J Appl Physiol
90: 657–664, 2001.
Reserve capacity for ATP consumption during isometric
contraction in human skeletal muscle fibers
YOUNG-SOO HAN, DAVID N. PROCTOR, PAIGE C. GEIGER, AND GARY C. SIECK
Departments of Anesthesiology and of Physiology and Biophysics,
Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55905
Received 9 June 2000; accepted in final form 31 August 2000
quantitative histochemistry; immunohistochemistry; muscle
biopsy; sodium dodecyl sulfate-polyacrylamide electrophoresis; adenosine triphosphate
fibers expressing fast MHC isoforms (MHC2A, MHC2X,
and MHC2B). However, ATPiso is submaximal, and
therefore this measure does not establish the maximum capacity for ATP hydrolysis (22, 23). It is well
established that ATP consumption increases with
power output and work performance (11, 12, 22). The
Vmax ATPase establishes the upper limit for ATP consumption during work performance for each fiber type
in skeletal muscle. In this respect, it is important to
establish the range of ATP consumption rates (from
ATPiso to Vmax ATPase) because this provides a measure of the reserve capacity for ATP consumption. In
the rat diaphragm muscle, we found that the reserve
capacity for ATP consumption [calculated as 1⫺ (ratio
of ATPiso to Vmax ATPase)] was ⬃64, ⬃54, and ⬃52%
for fibers expressing MHCslow, MHC2A, and MHC2X/2B,
respectively (23). Unfortunately, values obtained in
laboratory animals cannot be necessarily extrapolated
to human muscle fibers. Therefore, the purpose of the
present study was to determine the reserve capacity for
ATP consumption of single permeabilized fibers from
the human vastus lateralis (VL) muscle. We hypothesized that the reserve capacity for ATP consumption
varies across VL muscle fibers expressing different
MHC isoforms.
METHODS
(MHC) is the site of ATP hydrolysis
during cross-bridge cycling, and ATP consumption rate
during cross-bridge cycling is a major determinant of
the mechanical performance of skeletal muscle fibers.
This is evident by the close relationship between the
maximum velocity of the actomyosin ATPase reaction
(Vmax ATPase), measured biochemically, and the fibertype composition and contractile properties of various
skeletal muscles (3).
Several recent studies have used an NADH-linked
fluorometric technique to measure the rate of ATP
consumption in single permeabilized muscle fibers during maximum isometric activation (ATPiso) (6, 22, 23,
25). In both animal (6, 22, 23) and human (25) studies,
muscle fibers expressing the MHCslow isoform were
found to have a slower rate ATPiso compared with
MYOSIN HEAVY CHAIN
Address for reprint requests and other correspondence: G. C.
Sieck, Anesthesia Research, Mayo Clinic and Foundation, 200 First
St. SW, Rochester, MN 55905 (E-mail: [email protected]).
http://www.jap.org
Muscle biopsies. Needle-biopsy samples (⬃50–100 mg)
were obtained from the superficial portion of the VL muscle
in 12 healthy but sedentary volunteers (10 men, 2 women;
age 21–66 yr). Muscle samples used for measurement of
ATPiso were cleaned of visible fat and connective tissue and
placed in a relaxing solution, at 5°C for 24 h, consisting of 85
mM K⫹, 1 mM free Mg2⫹, 5 mM MgATP, 7 mM EGTA,
propionate as the major anion, and 10⫺9 M free Ca2⫹ [⫺log
Ca2⫹ concentration (pCa) 9.0]; imidazole was used to maintain the pH at 7.0 ⫾ 0.02 and to adjust the ionic strength to
150 mM. The fiber bundles were then transferred to relaxing
solution containing 50% glycerol (vol/vol) and stored at
⫺20°C for no more than 4 wk before subsequent analysis.
Muscle samples used for quantitative histochemical measurements of Vmax ATPase were cleaned of visible fat and
connective tissue, oriented vertically in embedding medium,
frozen in isopentane cooled by liquid nitrogen, and stored at
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.
8750-7587/01 $5.00 Copyright © 2001 the American Physiological Society
657
Downloaded from http://jap.physiology.org/ by 10.220.33.2 on June 15, 2017
Han, Young-Soo, David N. Proctor, Paige C. Geiger,
and Gary C. Sieck. Reserve capacity for ATP consumption
during isometric contraction in human skeletal muscle fibers.
J Appl Physiol 90: 657–664, 2001.—Maximum velocity of the
actomyosin ATPase reaction (Vmax ATPase) and ATP consumption rate during maximum isometric activation (ATPiso)
were determined in human vastus lateralis (VL) muscle
fibers expressing different myosin heavy chain (MHC) isoforms. We hypothesized that the reserve capacity for ATP
consumption [1 ⫺ (ratio of ATPiso to Vmax ATPase)] varies
across VL muscle fibers expressing different MHC isoforms.
Biopsies were obtained from 12 subjects (10 men and 2
women; age 21–66 yr). A quantitative histochemical procedure was used to measure Vmax ATPase. In permeabilized
fibers, ATPiso was measured using an NADH-linked fluorometric procedure. The reserve capacity for ATP consumption
was lower for fibers coexpressing MHC2X and MHC2A compared with fibers singularly expressing MHC2A and MHCslow
(39 vs. 52 and 56%, respectively). Tension cost (ratio of ATPiso
to generated force) also varied with fiber type, being highest
in fibers coexpressing MHC2X and MHC2A. We conclude that
fiber-type differences in the reserve capacity for ATP consumption and tension cost reflect functional differences such
as susceptibility to fatigue.
658
RESERVE CAPACITY FOR ATP CONSUMPTION RATE
ATPase
¡ ADP ⫹ P i
ATP O
(1)
PK
ADP ⫹ PEP O
¡ pyruvate ⫹ ATP
(2)
Pyruvate ⫹ NADH LDH lactate ⫹ NAD ⫹
O
¡
(fluorescent)
(nonfluorescent)
(3)
where, in reaction 1, ATP is hydrolyzed by actomyosin
ATPase to ADP and Pi during detachment of the myosin head
from the myosin-binding domain of actin. In reaction 2, ATP
is regenerated from ADP and PEP by PK. In reaction 3, the
resulting pyruvate is converted to lactate by LDH, which
results in stoichiometric conversion of fluorescent NADH to
nonfluorescent NAD⫹. For each mole of ADP produced by the
actomyosin ATPase-dependent hydrolysis of ATP, 1 mol of
NADH is converted to NAD⫹. Therefore, for a period of time
when perfusion of the cuvette was stopped (15 s), the amount
of ATP consumed by the ATPiso was determined by measuring the rate of extinction of the NADH fluorescence signal
(Fig. 1). Flow through the cuvette was then reinitiated for 1 s
and then stopped again, and extinction of the NADH fluores-
Fig. 1. Typical trace of the simultaneous measurement of force and
ATP consumption rate during maximum isometric activation
(ATPiso) using the NADH-linked fluorometric procedure in a single
permeabilized fiber from the human vastus lateralis muscle [slow
myosin heavy chain (MHCslow) expression]. Fiber size: length, 3.0
mm; diameter, 78.3 ␮m.
cence signal was remeasured. This cycling continued for ⬃10
min at each pCa condition. Calibration involved measurements of fluorescence intensity for known amounts of NADH.
On the basis of changes in the NADH fluorescence intensity,
ATPiso was determined and expressed as nanomoles per
cubic millimeter per second. The ATPiso was determined by
subtracting the ATP consumption rate measured at a pCa 9.0
from that obtained at pCa 4.0.
In a subset of permeabilized muscle fibers, the temperature dependence of the ATPiso was determined by obtaining
measurements at 15, 20, and 25°C. The temperature coefficient (Q10) for the ATPiso was then calculated over this
temperature range.
Gel electrophoretic determination of MHC isoform expression in single fibers. After completion of the force and ATPiso
measurements, the MHC isoform composition of the muscle
fiber was determined by SDS-PAGE using a previously described procedure (14, 15, 22, 23). Briefly, fibers were placed
in 25 ␮l of SDS sample buffer containing 62.5 mM Tris 䡠 HCl,
2% (wt/vol) SDS, 10% (vol/vol) glycerol, and 0.001% (wt/vol)
bromophenol blue at a pH of 6.8. The sample was denatured
by boiling for 2 min, and 10-␮l samples [⬃125 ng as determined by the Lowry method (19)] were loaded per lane. The
gels were silver stained to visualize the MHC migration
bands. Two mixed muscle fiber samples were run on each gel
to compare the migration patterns of identified MHC isoforms. In the case of coexpression of MHC isoforms within a
single fiber, the relative expression of each MHC isoform was
determined by densitometric analysis.
Quantitative histochemical measurement of fiber Vmax
ATPase. The quantitative histochemical procedure for measuring the Vmax ATPase in type-identified muscle fibers has
been previously described in detail (4, 24). Serial cross sections of muscle fibers were cut at 10-␮m thickness using a
cryostat kept at ⫺20°C, and alternate sections were used to
determine MHC isoform expression and the Vmax ATPase. In
four alternate transverse sections, immunoreactivity against
antibodies specific for anti-MHCslow (NCL), anti-MHC2A (SC71), and anti-MHCall-2X (BF-35) (MHCall-2X means all but the
MHC2X isoform) was evaluated. Primary antibodies were diluted in PBS (pH 7.4) containing 0.5% bovine serum albumin
and were then applied to the muscle sections for ⬃12 h at room
temperature in a humidified chamber. Slides were then washed
in PBS and incubated with a fluorescein-conjugated secondary
antibody (goat anti-mouse IgG) for ⬃60 min at room tempera-
Downloaded from http://jap.physiology.org/ by 10.220.33.2 on June 15, 2017
⫺80°C. No attempt was made to stretch the muscle sample to
optimal length before freezing.
Permeabilized single-fiber preparation. Glycerinated fiber
bundles were transferred to a relaxing solution containing 10
mM dithiothreitol (DTT) and dissected under a microscope.
The dissected single fibers were then transferred to a relaxing solution containing 10 mM DTT and 1% Triton X-100 for
20–30 min to permeabilize the plasma membrane. The permeabilized fibers were again transferred to 50% glycerol
relaxing solution before measurements of ATPiso. Permeabilized single fibers, ⬃3 mm in length, were mounted between
force and displacement transducers in a quartz cuvette that
was perfused with solutions containing free ionized Ca2⫹
concentrations of either 1 nM (pCa 9.0) or 100 ␮M (pCa 4.0)
maintained at 15°C. Muscle fiber length was adjusted so that
average sarcomere length was 2.5 ␮m.
Measurement of maximum isometric force and ATPiso.
Maximum isometric force (Fmax) and ATPiso were measured
concurrently at 15°C in a Gûth Scientific Instruments Muscle
Research System (16, 17, 20). The procedures for measuring
isometric force in single permeabilized muscle fiber has been
previously reported (14, 15, 20, 22, 23). In preliminary studies on human fibers, we confirmed that Fmax is obtained at
pCa 4.0 and that no active force was obtained at pCa of 9.0.
The NADH-linked fluorometric technique for measuring
ATPiso has been previously described in detail (16, 17, 20, 22,
23). Using this procedure, it was confirmed that mitochondrial ATPases and sarcoplasmic reticulum ATPase make no
detectable contribution to the observed ATPase activity (17).
Measurements of ATPiso were made while fibers were
mounted in the quartz cuvette and perfused with either
relaxing (pCa 9.0) or activating (pCa 4.0) solutions. NADH
fluorescence was excited at 340 nm using a mercury lamp
and an interposed band-pass filter. Emitted fluorescence was
measured at 450 nm using a photomultiplier tube. The ATP
solutions consisted of relaxing (pCa 9.0) and activating (pCa
4.0) solutions, both containing 5 mM phospho(enol)pyruvate
(PEP), 0.2 mM NADH, 100 U/ml pyruvate kinase (PK), 140
U/ml lactate dehydrogenase (LDH), and 0.2 mM P1,P5di(adenosine-5⬘)pentaphosphate. The NADH-linked enzymatic assay involves the following reactions
RESERVE CAPACITY FOR ATP CONSUMPTION RATE
Fig. 2. Lineweaver-Burke plot displaying the dependence of the
velocity (V) of the maximum velocity of the actomyosin ATPase
reaction (Vmax ATPase) on ATP concentration in the incubation
medium for 3 human vastus lateralis muscle fibers expressing different (MHC) isoforms. Slope and y-intercept of each line were
calculated by linear regression analysis, from which the Vmax
ATPase reaction was determined. OD570nm, optical density at 570
nm. Brackets denote concentration.
RESULTS
Electrophoretic determination of MHC isoform expression in single fibers. The VL muscle displayed
three distinct MHC migration bands. On the basis of
Western blot analysis, these MHC migration bands were
found to correspond with the expression of MHCslow,
MHC2A, and MHC2x isoforms. To evaluate MHC isoform
expression in single VL fibers, a larger number of fibers
were sampled in addition to those used in the mechanical
and energetic studies (see Classification of fiber types in
muscle cross sections). Among human VL fibers, MHCslow
(n ⫽ 64) and MHC2A isoforms (n ⫽ 65) were found to be
singularly expressed. However, the MHC2X isoform was
not found to be singularly expressed and was coexpressed
predominantly with the MHC2A isoform (n ⫽ 24; Fig. 3).
Within fibers coexpressing MHC2X and MHC2A, the relative expression of each isoform ranged from 20 to 80%,
but the mean relative expression was 54.8 ⫾ 1.2%
MHC2X and 45.2 ⫾ 1.2% MHC2A. In these preliminary
studies, it was determined that the fiber-type composition of the VL muscle could not be characterized from a
single biopsy. It was estimated that up to seven biopsies
would be required, and such repeated biopsies were not
possible in these subjects. In addition, there was a relatively low abundance of fibers expressing the MHC2X
isoform in the biopsies that were obtained. Indeed, it was
necessary to obtain biopsies from 12 subjects to obtain a
sufficient sample of fibers expressing the MHC2X isoform.
For these reasons, it was not possible to characterize the
overall population of this or other fiber types in the VL of
subjects. This raised the important issue of whether
across-subject variability may have influenced the results. For fibers expressing the MHC2X isoform, this issue
could not be addressed. However, in comparing values for
fibers expressing MHCslow and MHC2A isoforms, there
were no significant differences across subjects. The coefficient of variation for ATPiso of fibers expressing MHC2A
across subjects was 6.26%.
Downloaded from http://jap.physiology.org/ by 10.220.33.2 on June 15, 2017
ture in a humidified chamber. The slides were then washed in
PBS, coverslipped with Permount, and viewed through a microscope (model BH2, Olympus) equipped with epifluorescence.
An additional four alternate sections were stained for myofibrillar ATPase (mATPase) after preincubation at pH 4.3, 4.6, 9.0,
and 10.4 (after 4% paraformaldehyde fixation) (7, 24).
The Vmax ATPase was measured in a series of 28 alternate
sections of the same muscle fibers. The values for ATPase in
a given fiber were the average of measurements across the
four sections at a given ATP concentration, and the same
fiber was measured at each of seven ATP concentrations (see
below). In a previous study, our laboratory verified that the
quantitative histochemical method is specific for the Vmax
ATPase (4) and that the deposition of the reaction product is
localized at the site of cross-bridge cycling (i.e., A band). In
this procedure, an image-processing system (MegaVision
1024 XM), mounted on an Olympus BH-2 microscope and
calibrated for microdensitometry using a set of neutral density filters [0.1–2.0 optical density (OD) units], was used.
Microscopic images of muscle fiber cross sections were digitized at 8-bit resolution into a 1,024 ⫻ 1,024 picture element
(pixel) array using a video scanner and then stored in a
computer file. In the digitizing procedure, 16 separate scans
were averaged to reduce electronic noise, and a shading
algorithm was employed to reduce errors attributed to uneven illumination of the tissue. Previously, our laboratory
estimated that, when using this imaging system, the measurement error for microdensitometry was ⬍4% (4, 24). The
boundaries of individual fibers within the digitized images
were delineated, and the average OD of all pixels within the
fiber was calculated.
Four replicate sections of the same muscle fibers were
reacted at 22°C in an incubation medium containing one of a
series of ATP concentrations (0.0, 0.5, 0.75, 1.0, 2.0, 4.0, 5.0
mM ATP). The use of several ATP concentrations was necessary because sufficient ATP could not be dissolved in the
incubation medium to avoid substrate limiting the Vmax
ATPase (4). In the Vmax ATPase, the Pi ions produced by the
enzymatic hydrolysis of ATP were precipitated within the
muscle fiber cross section by complexing with lead ions (lead
ammonium citrate-acetate complex) to form a lead phosphate
precipitate. The precipitated lead ions were subsequently
exchanged for sulfide ions, by reaction with sodium sulfide, to
form a brown-colored lead sulfide precipitate. The concentration of the lead sulfide precipitate in the muscle section was
then measured by microdensitometry using the LambertBeer equation and a molar extinction coefficient of 1,450
mol/cm for lead sulfide (4, 24).
To determine the Vmax ATPase, a Lineweaver-Burke
transformation of the data was performed (Fig. 2). On the
basis of stoichiometric relationships of the Pi-sulfide exchange in the histochemical reactions, the Vmax ATPase was
expressed as millimoles of Pi precipitated per liter of tissue
(fiber cross-sectional area ⫻ 10-␮m thickness) per minute
(mM Pi/min). However, this unit of millimoles of Pi per
minute was converted to nanomoles per cubic millimeter per
second to be compatible with that used in the ATPiso. The
Vmax ATPase for at least 20 fibers singularly expressing each
MHC isoform was determined for each muscle biopsy. In the
case of MHC isoform coexpression, fewer fibers were sampled
due to the relative paucity of such fibers.
Statistical analysis. Values are reported as means ⫾ SE. A
one-way ANOVA with repeated-measures design was used to
independently assess fiber-type differences in histochemical
Vmax ATPase and ATPiso. Post hoc analysis (Neuman-Keuls)
was performed when appropriate. Statistical significance
was accepted when P ⬍ 0.05.
659
660
RESERVE CAPACITY FOR ATP CONSUMPTION RATE
Fig. 3. MHC isoform expression in human
vastus lateralis muscle fibers was identified by SDS-PAGE. A mixed muscle sample of human vastus lateralis muscle fibers was included for comparison of
electrophoretic migration patterns.
The ATPiso of single fibers singularly expressing
MHC2A was also significantly lower than that of fibers
coexpressing MHC2X and MHC2A (P ⬍ 0.05; Fig. 4).
Vmax ATPase (n ⫽ 525). The Vmax ATPase of VL
muscle fibers expressing MHCslow was significantly
lower than that of fibers expressing MHC2A either
alone or coexpressed with MHC2X (P ⬍ 0.05; Fig. 4).
The Vmax ATPase of fibers expressing MHC2A alone
was significantly lower than that of fibers coexpressing
MHC2X and MHC2A (P ⬍ 0.05; Fig. 4).
Reserve capacity of ATP consumption. For human VL
muscle fibers, the reserve capacity for ATP consumption was calculated as
Reserve capacity for ATP consumption ⫽ 1
⫺ 共ratio of ATPiso to Vmax ATPase兲
In fibers singularly expressing MHCslow or MHC2A, the
reserve capacity for ATP consumption was 56 and 52%,
respectively. For fibers coexpressing MHC2X and
MHC2A, the reserve capacity for ATP consumption was
only 39% (P ⬍ 0.05 compared with fibers singularly
expressing MHCslow or MHC2A).
Isometric tension cost (n ⫽ 46). For a measurement of
ATP cost for generating force, the isometric tension
Table 1. Cross-sectional area, maximum isometric
force, and tension cost of human vastus
lateralis muscle fibers at 15°C
MHC Isoform
Slow
2A
2X/2A
n
CSA, ␮m2
Fmax, N/cm2
Tension Cost,
pmol 䡠 mm⫺1 䡠 s⫺1 䡠 mN⫺1
19
21
6
4972 ⫾ 42
4368 ⫾ 261
4651 ⫾ 719
17.5 ⫾ 0.6
13.6 ⫾ 0.6*
15.7 ⫾ 0.1
2.0 ⫾ 0.1
5.5 ⫾ 0.2*
6.7 ⫾ 0.9*†
Values are means ⫾ SE; n, no. of muscle fibers. CSA, fiber crosssectional area; Fmax, maximum isometric force; MHC, myosin heavy
chain. * Significant difference from fibers expressing MHCslow, P ⬍
0.05. † Significant difference from fibers expressing MHC2A, P ⬍
0.05.
Fig. 4. Comparison of ATPiso and Vmax ATPase for human vastus
lateralis fibers expressing different MHC isoforms. * Significantly
from fibers expressing MHCslow, P ⬍ 0.05 ⫹ Significantly different
from fibers expressing MHC2A, P ⬍ 0.05.
Downloaded from http://jap.physiology.org/ by 10.220.33.2 on June 15, 2017
Classification of fiber types in muscle cross sections.
The patterns of immunoreactivity against specific
MHC antibodies in the VL muscle generally corresponded with the histochemical classification of fiber
types. VL fibers classified histochemically as type I
displayed immunoreactivity for the anti-MHCslow antibody, and fibers classified histochemically as type IIa
displayed immunoreactivity for the anti-MHC2A antibody. However, the anti-MHCsll-2X antibody (BF-35),
specific for all MHC isoforms except for MHC2X, was
less reactive with fibers classified histochemically as
type IIb, indicating expression of the MHC2X isoform.
In these fibers, immunoreactivity for the MHCall-2X
antibody varied from faint to moderate, and most of
these fibers were also immunoreactive for the antiMHC2A antibody in varying degrees. Therefore, these
immunohistochemical results were consistent with the
coexpression of MHC2A and MHC2X.
Fmax (n ⫽ 46). Fmax of human VL fibers was significantly lower for fibers expressing MHC2A (n ⫽ 21;
range 10–20 N/cm2) compared with fibers expressing
MHCslow (n ⫽ 19; range 12–22 N/cm2; Table 1; P ⬍
0.05). For fibers coexpressing MHC2X and MHC2A (n ⫽
6), there was a considerable range in Fmax (11–27
N/cm2). Unfortunately, there were an insufficient number of fibers sampled in this group to determine
whether Fmax depended on the relative expression of
MHC2X and MHC2A.
ATPiso (n ⫽ 46). The ATPiso of human VL fibers expressing the MHCslow isoform was significantly lower
than that of fibers expressing MHC2A as well as fibers
coexpressing the MHC2X and MHC2A (P ⬍ 0.05; Fig. 4).
RESERVE CAPACITY FOR ATP CONSUMPTION RATE
cost of human VL fibers was determined by dividing
the ATPiso by the corresponding isometric force. The
tension cost of human VL fibers expressing the
MHCslow isoform was significantly lower than that of
fibers expressing MHC2A (P ⬍ 0.05; Fig. 5). There were
an insufficient number of fibers sampled in this group
to determine whether tension cost depended on the
relative expression of MHC2X and MHC2A. Comparison
of tension cost determined at 15°C between human VL
fibers and rat diaphragm fibers in our laboratory’s
previous study (22) is shown in Fig. 5. The relationship
between Vmax ATPase and tension cost is displayed in
Fig. 6.
Temperature dependence of ATPiso (n ⫽ 6). The dependence of ATPiso was determined by comparing measurements at 15, 20, and 25°C. As temperature increased, ATPiso of fibers also increased. The Q10 value
of the ATPiso was 1.72 ⫾ 0.16 (Fig. 7).
Fig. 6. Comparison of Vmax ATPase and tension cost for human
vastus lateralis fibers expressing different MHC isoforms. * Significantly different from fibers expressing MHCslow, P ⬍ 0.05.
Fig. 7. Relationship between temperature and ATPiso was measured
in 6 human vastus lateralis muscle fibers. Values represent individual measurements on 6 human vastus lateralis muscle fibers from 2
subjects.
DISCUSSION
The present study examined both maximal (Vmax
ATPase) and submaximal (ATPiso) values for ATP consumption in human VL muscle fibers. The Vmax
ATPase establishes the upper limit for ATP consumption during work performance for each fiber type in
skeletal muscle (23). In this respect, it is important to
determine the range of ATP consumption rates (from
Vmax ATPase to ATPiso), because this provides a measure of the reserve capacity for ATP consumption. The
present study reports important new information in
this regard. The reserve capacity for ATP consumption
was lower for fibers expressing MHC2X (coexpressed
with MHC2A) compared with fibers expressing MHC2A
and MHCslow. This is consistent with the lower energy
efficiency of fibers expressing the MHC2X isoform as
reflected by the higher tension cost of these fibers.
Similar to previous reports (10, 30), we found that
three MHC isoforms were expressed in the VL muscle
of healthy adult humans, MHCslow, MHC2A, and
MHC2X. Other studies evaluating MHC isoform expression in the human VL muscle also found three
isoforms, although MHC2B expression was reported
rather than MHC2X (18, 25). Because there is now
strong evidence to indicate that MHC2B is not expressed in human fibers, it is likely that expression of
MHC2B was confused with MHC2X expression. On the
basis of single-fiber gel electrophoresis and Western
blot analysis, it appears that MHCslow and MHC2A are
singularly expressed in VL fibers, whereas the MHC2X
isoform is only coexpressed, predominantly with
MHC2A. The coexpression of MHC2X and MHC2A in
human VL muscle fibers has also been reported in
other studies (10, 30). The pattern of MHC isoform
expression in single human VL muscle fibers, as determined by SDS-PAGE, generally corresponded with the
pattern of immunoreactivity against specific MHC antibodies, as well as the histochemical classification of
fiber types based on the pH lability of myofibrillar
mATPase staining. These results are consistent with
other studies showing a relationship between histo-
Downloaded from http://jap.physiology.org/ by 10.220.33.2 on June 15, 2017
Fig. 5. Comparison of tension cost determined at 15°C between
vastus lateralis muscle fibers and rat diaphragm fibers expressing
MHCslow, MHC2A, and MHC2X/2A isoforms. Human vastus lateralis
fibers expressing MHC2A/2X were compared with the rat diaphragm
fibers expressing MHC2X/2B. * Significantly different from fibers expressing MHCslow, P ⬍ 0.05.
661
662
RESERVE CAPACITY FOR ATP CONSUMPTION RATE
MHCslow to ⬃52% for fibers expressing fast MHC isoforms (23). In the human VL, we found that fibers
singularly expressing MHCslow and MHC2A had a
greater reserve capacity (56 and 52%, respectively)
compared with fibers coexpressing MHC2X and MHC2A
(39%). It should be noted that both the ATPiso and the
Vmax ATPase for human VL fibers were substantially
lower than values for fibers expressing the same MHC
isoforms (MHCslow, MHC2A, and MHC2X) in the rat
(22–24). Together, these results indicate that ATPiso
represents only submaximal energy utilization compared with the Vmax ATPase in muscle fibers. The Vmax
ATPase in a muscle fiber is determined by the product
of the ATP consumed per MHC molecule (i.e., the
quantal contribution from a single myosin cross bridge)
times the number of available cross bridges (i.e., MHC
concentration). Similarly, ATPiso is determined by the
ATP consumed per MHC molecule times the number of
cross bridges in the force-generating state. In both
cases, MHC concentration becomes an important determinant of ATP consumption. Recently, our laboratory reported that, in the rat diaphragm muscle, fibers
expressing MHCslow and MHC2A have lower MHC concentrations compared with fibers expressing MHC2X
and MHC2B (14). Furthermore, our laboratory found
that, during maximum isometric activation, the fraction of cross bridges in the force-generating state was
comparable across fiber types (14). Thus, with a lower
MHC concentration, lower ATPiso and Vmax ATPase
would be expected for fibers expressing MHCslow and
MHC2A. However, when we normalized our Vmax
ATPase for previously reported myofibrillar volume
densities in the VL muscle (28), fiber-type differences
in ATPiso and Vmax ATPase persisted. Thus it is likely
that the lower ATPiso and Vmax ATPase values seen in
VL fibers expressing the MHCslow isoform primarily
reflect phenotypic differences in the capacity for ATP
consumption of MHCslow vs. MHC2A or MHC2X molecules. The lower ATPiso and Vmax ATPase of fibers
expressing MHCslow are also likely to be reflected by a
slower maximum rate constant for cross-bridge detachment compared with fibers expressing MHC2A or
MHC2X (23).
It is not surprising that the ATPiso of muscle fibers
was only a fraction of the Vmax ATPase (i.e., maximum
capacity for ATP consumption). In 1923, Fenn observed
that energy utilization of skeletal muscle increases in
proportion to work (Fenn effect; Refs. 11, 12). Thus, as
muscle fibers reach maximum power during shortening, ATP consumption rate should increase (22). In a
previous study on the rat diaphragm muscle, our laboratory found that the maximum rate of ATP consumption was achieved at a shortening velocity corresponding to peak power output of fibers (22). Although the
maximum rate of ATP consumption achieved at peak
power output was closer to the Vmax ATPase, it was
still less. This may reflect differences in the number of
cross bridges contributing to the measured ATP consumption rate during active shortening vs. those contributing to the measurements of Vmax ATPase.
Downloaded from http://jap.physiology.org/ by 10.220.33.2 on June 15, 2017
chemical fiber-type classification and MHC isoform
composition in human muscle fibers (1, 10, 30).
Similar to the previous results of Stienen and colleagues (25) for the human rectus abdominis and VL
muscles, we found that ATPiso varied across fibers
expressing different MHC isoforms in human VL muscle. These results are also in general agreement with
our laboratory’s previous observations in the rat diaphragm muscle (22, 23). However, the values for ATPiso
in human muscle fibers reported by Stienen and colleagues were lower than those found in the present
study. These investigators measured ATPiso at 20°C
rather than 15°C. The dependence of ATPiso on temperature was measured in both studies; a Q10 of 1.72
was found in the present study vs. a Q10 of 2.34 in the
study of Stienen et al. Even when corrected for differences in temperature, the ATPiso values found in the
present study were ⬃30–40% higher than those reported by Stienen et al. It should be noted that Stienen
and colleagues measured NADH concentration by absorbency rather than fluorometry, and these technical
differences may have accounted for the discrepancies
in reported values.
31
P-nuclear magnetic resonance (NMR) spectroscopy
has also been used to measure ATP consumption in
human muscle fibers in vivo on the basis of the dynamics
of creatine phosphate content. Using this procedure, Blei
and colleagues (5) reported an average ATP consumption
rate of 0.15 ⫾ 0.03 nmol䡠mm⫺3 䡠s⫺1 during single-twitch
stimulation in the human forearm flexor musculature,
whereas Turner and colleagues (27) reported an average
value of 4.4 ⫾ 0.4 nmol䡠mm⫺3 䡠s⫺1 in the human adductor
pollicis muscle during maximum isometric activation.
After corrections for the higher in vivo temperature were
made, the ATPiso for VL muscle fibers in the present
study would range from 1.02 ⫾ 0.07 nmol䡠mm⫺3 䡠s⫺1 for
fibers expressing MHCslow to 3.06 ⫾ 0.15 nmol䡠mm⫺3 䡠s⫺1
for fibers coexpressing MHC2X and MHC2A. Using the
NADH-linked fluorometric procedure, only the ATP consumption related to the actomyosin ATPase was measured, whereas in the 31P-NMR spectroscopy method the
total ATP consumption of activated fibers was measured,
including other membrane ATPases. These differences
may account for the slightly higher ATP consumption
measured using 31P-NMR spectroscopy, although several
other factors may have also contributed.
In the present study, Vmax ATPase was highest for
fibers coexpressing MHC2X and MHC2A and lowest for
fibers expressing MHCslow. These results are consistent with the previous study of Castro et al. (8) on
human VL muscle fibers using an identical technique.
These results are also generally consistent with differences in the Vmax ATPase of fibers expressing different
MHC isoforms in the rat diaphragm muscle (23).
The reserve capacity for ATP consumption in single
muscle fibers was estimated by the following calculation: 1 ⫺ [ratio of the ATPiso to the Vmax ATPase]. In a
previous study in the rat diaphragm muscle, our laboratory found that the reserve capacity for ATP consumption ranged from ⬃64% for fibers expressing
RESERVE CAPACITY FOR ATP CONSUMPTION RATE
We thank Dr. Janet L. Vittone, Y. H. Fang, Heidi M. Hinderaker,
A. Iyanoye, Rebecca Macken, and J. Sieck for their assistance in
these studies.
This research was supported by National Heart, Lung, and Blood
Institute Grants HL-34817 and HL-37680.
REFERENCES
1. Adams GR, Hather BM, Baldwin KM, and Dudley GA.
Skeletal muscle myosin heavy chain composition and resistance
training. J Appl Physiol 74: 911–915, 1993.
2. Ameredes BT, Zhan WZ, Vanderboom R, Prakash YS, and
Sieck GC. Power fatigue of the rat diaphragm muscle. J Appl
Physiol 89: 2215–2219, 2000.
3. Barany M. ATPase activity of myosin correlated with speed of
muscle shortening. J Gen Physiol 50: 197–216, 1967.
4. Blanco CE and Sieck GC. Quantitative determination of calcium-activated myosin adenosine triphosphatase activity in rat
skeletal muscle fibres. Histochem J 24: 431–444, 1992.
5. Blei ML, Conley KE, and Kushmerick MJ. Separate measures of ATP utilization and recovery in human skeletal muscle.
J Physiol 465: 203–222, 1993.
6. Bottinelli R, Canepari M, Reggiani C, and Stienen GJM.
Myofibrillar ATPase activity during isometric contraction and
isomyosin composition in rat single skinned muscle fibres.
J Physiol (Lond) 481: 663–675, 1994.
7. Brooke MH and Kaiser KK. Three “myosin adenosine triphosphatase” systems: the nature of their pH lability and sulfhydryl
dependence. J Histochem Cytochem 18: 670–672, 1970.
8. Castro MJ, Apple DFJ, Melton-Rogers S, and Dudley GA.
Muscle fiber type-specific myofibrillar Ca(2⫹) ATPase activity
after spinal cord injury. Muscle Nerve 23: 119–121, 2000.
9. Eddinger TJ, Gassens RG, and Moss RL. Mechanical and
histochemical characterization of skeletal muscles from senescent rats. Am J Physiol Cell Physiol 251: C421–C430, 1986.
10. Ennion S, Sant’Ana Pereira J, Sargeant AJ, Young A, and
Goldspink G. Characterization of human skeletal muscle fibres
according to the myosin heavy chains they express. J Muscle Res
Cell Motil 16: 35–43, 1995.
11. Fenn WO. A quantitative comparison between the energy liberated and the work performed by the isolated sartorius muscle
of the frog. J Physiol (Lond) 58: 175–203, 1923.
12. Fenn WO. The relation between the work performed and the
energy liberated in muscular contraction. J Physiol (Lond) 58:
373–395, 1924.
13. Fukunaga T, Roy RR, Shellock FG, Hodgson JA, and Edgerton VR. Specific tension of human plantar flexors and dorsiflexors. J Appl Physiol 80: 158–165, 1996.
14. Geiger PC, Cody MJ, Macken RL, and Sieck GC. Maximum
specific force depends on myosin heavy chain content in rat
diaphragm muscle fibers. J Appl Physiol 89: 695–703, 2000.
15. Geiger PC, Cody MJ, and Sieck GC. Force-calcium relationship depends on myosin heavy chain and troponin isoforms in rat
diaphragm muscle fibers. J Appl Physiol 87: 1894–1900, 1999.
16. Guth K and Wojciechowski R. Instruments and techniques:
perfusion cuvette for the simultaneous measurement of mechanical, optical and energetic parameters of skinned muscle fibres.
Pflügers Arch 407: 552–557, 1986.
17. Kerrick WG, Potter JD, and Hoar PE. The apparent rate
constant for the dissociation of force generating myosin crossbridges from actin decreases during Ca2⫹ activation of skinned
muscle fibres. J Muscle Res Cell Motil 12: 53–60, 1991.
18. Larsson L and Moss RL. Maximum velocity of shortening in
relation to myosin isoform composition in single fibers from
human skeletal muscles. J Physiol 472: 595–614, 1993.
19. Lowry OH, Rosenbrough NJ, Farr AL, and Randall RJ.
Protein measurement with the Folin phenol reagent. J Biol
Chem 193: 265–275, 1951.
20. Perkins WJ, Han YS, and Sieck GC. Skeletal muscle force
and actomyosin ATPase activity reduced by nitric oxide donor.
J Appl Physiol 83: 1326–1332, 1997.
21. Sieck GC and Fournier M. Diaphragm motor unit recruitment
during ventilatory and nonventilatory behaviors. J Appl Physiol
66: 2539–2545, 1989.
22. Sieck GC, Han YS, Prakash YS, and Jones KA. Cross-bridge
cycling kinetics, actomyosin ATPase activity and myosin heavy
chain isoforms in skeletal and smooth respiratory muscles.
Comp Biochem Physiol B Biochem Mol Biol 119: 435–450, 1998.
23. Sieck GC and Prakash YS. Cross bridge kinetics in respiratory muscles. Eur Respir J 10: 2147–2158, 1997.
Downloaded from http://jap.physiology.org/ by 10.220.33.2 on June 15, 2017
The Fmax values for human VL muscle fibers measured in the present study were comparable to those
reported in previous studies in single fibers (9, 18, 25,
29) as well as whole human muscle in vivo (13). We
found that the Fmax of VL fibers expressing MHCslow
was slightly greater than that for fibers expressing
MHC2A. In contrast, Stienen et al. (25) reported that
VL fibers expressing MHCslow generated lower Fmax
compared with fibers expressing fast MHC isoforms.
Larsson and Moss (18) reported no significant differences in Fmax across human VL muscle fibers expressing different MHC isoforms. In the rat diaphragm
muscle, our laboratory found that fibers expressing
MHCslow generated lower Fmax compared with fibers
expressing fast MHC isoforms (14, 15, 22, 23).
The tension cost (the ratio of ATPiso to isometric
force; Fig. 5) of human VL muscle fibers reported in the
present study is in general agreement with that reported by Steinen et al. (25). Fibers expressing
MHCslow had the lowest values of tension cost followed
by fibers expressing MHC2A and fibers coexpressing
MHC2X and MHC2A. Therefore, fibers expressing
MHCslow are the most energy efficient. Compared with
values of tension cost reported for rat diaphragm muscle fibers (22), the tension cost of human VL muscle
fibers was significantly lower. These results generally
agree with the principle that energetic costs of generating muscular force are lower in larger animals (26).
In conclusion, measurement of submaximal and
maximal rates of ATP consumption in the present
study indicates that a substantial reserve capacity for
ATP consumption exists in human muscle fibers. In
addition, fiber-type differences in the reserve capacity
for ATP consumption exist, with fibers expressing
MHC2X (coexpressed with MHC2A) displaying a significantly lower reserve capacity compared with fibers
singularly expressing MHC2A and MHCslow. These
measurements provide new information that is important in determining the balance between energy supply
and demand. Certainly this reserve capacity for ATP
consumption becomes important under conditions
where ATP production may be insufficient to meet the
demands for cross-bridge cycling. Such an energetic
imbalance has been suggested as an underlying mechanism of muscle fatigue. The lower reserve capacity for
ATP consumption, together with the higher ATP consumption rates, may explain, at least in part, the
greater fatigue susceptibility of fibers expressing
MHC2X (21). Under conditions of greater workloads, as
energy utilization increases in proportion to work
(Fenn effect; Refs. 11, 12), reserve capacity for ATP
consumption decreases and susceptibility to fatigue
increases (2). Therefore, these novel results regarding
the reserve capacity of ATP consumption in human VL
muscle fibers have important functional implications.
663
664
RESERVE CAPACITY FOR ATP CONSUMPTION RATE
24. Sieck GC, Zhan WZ, Prakash YS, Daood MJ, and Watchko
JF. SDH and actomyosin ATPase activities of different fiber
types in rat diaphragm muscle. J Appl Physiol 79: 1629–1639,
1995.
25. Stienen GJM, Kiers JG, Bottinelli R, and Reggiani C.
Myofibrillar ATPase activity in skinned human skeletal muscle
fibres: fibre type and temperature dependence. J Physiol (Lond)
493: 299–309, 1996.
26. Taylor CR, Heglund NC, McMahon TA, and Looney TR.
The energetic cost of generating muscular force during running.
J Exp Biol 86: 9–18, 1980.
27. Turner DL, Jones DA, McIntyre DB, and Newham DJ. ATP
turnover measured by 31P-magnetic resonance spectroscopy in
human adductor pollicis during isometric and shortening contractions (Abstract). J Physiol 459: 154P, 1993.
28. Wang N, Hikida R, Staron R, and Simoneau J. Muscle fiber
types of women after resistance training— quantitative ultrastructure and enzyme activity. Pflügers Arch 424: 494–502,
1993.
29. Wood DS, Zollman J, and Reuben JP. Human skeletal muscle: properties of the “chemically skinned” fiber. Science: 1075–
1076, 1974.
30. Zhou MY, Klitgaard H, Saltin B, Roy RR, Edgerton VR,
and Gollnick PD. Myosin heavy chain isoforms of human
muscle after short-term spaceflight. J Appl Physiol 78: 1740–
1744, 1995.
Downloaded from http://jap.physiology.org/ by 10.220.33.2 on June 15, 2017