (
6.3 ± 11.9%, n = 3). The
change in ATPase activity from pre- to postincubation with MLCK was 1.8 ± 5.5% at pH 7, 0% dextran (n = 6), and 6.2 ± 4.5% (n = 7) in the
presence of 5% dextran. At pH 6.2, incubation with MLCK decreased
ATPase activity by 12.5 ± 4.9%,
(n = 2) without dextran. With 5%
dextran, ATPase activity increased by 4.4 ± 8.2%
(n = 7). The above data show that
phosphorylation of skinned fibers by incubation with a kinase does not
greatly alter ATPase activity in either the absence or the presence of dextran (P > 0.1).
We also examined the effects of increasing
Pi in phosphorylated fibers.
Increasing Pi concentrations from
3 to 25 mM in phosphorylated fibers resulted in a decrease in the
tension of between 29 and 37%. There was essentially no difference in
the response to Pi at pH 7 or 6.2, with or without dextran, and the mean decrease for these conditions was
32 ± 3%, which is similar to that observed in unphosphorylated
fibers. Increased Pi generated
small increases in velocity at pH 7 (9 ± 4 and 21 ± 6%,
without and with dextran, respectively) and small decreases at pH 6.2 (12 ± 3 and 6 ± 6%, without and with dextran,
respectively). These changes were not significantly different from
those in unphosphorylated fibers (see Fig. 3). We conclude that
phosphorylation has little effect on the mechanics of fully activated
fibers and that this result is not affected by increased
Pi, decreased pH, or fiber
compression.
Effect of IMP on fiber mechanics in control and phosphorylated
fibers.
The effect of IMP on fiber mechanics was determined in control fibers
under four conditions (see Fig. 5). IMP did
not alter force production or
V10 in control or
phosphorylated fibers at either pH 7 and 3 mM
Pi or at pH 6.2 and 25 mM
Pi with or without dextran. Mean
values for the change from 0-5 mM IMP ranged between 4 and
+4% and were not significantly different
(P > 0.1). The results obtained from
fibers phosphorylated in the skinning procedure were not different from
those obtained from control fibers, and the results of both types of
preparation were averaged to produce the data in Fig. 5.
-monophosphate (IMP) to fibers activated under 4 different
conditions: 0% dextran, pH 7, 3 mM
Pi (hatched bars); 5% dextran, pH
7, 3 mM Pi (crosshatched bars);
0% dextran, pH 6.2, 25 mM Pi
(solid bars); 5% dextran, pH 6.2, and 25 mM
Pi (open bars;
n = 3-6 fibers). Mean baseline
values for Po and
V10 at pH 7 without dextran were 0.15 ± 0.02 N/mm2 and 1.19 ± 0.07 lengths/s, respectively. None of measurements is statistically
different from 0, indicating that addition of IMP had no effect on
fiber mechanics under any of conditions tested (P > 0.10).
Combined conditions. Because some of the above conditions may act in concert in modifying the activity of muscle cross bridges, we assayed fibers under conditions in which many of the possible modifiers were combined. Each fiber was activated in a solution simulating the intracellular medium of an unfatigued fiber: rigor buffer plus 4 mM ATP, 3 mM Pi, and 5% dextran at pH 7. The fiber was then switched to a solution simulating a fatigued fiber: rigor buffer plus (in mM) 1 ATP, 30 Pi, 1 ADP, and 4 IMP, as well as 5% dextran at pH 6.2. The velocity of contraction was inhibited by 30 ± 4% under the conditions simulating the fatigued state (Fig. 6). However, Po was also inhibited in the fatigue conditions by 52 ± 2%.
)] or in 1 mimicking a fatigued fiber [ (in mM) 1 ATP, 30 Pi, 1 ADP, and 4 IMP, pH 6.2 ()]. Unfatigued Po was
0.17 ± 0.03 N/mm2 and was
inhibited in fatigue solution (52 ± 2%). Fits to data by
using Hill equation gave maximal velocity of 1.4 and 1.15 for unfatigued and fatigued conditions, respectively. Curvature is described by a parameter
a/Po
in this equation and was determined as 0.18 0.17.
We also measured the ATPase activity of the fibers (n = 14), again contrasting two conditions: simulating 1) unfatigued fibers and 2) fatigued fibers, with 5% dextran included in both solutions. One difference between these experiments and the mechanical experiments described in the paragraph above was that we kept Pi low in solutions simulating both fresh and fatigued conditions so that we could assess the ATPase activity by using the sensitive Malachite Green assay for phosphate. The two solutions contained either 1) rigor solution + 4 mM ATP, pH 7 (unfatigued condition), or 2) rigor solution + (in mM) 1 ATP, 1 ADP, and 4 IMP, pH 6.2 (fatigued condition). We found that the ATPase activity of the fibers in the presence of dextran was inhibited only slightly on transfer from unfatigued to fatigued solutions (0.69 ± 0.05 to 0.57 ± 0.03 s). However, in a parallel set of measurements made with the same solutions and fibers from the same preparations, we found that the tension was inhibited by 35 ± 4% (n = 10). Thus tension is inhibited to a greater extent than ATPase activity, showing that the fibers with in vivo lattice spacings also display a decrease in tension economy when they are shifted into conditions that simulate fatigue. The inhibition in the ATPase activity was about the same as was observed for the inhibition of velocity under slightly different conditions (from 1.4 to 1.15 lengths/s). Together these results suggest that tension economy of skinned fibers was not increased by conditions that simulate fatigue, that contraction velocity is inhibited less than force, and that a synergistic action of the various conditions did not occur.
DISCUSSION
Although the basic contractile machinery is clearly functional in skinned fibers, there remain a number of conditions in which it appears to respond differently than it does in vivo. The most dramatic difference occurs in the tension economy, which appears to increase in vivo during fatigue but to decrease in the skinned fibers under conditions that simulate fatigue.
There appears to be definitive evidence that the tension economy increases at least twofold during fatigue in vivo. The economy of tension production, calculated from changes in either force production or energy consumption, or both, has been shown to improve with fatigue in frogs (13), mice (4, 11, 12), and in human subjects (7, 15). This issue is, however, somewhat complex. For example, increases in economy have been shown predominantly with continuous isometric stimulation protocols (7) and in muscle containing predominantly fast-twitch fibers (11), although it is also present in muscle of mixed fiber type (8, 15). In the skinned muscle fiber model, economy is calculated as the ratio of force produced to the ATPase rate. In vivo, the lower ATPase activity, which produces the improved economy, is correlated temporally with a decrease in the velocity of contraction (3), and both parameters are inhibited at a point where tension remains relatively high. In skinned fibers, however, increases in the concentrations of metabolic products inhibit tension more than either velocity or ATPase activity, leading to a decrease in the tension economy. A major purpose of this study was to attempt to understand these differences between skinned and intact fibers.
One observation made here, and by other investigators, is that compression of skinned fibers to physiological widths increases their force production, decreases their ATPase activity, and thus increases their tension economy under conditions simulating the unfatigued state (22, 35). We hypothesized that a further improvement in tension economy might be obtained under conditions simulating fatigue. If our hypothesis proved correct, it could be concluded that increased myofilament lattice spacing in uncompressed skinned fibers was a factor that prevented them from showing an increased economy of tension production under conditions that simulate fatigue. In the absence of compression, skinned fibers in our study responded predictably to increases in the metabolites of fatigue: either phosphate, H+, or both decreased force production and velocity of contraction, and increased ADP concentration increased force production and decreased velocity of contraction. These effects have previously been observed in our and other laboratories (see Ref. 26 for review). However, contrary to the above hypothesis, compression did not alter the mechanical response of skinned fibers to increased concentrations of either Pi, ADP, or H+. Nor was the economy of tension production increased by a combination of compression and conditions that simulate fatigue. Although ATPase activity was not measured at high Pi concentration, it is unlikely that the economy would be different under conditions of high Pi concentration because velocity, which correlates with ATPase activity (3), was similar in compressed and uncompressed fibers under those conditions.
An additional difference between fatigued intact fibers and the skinned
fiber model is that intact fibers become phosphorylated with fatigue
(31). Although myosin phosphorylation has been found to have little
effect (
5%) on fully activated skinned fiber mechanics or economy
(20, 28), it could be that the effects of phosphorylation are enhanced
under metabolic conditions simulating fatigue or when the myofilament
lattice spacing is at physiological widths, or both. However, the
responses of phosphorylated fibers in the present study followed
similar patterns to those of unphosphorylated fibers on changes in
either lattice compression or metabolite concentrations, or both.
The large changes in IMP concentration from rest to fatigue (70- to 80-fold) (14, 33) make it a reasonable effector of muscle mechanics and energetics, as previously suggested by Westra et al. (33), who found an excellent correlation between increasing IMP concentration and decreasing Po in rat muscle in situ. However, we did not observe any changes in skinned fiber mechanics on addition of 5 mM IMP (approximately equal to that obtained in vivo) to fibers under a variety of conditions, suggesting that previous results were due to other factors.
Although the skinned fiber model has been extensively used to investigate the effects of potential effectors on the mechanics of contraction, these effectors have been studied separately or in pairs. Because there is a possibility that such effectors may act in concert, we have investigated several additional combinations. In all of these investigations, we found little synergistic effect between various factors.
In conclusion, our data confirm that compression of skinned fibers to physiological widths increases their force production, decreases their ATPase activity, and thus increases their tension economy under conditions simulating the unfatigued state (22, 35). From the present study, we also conclude that the different response to fatigue of fully activated, skinned fibers compared with intact fibers is not due to unphysiological, expanded lattice spacing or myosin phosphorylation or to high IMP concentration. Thus the discrepancies between the mechanics of intact fatigued fibers and skinned fibers in conditions that simulate fatigue remain an enigma.
It is possible that the improved economy of intact fibers is due to different Ca2+ handling during fatigue, as suggested by several recent reports (23, 24). These studies have described, in particular, decreased calcium release, subsequent to decreased capacity for calcium reuptake by the sarcoplasmic reticulum (for review, see Ref. 2). However, exactly how and when the alterations in calcium handling might affect economy of force production during fatigue are unclear. Alternatively, economy of force production during fatigue could be due to another factor, such as an additional metabolite or phosphorylation of proteins other than the myosin light chain, which have not yet been investigated in skinned fibers. The observation that the mechanical economy in skinned fibers improves as a result of compression to physiological widths suggests that all future experiments, and particularly those relating to fatigue, should be done with fibers with physiological lattice spacings.
ACKNOWLEDGEMENTS
The authors thank Dr. James Stull for the gift of myosin light chain kinase and Dr. Edward Pate for useful discussions of the results. Charles Day, Jack Maypole, Meenesh Bhimani, and Janet Daijo all provided excellent technical assistance at various times in the measurements of fiber mechanics.
FOOTNOTES
Address for reprint requests: R. Cooke, Dept. of Biochemistry and Biophysics and the Cardiovascular Research Institute, Box 0448, Univ. of California at San Francisco, San Francisco, CA 94143.
Received 7 August 1996; accepted in final form 25 November 1996.
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