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Muscle Biology Laboratory, Texas A&M University, College Station, Texas 77843, and Muscular Function Laboratory, Virginia Polytechnic Institute, Blacksburg, Virginia 24061
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
ACKNOWLEDGEMENTS
FOOTNOTES
REFERENCES
ABSTRACT 
Warren III, Gordon L., Jay H. Williams, Christopher W. Ward,
Hideki Matoba, Christopher P. Ingalls, Karl M. Hermann, and R. B. Armstrong. Decreased contraction economy in mouse EDL muscle
injured by eccentric contractions. J. Appl.
Physiol. 81(6): 2555-2564, 1996.
The objective of
this study was to find out whether basal and/or active energy
metabolism are altered in isolated mouse extensor digitorum longus
muscle injured by eccentric (Ecc) contractions. Measurements of basal
O2 consumption and isometric tetanus O2 recovery cost were made
at 25°C on muscles that had done either 10 Ecc, 10 isometric (Iso),
or no contractions (No). In parallel experiments, rates of lactate and
pyruvate production were measured to estimate the anaerobic
contribution. Basal O2 consumption
was unaffected by the type of protocol performed
(P = 0.07). However, the tetanus
O2 cost per force-time integral was elevated by 30-36% for the Ecc protocol muscles over that for
the Iso and No protocol muscles. When including the increased lactate
production by the Ecc protocol muscles, the total energetic cost per
force-time integral was 53% higher than that for the Iso protocol
muscles [2.35 ± 0.17 vs. 1.54 ± 0.18 µmol
O2/(N · m · s)].
The decreased economy was attributed to two factors. First, in skinned
fibers isolated from the injured muscles, the ratio of maximal
actomyosin adenosinetriphosphatase activity to force production was up
by 37.5%, suggesting uncoupling of ATP hydrolysis from force
production. Second, increased reliance on anaerobic metabolism along
with the fluorescent microscopic study of mitochondrial membrane
potential and histochemical study of ATP synthase suggested an
uncoupling of oxidative phosphorylation in the injured muscles.
oxygen consumption; lactate release; actomyosin
adenosinetriphosphatase; confocal microscopy; ATP synthase
INTRODUCTION THERE ARE SEVERAL indications that energy metabolism
may be disturbed in muscle injured by eccentric contractions. First, the ratio of Pi to phosphocreatine
contents as determined by nuclear magnetic resonance spectroscopy is
increased by 45-125% in the days after initiation of the injury
(1, 20, 26). Second, glycogen levels remain depressed in injured muscle
for several days after the exercise bout (24, 33). Third, skeletal
muscle mitochondrial Ca2+ content
increases 2- and 4.5-fold immediately and 48 h, respectively, after a
bout of downhill walking in rats (10). Although low to moderate levels
of Ca2+ stimulate mitochondrial
respiration, high levels lead to uncoupling of oxidative
phosphorylation (34). Finally, the rate of
O2 consumption ( The first objective of this study was to find out whether basal
and/or active muscle energy metabolism is altered in isolated mouse extensor digitorum longus (EDL) muscle injured by eccentric contractions. Measurements of basal
On finding a decreased contraction economy in the injured muscle, the
second objective of this study was to determine whether the decreased
economy could be attributed to 1) an
increased ATP hydrolysis relative to force production and/or
2) an increased substrate
consumption (e.g., The possibility of increased
METHODS Animals
O2) drifts upward by
0.3-0.5%/min during prolonged downhill running (9, 32). Dick and
Cavanagh (9) suggested that the
O2 increase resulted from
increased muscle recruitment in an attempt to maintain power output as
fibers are injured over time.
O2 and isometric tetanus O2 recovery costs were made along
with estimates of the anaerobic contribution from measurements of
lactate and pyruvate release.
O2)
relative to ATP resynthesis (e.g., through mitochondrial uncoupling).
The first of these two possibilities was probed with two sets of
experiments, one at the whole muscle level and one at the single fiber
level. We hypothesized that a selective damage may have occurred to the
more economical fibers in the injured muscle. This was investigated by
measuring muscle maximal unloaded shortening velocity
(Vo) and maximal
shortening velocity
(Vmax) determined
from the Hill equation and by using the ratio of these two velocities
as an indicator of the heterogeneity of fiber economy within the
muscle; a ratio close to one would indicate a homogenous muscle in
terms of fiber economy. In the second set of experiments, we measured
actomyosin adenosinetriphosphatase (ATPase) activity relative to force
production in skinned single fibers taken from injured and uninjured
muscles. This allowed us to determine whether uncoupling of ATP
hydrolysis from force production occurred, possibly resulting from
impaired force transmission somewhere along the length of the fiber.
O2 relative to ATP
resynthesis was probed by using two tests for mitochondrial uncoupling
in injured muscle. In the first test, mitochondrial membrane potential was assessed by using confocal laser scanning microscopy (CLSM) of
rhodamine 123-loaded muscles in an in vitro preparation. In the second
test, mitochondrial uncoupling was evaluated by using histochemical
analysis of ATP synthase activity in injured and uninjured muscle.
Experimental Procedures
In vitro muscle preparation. Except for the experiments that involvedO2 measurements, the mouse
EDL muscle preparation was identical to that previously described
(e.g., Refs. 18, 30) with the following differences. In these
experiments, the bath chamber was maintained at 25°C instead of
37°C. Also, bovine serum albumin was not included in the
Krebs-Ringer buffer.
For the experiments that involved O2 measurements, a modified bath assembly was used. The assembly was modeled after that used by Moerland and Kushmerick (22). It consisted of a sealed chamber fitted with a polarographic electrode (model 5331 Pt/AgO2 probe, Yellow Springs Instruments) and a magnetic stirrer. There were two ports into the chamber. One was 0.8 × 31 mm (diameter × length) and permitted externalization of the suture tied to the proximal EDL tendon so that the suture could be tied to the servomotor arm. The second port (0.7 × 31 mm) was used for injection of oxygenated Krebs-Ringer buffer into the chamber. The long diffusion paths provided by these two ports acted as an effective gas seal between the muscle-bathing solution and the external environment of the chamber. Ag/AgCl electrodes (32 gauge) with monophasic pulses were used for stimulation of the muscle. When the AgCl electrode is used as the cathode, no Cl2 gas is evolved. This is important because the sensitivity of the O2 probe to Cl2 is two-thirds of that to O2. We found that platinum electrodes that use monophasic pulses cause considerable Cl2 evolution, and we were unable to elicit maximal eccentric contraction force by using platinum electrodes and biphasic pulses.
Experimental protocols. After it was
placed in the bath assembly, the muscle remained quiescent for 23 min
in experiments in which O2
and release of lactate and pyruvate measurements were made. In all
other experiments, this preincubation period was 10 min. During this
time, muscle resting force was set to 4.9 mN and muscle length
(Lo) was
measured.
At the end of the preincubation, two isometric twitches (0.2-ms pulse duration) were done 30 s apart. Thirty seconds after the second twitch, an isometric tetanus (200-ms train at 179 Hz) was performed. A second tetanus was done 2 min later. These contractions are termed the preprotocol contractions. The stimulator (models 8800 and SIU-5, Grass Instruments) and Lo were controlled by computer (Pentium 60 MHz) using a Cambridge Technology 300B servomotor, Keithley-MetraByte DAS-1602 interface board, and Laboratory Technologies NOTEBOOKpro software (version 8.03). Force and length outputs from the servomotor were sampled at 1,000 Hz.
Next, one of three contraction protocols was done. The protocols
consisted of 10 eccentric contractions (Ecc), 10 isometric contractions
(Iso), or no contractions (No). The No protocol was conducted only in
the experiment in which O2
was measured. There was no difference between the Iso and No protocol
muscles for either of the two
O2 measurements or for the
contractile decrement. For this reason, the Iso protocol served as the
control condition in later experiments.
The contraction protocol began 4 min after the second preprotocol
tetanus in all experiments except in those in which
O2 or release of lactate
and pyruvate was measured; for these experiments, an additional 20-min
delay was required for equilibration and measurement of basal
O2 rates. For the eccentric
contractions, the muscle was passively shortened from
Lo to 0.9 Lo over 3 s, stimulated to produce a tetanus of 133-ms duration while lengthened at
1.5 Lo /s to
1.1 Lo, then
returned passively to
Lo over 3 s. For
the isometric contractions, the muscle remained at
Lo and was
stimulated for 133 ms. Peak force produced on the first contraction was
198.6 ± 1.6 and 96.4 ± 0.5% of maximal isometric tetanic force (Po) for the Ecc and Iso
protocols, respectively. There were 3 min between contractions; thus
the protocol duration was 30 min. One minute after the end of the
protocol, a 200-ms isometric tetanus (postprotocol tetanus) was done.
Measurement of
O2. The
probe (model 5331, Yellow Springs Instruments) inserted into the
chamber was connected to a biological oxygen monitor (model 5300, Yellow Springs Instruments). Output from the monitor was low-pass
filtered at 0.5 Hz and subsequently acquired at 1 Hz via an interface
board (model DAS 801, Keithley-MetraByte), and the computer and
software were used for force and length measurements. PO2 in the bath chamber was
maintained at >500 Torr. At this
PO2, a sustained
O2 of more than three times the basal rate could be maintained as estimated by using a Krogh cylinder model of the muscle and a conservative diffusion coefficient of 1.98 cm2 · min
1 · Torr1
(12).
In the 4 min after the second preprotocol tetanus, additional
oxygenated Krebs-Ringer buffer was injected into the chamber. Ten
minutes were allowed to elapse for equilibration before measurement of
preprotocol basal O2 over
the next 10 min. One of the three contraction protocols was then
carried out. After the postprotocol tetanus, oxygenated Krebs was
injected into the chamber with 10 min elapsing again before starting
measurement of basal O2
over 10 min. A 2- to 8-s isometric tetanus was then done with a
stimulation frequency of 80 Hz. The lower frequency was used to reduce
fatigue and elicited a force equal to 89.5 ± 1.7% of
Po in normal EDL muscle. Stimulus
duration for the Ecc muscles was ~80% longer on average to yield
force-time integrals similar to those for the Iso and No protocol
muscles. Twenty-five minutes was allowed to elapse before another 2- to
8-s isometric tetanus was done. Basal
O2 was measured in the 10 min before this tetanus. Twenty minutes after this second tetanus,
oxygenated Krebs-Ringer solution was injected into the chamber over 5 min and the sequence of two basal
O2 and two tetanus
O2 recovery cost measurements was
repeated. Thus basal O2 was
measured beginning at 15, 40, 85, and 110 min after the end of the
contraction protocol. Isometric tetanus O2 recovery cost was measured
beginning at 25, 50, 95, and 120 min after the end of the contraction
protocol. Measurements were made on nine muscles each for the Ecc, Iso,
and No protocols.
O2 was calculated from the
net decrease in chamber PO2, chamber
volume, and solubility of O2 in
Krebs-Ringer buffer. The chamber volume (3.962 ± 0.019 ml;
n = 41) was determined from the
dilution of a known quantity of
[3H]-methoxyinulin injected into
the chamber with 5 min remaining in the experiment. The solubility of
O2 in Krebs-Ringer buffer at
25°C was assumed to be 1.52 nmol · ml1 · Torr1
(28).
The estimate of basal O2
required measurement of the O2
loss/consumption in the absence of a muscle (termed baseline
O2). Ten
experimental trials were done in which the baseline
O2 measured over the five
10-min intervals was coinciding temporally with the times when muscle
basal O2 measurements were
made. The muscle basal
O2 was determined from the
linear regression equation slope for the chamber
O2 content (after correction for
baseline O2) vs. time data.
The analysis of the 10-min intervals was over 600 data points, and the
correlation coefficient exceeded 0.99 in all but one instance out of
the 135 measurements.
The tetanus O2 recovery costs were
calculated by using an analysis program that searched for the 5-min
interval after the tetanus when the slope of the chamber
O2 content decrease over time best
matched the pretetanus slope; the search began 3 min after the tetanus.
The analysis was done by a least-squares fit of the data, forcing the
posttetanus slope to equal the pretetanus slope while allowing the
regression equation intercept to vary. The difference between the pre-
and posttetanus intercepts equaled the
O2 cost of the contraction. The
correlation coefficient for the fit of the posttetanus data exceeded
0.99 in all but two instances. Figure 1
depicts a typical data record used in determination of basal
O2 and tetanus
O2 recovery cost.
O2) and tetanus
O2 recovery cost are calculated.
Data shown are from extensor digitorum longus muscle that had performed
isometric (Iso) protocol. At 10 min, muscle was stimulated at 80 Hz for 3 s. Inset, force record. Dashed line, extension of
regression line for pretetanus change in bath
O2 saturation over time. When rate
of change in bath O2 saturation
returned to pretetanus value (i.e., 22.4 min), vertical distance
between dashed and solid lines is proportional to
O2 cost of contraction.
The tetanus O2 recovery cost was
normalized to the force-time integral (in
N · m · s/g) of the contraction.
The force-time integral (in N · s) was divided by the
muscle anatomic cross-sectional area (i.e., muscle
weight/Lo)
as described in the study of Moerland and Kushmerick (22). In
preliminary experiments with force-time integrals over the narrow range
used in this study (i.e., 0.341-1.442 N · m · s/g), we found no tendency
for an increased contraction economy at higher force-time integrals as
observed previously (8). We found
O2 cost (in µmol/g) to be
correlated to force-time integral (in
N · m · s/g)
(O2 cost = 
0.10 + 1.4
Fdt, where F
is force; r = 0.71;
P = 0.001) with the
y-intercept not different from zero
(P = 0.74). Therefore, we
opted to use the ratio of O2 cost
to force-time integral to describe contraction economy.
Lactate and pyruvate release
measurements. Forty experiments were conducted in
parallel with the O2
measurement experiments to assess the anaerobic energy metabolism
contribution. In 16 experiments (8 each for the Ecc and Iso protocols),
the four 2- to 8-s isometric tetani were done at the same times as in
the O2 measurement
experiments. In 16 experiments (8 each for the Ecc and Iso protocols),
the muscles remained quiescent during the period the four 2- to 8-s
isometric tetani would have been done (termed basal). Samples (100 µl) of the bath media were taken 5 min after the end of the Ecc or
Iso protocol and 135 min later at the end of the experiment. Muscles
were quick frozen at the end of the experiment and stored at
80°C along with the bath media samples. In addition, in
eight experiments (4 each for the Ecc and Iso protocols), the
experiments were terminated coinciding with the time of the first bath
media sampling; these experiments were done to help determine whether
muscle lactate or pyruvate content changed over time.
Measurements of lactate and pyruvate in the muscles and bath media were
performed by using the direct fluorometric assays of Lowry and
Passonneau (19). Muscle lactate content was unaffected by the time of
sampling (P 
0.92) or by the type of
contraction protocol (P 0.46).
Because of these observations and the fact that the muscle lactate
content was <0.3% of the bath lactate content, muscle lactate
content changes were not considered in the calculation of lactate
production by the muscle. The rate of pyruvate release from the muscle
was not significantly affected by the time of sampling or by the type
of contraction protocol (P 0.91).
In addition, because the mean pyruvate release rate (i.e., 23.8 ± 12.0 nmol ·
min1 · g1)
was 
15% of the lowest lactate release rate, pyruvate release was not
included in the energy use calculations nor were additional anaerobic
end-products pursued.
Lactate release per force-time integral was calculated by subtracting the basal release rate from the respective release rate for the protocol with the four 2- to 8-s tetani, multiplying by the time between samples, and dividing through by the mean sum of the four 2- to 8-s force-time integrals. Lactate release was converted into O2 equivalents by using 0.1579 mol O2/mol lactate. This number assumes a malate-aspartate NADH shuttle and coupled mitochondria, with exogenous glucose as the substrate. This latter assumption was justified because insufficient glycogen is present in the EDL muscle to support the lactate release rates observed for this muscle and because removal of insulin and glucose from the Krebs-Ringer buffer reduced lactate release by 80-85% (n = 2).
Muscle shortening velocity measurements. Vo is most likely determined by the shortening velocity of the single fastest fiber in a muscle (7, 23). Vmax as determined from extrapolating the force-velocity curve to zero load is dependent on the shortening velocity characteristics of all fibers in a muscle (7). Vmax is less than Vo in a muscle because of the heterogeneity of fiber-shortening speeds in the muscle; the greater the heterogeneity, the greater the difference is between Vmax and Vo. We hypothesized that the decreased contraction economy might result from a selective damage to the more economical fibers in a muscle. It is known that the energetic cost of an isometric contraction is 50-190% greater in the fast-twitch mouse EDL muscle than in the predominantly slow-twitch mouse soleus muscle (8, 22). If contraction economy is inversely related to Vmax (25), then a selective loss of slower contracting fibers could explain the decreased economy in the present study. We predicted that Vmax would approach Vo because the muscle would be composed of a more homogenous population of faster contracting fibers; a corollary was that Vo would remain unchanged.
In these experiments, after the postprotocol tetanus, physiological
Lo and the
stimulation frequency eliciting the maximal rate of force development
were determined because of their influence on the shortening velocity
measurement. Physiological
Lo was determined by using isometric twitches;
Lo was changed by
0.9 ± 0.8 and 6.6 ± 0.4% of the preprotocol
Lo for the Iso
and Ecc protocol muscles, respectively. The stimulation frequency that
elicited the maximal rate of force development was determined by using 20-ms stimulations at frequencies between 149 and 400 Hz. In all experiments, either a frequency of 303 or 345 Hz was found to be
optimal.
Vo was determined by using the step-release procedure described by Claflin and Faulkner (7). Measurements began 20 min after the end of the contraction protocol. Step releases from Lo of 12, 11, 10, 9, 8, and 7% Lo were made (in that order) at 3-min intervals. Stimulations of 250-ms duration were used; the first 200 ms was isometric. Force and length outputs from the servomotor were sampled at 5,000 Hz. The time for the onset of force recovery was determined by using change-point linear regression (18) of the force vs. time data.
Vmax was determined by using the procedures described by Brooks and Faulkner (6), except that after-loaded isotonic contractions were not used. Instead, quick releases were made from Po to the desired isotonic load. Measurements began 40 min after the end of the contraction protocol (i.e., 4 min after the last step release in the Vo determination). Isotonic loads of 7.5, 10, 12.5, 15, 17.5, 20, 30, 40, and 50% of Po were used in order of increasing load. Stimulations of 300-ms duration were used; the first 200 ms was isometric. There were 3 min between contractions. The declines in Po over the Vo and Vmax measurements were small: 7.4 ± 0.6 and 8.4 ± 0.8% for Ecc (n = 8) and Iso (n = 8) protocol muscles, respectively.
Shortening velocity for a given isotonic load was determined by using
linear regression analysis of the length vs. time data beginning when
the load was within 0.98 mN of the target load. The analysis program
searched for the 10-ms interval (i.e., 50 data points), giving the
highest velocity while constraining the correlation coefficient to 0.99.
Skinned single fiber measurements of actomyosin ATPase activity and force. The actomyosin ATPase activity of skinned fibers was determined by using the NADH fluorescence method (16) in which ATP is regenerated from ADP and phosphoenolpyruvate through the reaction catalyzed by pyruvate kinase. This reaction is coupled to the conversion of pyruvate to lactate by lactate dehydrogenase with the oxidation of NADH (fluorescent) to NAD (nonfluorescent).
Immediately after the postprotocol tetanus, the muscle was placed in
ice-cold relaxing solution [in mM: 125 potassium proprionate, 2 ethylene glycol-bis(
-aminoethyl
ether)-N,N,N
,N-tetraacetic acid (EGTA), 4 Na2ATP, 1 MgCl2, and 20 imidazole].
Fiber bundles (50-100 fibers) were dissected from the muscle at
random and tied to glass capillary tubes with 6-0 silk suture. The
fiber bundles were stored at 20°C in a skinning solution
containing a 1:1 mixture of relaxing solution and glycerol. Single
fiber experiments were conducted within 4 wk after the isolation. To
remove membrane-bound ATPases, fibers were treated with 1% Triton
X-100 in relaxing solution for 20 min at room temperature immediately
before a single-fiber experiment. In preliminary work, addition of
cyclopiazonic acid was found to have no effect on maximal ATPase
activity, indicating elimination of sarcoplasmic reticulum (SR)
Ca2+-ATPase activity by the Triton
X-100 treatment. In the present experiment, basal ATPase activities
were 5.2 ± 0.2 and 5.4 ± 0.3% of maximal activities for the
fibers from Iso and Ecc muscles, respectively. The reported actomyosin
ATPase rates reflect subtraction of these basal values.
Single fibers were dissected at random from the bundles. From each fiber, a 2- to 3-mm segment was mounted between a force transducer and a length controller (Scientific Instruments) by using microtweezers. The tweezers and fiber were placed in a quartz capillary cuvette with a cross-sectional area of 1 mm2 and length of 1 cm (13). Sarcomere length was adjusted to 2.50 µm with the aid of a helium-neon laser. Force- and actomyosin ATPase-Ca2+ relationships were determined by perfusing increasing free [Ca2+] [negative log of [Ca2+] (pCa) of 9-4.5] through the cuvette. The solution surrounding the fiber in the cuvette was illuminated at 340 nm, and the decrease in NADH concentration was determined from the decrease in fluorescence at 470 nm. Solutions were changed every 15 s, which allowed sufficient time for force and ATPase activity to reach a steady state. This resulted in the exposure of the fiber to 60-80 different free [Ca2+].
After the experiment, the fiber was briefly exposed to air and imaged
with a calibrated video system. The largest and smallest fiber
diameters were measured; the average of the two was used to calculate
fiber cross-sectional area assuming cylindrical geometry. The
fluorescence signal was then calibrated against NADH standards. The
actomyosin ATPase rate per unit fiber volume (µM/s) was calculated by
dividing the change in NADH over time by the product of fiber cross-sectional area and fiber segment length.
[Ca2+] required for
one-half-maximal actomyosin ATPase activity/force ([Ca2+]50)
was determined by fitting the actomyosin ATPase/force data to
the modified Hill equation by using a nonlinear curve fitting routine
(i.e., for the force data, F/Fo = [Ca2+]N · ([Ca2+]50N + [Ca2+]N)1,
where Fo is maximal
Ca2+-activated force and
superscript N is steepness of
force-Ca2+ relationship). Figure
2 depicts typical force- and actomyosin ATPase-pCa relationships determined for a fiber isolated from an Iso
protocol muscle. Measurements were made on 21 fibers each from the Ecc
and Iso protocol muscles (n = 3 each).
)- and actomyosin ATPase (
)-pCa
relationships determined for skinned single fiber isolated from muscle that had performed the Iso protocol. Fiber diameter was 66.9 µm and
Fo was 9.70 N/cm2.
[Ca2+] required for
one-half-maximal force, and actomyosin ATPase activity was 2.08 and
1.03 µM, respectively.
For the skinned fiber experiments, solutions contained 85 mM
K+, 85 mM
Na+, 2 mM
MgATP2, 1 mM
Mg2+, 1 mM EGTA, and
109 to
104.5 M
Ca2+ with proprionate as the major
anion. To measure actomyosin ATPase activity, the solutions also
contained 0.4 mM NADH, 0.2 mM
P1,P5-di(adenosine-5)pentaphosphate
(to inhibit myokinase activity), 5 mM phosphoenolpyruvate,
100 U/ml pyruvate kinase, and 140 U/ml lactate dehydrogenase. Ionic
strength was adjusted to 0.15 M, and pH was maintained at 7.0 with
imidazole proprionate. All experiments were performed at 21°C.
Assessment of mitochondrial membrane
potential. Rhodamine 123 has been widely used to assess
mitochondrial viability and mitochondrial membrane potential in
particular (see Refs. 14, 15). A 25 mM stock of rhodamine 123 (Molecular Probes) was prepared in dimethyl sulfoxide and stored at
20°C. Immediately after the postprotocol tetanus, the muscle
was incubated with 3.3 µg/ml rhodamine 123 in oxygenated Krebs-Ringer
solution for 10 min. The muscle was then mounted in a superfused
preparation for acquisition of longitudinal optical sections as
previously described (31). The superfused preparations were observed on
a Zeiss Axioskop upright microscope with a NORAN Odyssey XL CLSM
equipped with a 50-mW argon-krypton laser. A 488-nm excitation was used
with a 515-nm long-pass emission filter. A Zeiss Achroplan ×40
H2O-immersion objective (numerical aperture of 0.75) was used along with an electronic zoom of 0.8.
At four evenly spaced distances along the length of the EDL muscle, all visible fibers (i.e., up to 4 fibers deep) were categorized as staining normal or abnormal for rhodamine 123. Abnormal staining was anything different from the normal regularly spaced transverse bands. Most often the abnormal staining consisted of a diffuse stain throughout the cytosol, but rarely was the abnormal staining observed more than a few hundred micrometers along the length of a fiber. In this preparation, exposure to the uncoupler, 2,3-dinitrophenol (1 mM), elicits a diffuse cytosolic stain within 5 min and a complete loss of transverse banding by 15 min. Observations were made on six muscles each from the Ecc and Iso protocols in addition to seven muscles taken directly from the animal. Observations on a muscle were completed within 30-45 min.
Histochemical assessment of mitochondrial uncoupling. The technique described by Meijer and Vloedman (21) was used to assess the coupling state of mitochondria in frozen sections. The technique is based on forcing the ATP synthase reaction to run "in reverse" with visualization of the Pi product. Dinitrophenol (DNP) produces dark staining of the fibers, whereas oligomycin blocks the specific staining in the presence of DNP.
Ten pairs of muscles were used in this experiment. On a given day, one
muscle underwent the Ecc protocol and the contralateral muscle
underwent the Iso protocol. After the postprotocol tetanus, the muscle
was removed from the bath, cut in half transversely, mounted alongside
a block of tibialis anterior muscle, embedded in OCT (Miles), and
frozen in melting isopentane. When the muscle had warmed to cryostat
temperature (i.e., 25°C), 10-µm-thick serial sections
(n = 20-30 per muscle) were cut
and picked up on chrom-alum-coated coverslips. Immediately after a
section was picked up on a coverslip, the section was fixed for 3 min
in 4% formaldehyde-macrodex at 4°C and subsequently washed in
saline. Serial sections were then incubated for 45 min at 37°C in
one of three solutions. One was the normal incubation medium described by Meijer and Vloedman (21), while the other two also contained 1 mM
DNP. One of the two containing DNP also contained 20 µg/ml oligomycin. Sections were then washed in
H2O, immersed in 1% ammonium sulfide, washed, and mounted on a slide with glycerin jelly.
Images were captured from a model BH-2 Olympus microscope (×10
objective) with a model XC-77 charge-coupled device Hamamatsui camera
and Optimas image-analysis software. Image analysis was performed with
Jandel SigmaScan/Image software (version 1.20). Optical densities
(OD) were determined for individual fibers from serial sections stained
with the normal incubation medium and from adjacent sections stained
with the medium containing DNP. The ratio of OD for the two sections
(i.e., normal incubation medium OD/DNP medium OD) was used as an
indicator of the degree of mitochondrial uncoupling. A ratio
approaching 1.0 would suggest extreme uncoupling in a fiber. Because
the sectioning process itself induces damage to the mitochondria and
because the OD of sections exposed to DNP with oligomycin is
35-40% that of DNP-stained sections, even control muscle sections
exhibit a ratio of 0.4. Ratios were obtained on 50 randomly selected
fibers from each muscle; thus 500 fibers each for the Ecc and Iso
protocols were analyzed.
Statistical Analyses
Comparisons among the three contraction protocol muscle groups for basalO2 and
O2 cost per force-time integral
were made by using a two-way (protocol type × time) analysis of
variance (ANOVA) with repeated measures on the time factor. Comparisons between the Ecc and Iso protocol muscle groups for the lactate and
pyruvate release data were made by using a two-way (protocol type × basal or 2- to 8-s tetani) ANOVA. Comparisons between muscle groups for contractile measurements (including the
Vmax) and single-fiber force and actomyosin
ATPase measurements were made by using a one-way (protocol type) ANOVA.
To test for significant differences between protocols in the total
energetic cost per force-time integral, error propagation assuming
uncorrelated variables was applied to the lactate release data during
the calculation of the lactate cost per force-time integral. If
assumptions of normality or equal variance were violated, a
Kruskal-Wallis one-way ANOVA on ranks was used. When significant
differences were detected, Student-Newman-Keuls post hoc tests were
used.
The ATP synthase OD ratio data on the 500 fibers each from the Ecc and
Iso protocols were analyzed by using a Kruskal-Wallis one-way (protocol
type) ANOVA on ranks. To test whether a difference in the proportion of
abnormal rhodamine 123-staining fibers existed between the Ecc and Iso
protocol muscles and between the contraction protocol muscles and the
muscles taken directly from the animal, 
2 analyses
were done. All statistical testing was performed by using Jandel
SigmaStat (version 1.02/2.0). An 
level of 0.05 was
used for all statistical tests. Values presented are means ± SE except where noted.
RESULTS
The mean preprotocol Po for the
EDL muscles was 362.3 ± 5.0 mN (n = 121). Mean Lo
was 15.05 ± 0.04 mm, and mean muscle weight was 10.69 ± 0.11 mg. Po was minimally decreased by
the No (n = 9) and Iso
(n = 56) protocols (i.e., 2.7 ± 1.3 and 2.8 ± 0.5%, respectively). The decrement in
Po (i.e., 31.6 ± 0.4%) for the Ecc protocol muscle group
(n = 56) was significantly greater
than that for the other two groups (P 0.0001).
Basal and Active Muscle O2
O2 equaled
240.4 ± 26.2 nmol · min1 · g1
(n = 27; Fig.
3) and matches that predicted from the 150 nmol · min1 · g1
value observed at 20°C by Crow and Kushmerick (8) with a
Q10 temperature
coefficient of 2.5. No effect of protocol type on basal
O2 was observed at any time
point (P = 0.07). There was a
significant overall time effect (P = 0.002) in which the basal O2
measured at 40- and 85-min postprotocol was lower than the preprotocol
level. We have no good explanation for this observation other than the
possibility that the baseline
O2 may have been overestimated at those time points.
O2. No effect of protocol
type was observed (P = 0.07), but
basal O2 was significantly
lower at 40 and 85 min (as indicated by *) for all protocols.
Measurements were made on 9 muscles for each of 3 protocols. , no
contractions protocol; , 10 isometric contractions protocol; 
, 10 eccentric contractions protocol.
After a tetanus, the time required for
O2 to return to the
pretetanus value was unaffected by protocol type (9.66 ± 0.73, 8.89 ± 0.65, and 8.72 ± 0.62 min for Ecc, Iso, and No protocol muscles, respectively; P = 0.57). The O2 cost per force-time
integral was elevated by 30-36% in the Ecc protocol muscles over
that in the Iso and No protocol muscles
(P = 0.002; Table
1). The 1.27-1.33 µmol
O2/(N · m · s)
values observed for the Iso and No protocol muscles are intermediate to
the 1.8 and 0.8 µmol
O2/(N · m · s) values predicted for mouse EDL and soleus muscles, respectively, from
the data of Moerland and Kushmerick (22) when using the mean force-time
integral of the present study. One might predict a greater
O2 cost per force-time integral in
the present study compared with that in the study of Moerland and
Kushmerick because of our 5°C higher temperature (27). However, the
temperature dependence of contraction economy has not been tested in
mammalian muscle.
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Basal and Active Muscle Lactate Release
The four 2- to 8-s tetani produced a significant elevation in lactate release over basal release only for the Ecc protocol muscles (Table 1). Baseline release tended to be higher (by 27%) for the Ecc protocol muscles over that for the Iso protocol muscles. The lactate release elicited by the four 2- to 8-s tetani was 58.3% higher for the Ecc protocol muscles than that for the Iso protocol muscles.Total Energetic Cost Per Force-Time Integral
Table 1 shows the sum of the O2 and lactate energetic costs per force-time integral for the Ecc and Iso protocol muscles. Although the basal lactate release rate and lactate release rate elicited by the four 2- to 8-s tetani were not significantly different in the Iso protocol muscles, the lactate contribution was included in the total energetic cost estimate to err on the conservative side. The total energetic cost per force-time integral was 53.2% higher for the Ecc protocol muscles compared with the Iso protocol muscles (P = 0.001). The fraction of the total cost coming from anaerobic sources was approximately twofold greater for the Ecc protocol muscles (i.e., 0.27 vs. 0.14). It is important to remember that in the conversion of lactate to O2 energy equivalents, it is assumed that no mitochondrial uncoupling has occurred.Ratio of Vmax to Vo
As hypothesized, Vo was not significantly reduced in the Ecc protocol muscles compared with the Iso muscles (10.8%; P = 0.13) (Table 1). However,
Vmax was significantly
lower by 34.3% for the Ecc protocol muscles
(P 0.0001). The EDL muscle
Vmax observed by Brooks
and Faulkner (6) (9.8-10.4 fiber lengths/s) is similar to that
observed for the Iso protocol muscles (i.e., 9.6 fiber lengths/s) if a
fiber
length-to-Lo
ratio of 0.45 is assumed. The ratio of
Vmax to
Vo was not increased as
hypothesized in the Ecc protocol muscles; it was in fact decreased from
0.75 ± 0.03 in the Iso protocol muscles to 0.56 ± 0.03 in the
Ecc protocol muscles (P = 0.0004).
Ratio of Fiber Actomyosin ATPase Activity to Force
Fo was reduced by 35.6% on average in the fibers taken from the Ecc protocol muscles compared with in those taken from the Iso protocol muscles (P = 0.002) (Table 1). This decrement is comparable to the Po decrement observed in the Ecc protocol muscles (i.e., 32%). However, mean maximal actomyosin ATPase activity was only down by 17.6% in these same fibers from the Ecc protocol muscles (P = 0.014). The maximal actomyosin ATPase activities observed were marginally higher (i.e., 20-46%) than those reported for rat IIb fibers (4) after correcting for temperature differences between the two studies. The ratio of maximal actomyosin ATPase activity to Fo was increased by 37.5% in the fibers from the Ecc protocol muscles compared with that in fibers from the Iso protocol muscles (i.e., 103.7 ± 5.4 vs. 75.4 ± 2.9 µM · cm2 · N1 · s1)
(P 0.0001). If the injured fibers
were active during stimulation of the whole muscle (which they may not
be; Refs. 3, 30), then ~70% of the increase in total energetic cost
per force-time integral for the Ecc protocol muscles could be accounted
for by an elevated actomyosin ATPase activity relative to force
production.
A small decrease in
Ca2+-sensitivity was observed for
both actomyosin ATPase activity and force production.
[Ca2+]50
was 1.10 ± 0.03 and 2.04 ± 0.05 µM for actomyosin ATPase
activity and force, respectively, for the Iso protocol muscles. The
[Ca2+]50
increased to 1.45 ± 0.07 (P 0.0001) and 2.33 ± 0.08 µM
(P = 0.02) for actomyosin ATPase and
force, respectively, for the Ecc protocol muscles.
Mitochondrial Uncoupling
Figure 4 shows two CLSM images of rhodamine 123-stained muscle fibers; Fig. 4A shows a control muscle, and Fig. 4B shows an injured muscle. Of the 662 fibers observed from the Ecc protocol muscles, 31.7% stained abnormally for rhodamine 123. This percentage was significantly greater (P 0.0001) than that for the Iso
protocol muscles (i.e., 17.4% of 768 fibers). These observations
suggest a decreased mitochondrial membrane potential and viability in the injured muscles. Unexpectedly, the percentage of abnormally stained
fibers in the Iso protocol muscles was significantly higher (P = 0.001) than the percentage in
muscles taken directly from the animal (i.e., 4.8% of 856 fibers). In
the histochemical assessment of mitochondrial uncoupling, the ratio of
OD for the normal ATP synthase-stained sections to that for the
sections incubated in the presence of DNP was significantly shifted to
higher values in the Ecc protocol muscles
(P 0.0001). The median
ratios were 0.912 and 0.874 for the Ecc and Iso protocol muscles,
respectively. This equates to an 8% increase in the OD ratio for the
Ecc protocol muscles if the true range of the OD ratio is considered
(i.e., ~0.4 to 1). These OD ratio data are indicative of a decreased mitochondrial coupling of O2
to ATP resynthesis in the injured muscles.
DISCUSSION
The data from the present study suggest that the altered metabolite
contents (e.g., Pi, glycogen)
observed in muscles injured by eccentric contractions may result from
an abnormal active muscle metabolism rather than from an abnormal basal
metabolism. We failed to observe any significant elevation in basal
O2 or in the basal rate of
lactate release for the muscles injured by eccentric contractions. In
the past, we had hypothesized that elevated basal metabolism might
occur due to increased SR and plasmalemmal
Ca2+-ATPase activities
after influx of extracellular Ca2+
through damaged plasmalemma (2). However, it must be kept in mind that
the observations of the present study extend to only 2 h past
initiation of the injury. An elevated basal metabolism occurring at
later times remains a possibility.
Our failure to observe a basal
O2 increase may have been due
in part to methodological problems. We observed a relatively high
O2 loss/consumption rate from the
chamber in the absence of muscle (i.e., baseline rate equal to 65.8%
of the total rate). However, the SE of the baseline
O2 measurement was only 4.0% of the mean value, so we should have detected any meaningful
elevation in O2. Yet no hint
of an increased basal O2 was
observed at any postprotocol time. There was a trend for an increased
basal lactate release in the injured muscles. However, when this
increased release is converted into
O2 equivalents (i.e., 6.7 nmol
O2 · g1 · min1),
it is obviously too small to be of any metabolic consequence.
These observations are in line with our previous observations in which we failed to observe elevated free cytosolic [Ca2+] ([Ca2+]i) in injured muscles except in focal areas within a small number of fibers (31). Recently, Balnave and Allen (3) observed increased resting [Ca2+]i in single intact mouse fibers injured by eccentric contractions. However, the elevations were small, ranging from ~20 nM to no higher than 40 nM. Using data from isolated rat SR vesicles at 25°C (11), one would predict the 20 nM increase in [Ca2+]i to have a negligible effect on SR uptake or Ca2+-ATPase activity (i.e., increase from 0.01 to 0.03 of maximal velocity).
Even if basal metabolism is not elevated, the altered metabolite contents in injured muscles could be explained by decreased contraction economy. The energetic cost of doing an isometric contraction was increased by 53% in the injured muscle. We believe that this increase was not due to a selective loss of the more economical fibers in the EDL muscle. The results from the Vmax experiment suggest just the opposite. The reduction in Vmax relative to Vo suggests a preferential loss of faster, less economical fibers. This conclusion is in agreement with observations of a preferential injury to fast twitch-glycolytic fibers in rabbit tibialis anterior muscle injured by eccentric contractions (17).
Most of the decrease in contraction economy in the injured muscle can be attributed to an increased oxidative metabolism relative to force production with the O2 cost per force-time integral up by 30-36%. The anaerobic fraction of the total energetic cost doubled as a result of the injury but still made up only a small percentage (i.e., 27%) of the total energetic cost. An increased use of glucose as substrate could account (at least partially) for both of these observations. A shift from oxidation of primarily free fatty acids to glucose would increase the moles of O2 consumed per mole of ATP produced. For example, oxidation of glucose consumes 27% more O2 per mole of ATP produced than does the oxidation of palmitate. However, we consider an extreme substrate shift an unlikely possibility. Furthermore, Crow and Kushmerick (8) observed a respiratory quotient of ~1 in their isolated mouse muscle preparation, indicating that the substrate supporting recovery oxidation was glycogen or glucose. An increased reliance on glucose as an aerobic or anaerobic substrate could contribute to the depressed muscle glycogen content observed after injury (24, 33).
However, a decreased contraction economy should not result in a persistent alteration in phosphagen content unless ATP resynthesis processes were adversely affected. Our observations of an increased incidence of mitochondrial membrane depolarization and of histochemical signs of mitochondrial uncoupling in the injured muscle are suggestive of impaired ATP resynthesis processes. This thought is also in congruence with our previous observations of increased mitochondrial [Ca2+] in injured muscle (10); Ca2+ overload in mitochondria is known to cause uncoupling of oxidative phosphorylation (34).
It does seem unusual that a mitochondrial abnormality would manifest
itself only during or immediately after a contraction and not during a
basal condition. Perhaps the fraction of mitochondria exhibiting
uncoupling is offset by a fraction of nonfunctional mitochondria so the
net effect would be no net change in basal O2 on a per unit
mass basis. It is also possible (although unlikely) that the coupled
mitochondrial fraction could handle the respiratory requirements under
basal conditions but that additional uncoupled mitochondria are
"recruited" as a result of contractile activity.
Two observations argue against the existence of significant mitochondrial uncoupling in the present study. First, the time required for O2 recovery after a contraction was not significantly longer for the Ecc protocol muscles as one might expect if mitochondrial uncoupling had occurred. Second, if mitochondrial uncoupling occurs only after a contraction, then why did we observe the mitochondrial abnormalities under basal conditions? However, the elevated lactate release by the Ecc muscles under active but not basal conditions does suggest that oxidative phosphorylation was capable of supplying ATP resynthesis requirements under basal conditions but not after a tetanus.
The rate of ATP hydrolysis in skinned fibers from the injured muscles was "excessive" in relation to the amount of force produced. If the increase in actomyosin ATPase activity relative to force production also occurs in the intact muscle, then this increase would act to exacerbate any existing defect in the ATP resynthesis processes by further reducing phosphagen content. However, we cannot assume that all fibers in an injured muscle are active during a contraction. Observations that excitation-contraction coupling failure occurs in the eccentric contraction-injured muscle (29) and fiber (3) argue against such an assumption. However, in the present study, we see no evidence for excitation-contraction failure. Unlike in the past (i.e., Ref. 30), the injured muscle Po decrement can be completely explained by the average decrement in the single fiber Fo. Perhaps a temperature dependence exists for the relative contribution of factors responsible for the muscle Po decrement; all previous experiments in this laboratory have been conducted at 37°C.
It is interesting to speculate on the cause of the increased maximal
actomyosin ATPase activity relative to
Ca2+-activated force in the
skinned fiber. A logical explanation would be that transmission of
force to the fiber segment ends is impaired. Force could be
"generated," but due to disruption of some force-bearing proteins
(e.g., myosin rod, nebulin, -actinin, titin), the force is not
efficiently transmitted in the direction parallel to the long axis of
the fiber. An alternative explanation is based on the model of
cross-bridge cycling kinetics described by Brenner (5). In Brenner's
model, the ratio of actomyosin ATPase activity to force is equal to the
rate constant of cross-bridge dissociation divided by the average force
per cross bridge (Favg). Our
data show that the actomyosin ATPase activity-to-force ratio was
increased under conditions of maximal
Ca2+ activation. They also
indicate that although there were decreases in
Ca2+ sensitivity for both force
and actomyosin ATPase activity in the injured fibers, the actomyosin
ATPase activity-to-force ratio of the injured fibers was consistently
increased by ~38% across all levels of free
Ca2+. This suggests two possible
mechanisms accounting for alterations in contractile apparatus function
in injured fibers. First, it is possible that
Favg is decreased as a result of
muscle injury. This could result from some population of myosin heads
becoming "dysfunctional" or from a loss of some regulatory
protein required for maximal cross-bridge force production. In either
case, diminished Favg would lead
to reduced Fo and increased
actomyosin ATPase activity-to-force ratio. Second, it is possible that
the rate constant of cross-bridge dissociation is also accelerated.
This would result in a decrease in the time each cross bridge spends in
the force-generating state and hence lower both force output and
actomyosin ATPase activity. Although this effect is not likely to
markedly alter Fo, it could change
force at submaximal levels of free
Ca2+ and lower
Ca2+ sensitivity.
In conclusion, the data from this study indicate that contraction
economy is decreased but basal energy metabolism is not elevated in
muscles during the ~2 h after injury initiation. Most of the decrease
in contraction economy may be explained by an increased actomyosin
ATPase activity relative to the force produced, but we cannot rule out
the possibility that increased
Ca2+-ATPase and/or
Na+-K+-ATPase
activities contribute to the decreased contraction economy. However, we
do not favor this latter possibility because these two ATPases normally
account for 30% of the energetic cost of a contraction (25).
Finally, the data suggest that mitochondrial uncoupling occurs in
injured muscles, although this needs to be confirmed by additional
experiments.
ACKNOWLEDGEMENTS
The authors thank Dr. T. Moerland for help in the design of the experiments and in interpretation of the data.
FOOTNOTES
Address for reprint requests: G. L. Warren, 158 Read Bldg., Texas A&M Univ., College Station, TX 77843-4243.
Received 22 May 1996; accepted in final form 2 August 1996.
REFERENCES
| 1. | Albridge, R., E. B. Cady, D. A. Jones, and G. Obletter. Muscle pain after exercise is linked with an inorganic phosphate increase as shown by 31P-NMR. Biosci. Rep. 6: 663-667, 1986. |
| 2. | Armstrong, R. B., G. L. Warren, and J. A. Warren. Mechanisms of exercise-induced muscle fibre injury. Sports Med. 12: 184-207, 1991. |
| 3. | Balnave, C. D., and D. G. Allen. Intracellular calcium and force in single mouse muscle fibres following repeated contractions with stretch. J. Physiol. Lond. 488: 25-36, 1995. |
| 4. | Bottinelli, R., M. Canepari, C. Reggiani, and G. J. Stienen. Myofibrillar ATPase activity during isometric contraction and isomyosin composition in rat single skinned muscle fibres. J. Physiol. Lond. 481: 663-675, 1994. |
| 5. | Brenner, B. The effect of Ca2+ on cross-bridge turnover kinetics on skinned single rabbit psoas fibers: implications for regulation of muscle contraction. Proc. Natl. Acad. Sci. 85: 3265-3269, 1986. |
| 6. | Brooks, S. V., and J. A. Faulkner. Contractile properties of skeletal muscles from young, adult and aged mice. J. Physiol. Lond. 404: 71-82, 1988. |
| 7. | Claflin, D. R., and J. A. Faulkner. The force-velocity relationship at high shortening velocities in the soleus muscle of the rat. J. Physiol. Lond. 411: 627-637, 1986. |
| 8. | Crow, M. T., and M. J. Kushmerick. Chemical energetics of slow- and fast-twitch muscles of the mouse. J. Gen. Physiol. 79: 147-166, 1982. |
| 9. | Dick, R. W., and P. R. Cavanagh. An explanation of the upward drift in oxygen uptake during prolonged sub-maximal downhill running. Med. Sci. Sports Exercise 19: 310-317, 1987. |
| 10. | Duan, C., M. D. Delp, D. A. Hayes, P. D. Delp, and R. B. Armstrong. Rat skeletal muscle mitochondrial [Ca2+] and injury from downhill walking. J. Appl. Physiol. 68: 1241-1251, 1990. |
| 11. | Ferrington, D. A., J. C. Reijneveld, P. R. Bar, and D. J. Bigelow. Activation of the sarcoplasmic reticulum Ca2+-ATPase induced by exercise. Biochem. Biophys. Acta-Biomembranes 1279: 203-213, 1996. |
| 12. | Groebe, K., and G. Thews. Role of geometry and anisotropic diffusion for modeling PO2 profiles in working red muscle. Respir. Physiol. 79: 255-278, 1990. |
| 13. | Guth, K., and R. Wojciechowski. Perfusion cuvette for the simultaneous measurement of mechanical, optical, and energetic parameters of skinned muscle fibres. Pfluegers Arch. 407: 552-557, 1986. |
| 14. | Jiang, T., R. L. Grant, and D. Acosta. A digitized fluorescence imaging study of intracellular free calcium, mitochondrial integrity and cytotoxicity in rat renal cells exposed to ionomycin, a calcium ionophore. Toxicology 85: 41-65, 1993. |
| 15. | Johnson, L. V., M. L. Walsh, and L. B. Chen. Localization of mitochondria in living cells with rhodamine 123. Proc. Natl. Acad. Sci. USA 77: 990-994, 1980. |
| 16. | Kerrick, W. G. L, J. D. Potter, and P. E. Hoar. 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. |
| 17. | Lieber, R. L., and J. Fridén. Selective damage of fast glycolytic fibres with eccentric contraction of the rabbit tibialis anterior. Acta Physiol. Scand. 133: 587-588, 1988. |
| 18. | Lowe, D. A., G. L. Warren, C. P. Ingalls, D. B. Boorstein, and R. B. Armstrong. Muscle function and protein metabolism after initiation of eccentric contraction-induced injury. J. Appl. Physiol. 79: 1260-1270, 1995. |
| 19. | Lowry, O. H., and J. V. Passonneau. A Flexible System Of Enzymatic Analysis. New York: Academic, 1972, p. 195-196. 213. |
| 20. | McCully, K. K., A. Zohar, B. P. Boden, R. L. Brown, W. J. Bank, and B. Chance. Detection of muscle injury in humans with 31P-magnetic resonance spectroscopy. Muscle Nerve 11: 212-216, 1988. |
| 21. | Meijer, A. E. F. H., and A. H. T. Vloedman. The histochemical characterization of the coupling state of skeletal muscle mitochondria. Histochemistry 69: 217-232, 1980. |
| 22. | Moerland, T. S., and M. J. Kushmerick. Contractile economy and aerobic recovery metabolism in skeletal muscle adapted to creatine depletion. Am. J. Physiol. 267 (Cell Physiol. 36): C127-C137, 1994. |
| 23. | Nelson, A. G., and W. J. Thompson. Contractile properties and myosin phenotype of single motor units from neonatal rats. Am. J. Physiol. 266 (Cell Physiol. 35): C919-C924, 1994. |
| 24. | O'Reilly, K. P., M. J. Warhol, R. A. Fielding, W. R. Frontera, C. N. Meredith, and W. J. Evans. Eccentric exercise-induced muscle damage impairs muscle glycogen repletion. J. Appl. Physiol. 63: 252-256, 1987. |
| 25. | Rall, J. A. Energetic aspects of skeletal muscle contraction: implications of fiber types. Exercise Sport Sci. Rev. 13: 33-74, 1985. |
| 26. | Rodenburg, J. B., R. W. Deboer, P. Schiereck, C. J. Van Echteld, and P. R. Bar. Changes in phosphorus compounds and water content in skeletal muscle due to eccentric exercise. Eur. J. Appl. Physiol. Occup. Physiol. 68: 205-213, 1994. |
| 27. | Rome, L. C., and M. J. Kushmerick. Energetics of isometric contractions as a function of muscle temperature. Am. J. Physiol. 244 (Cell Physiol. 13): C100-C109, 1983. |
| 28. | Umbreit, W. W., R. H. Burris, and J. F. Stauffer. Manometric Techniques (4th ed.). Minneapolis, MN: Burgess, 1964, p. 5. |
| 29. | Warren, G. L., D. A. Hayes, D. A. Lowe, C. J. Karwoski, B. M. Prior, and R. B. Armstrong. Excitation failure in mouse soleus muscle injured by eccentric contractions. J. Physiol. Lond. 468: 487-499, 1993. |
| 30. | Warren, G. L., D. A. Hayes, D. A. Lowe, J. H. Williams, and R. B. Armstrong. Eccentric contraction-induced injury in normal and hindlimb-suspended mouse soleus and EDL muscles. J. Appl. Physiol. 77: 1421-1430, 1994. |
| 31. | Warren, G. L., D. A. Lowe, D. A. Hayes, M. A. Farmer, and R. B. Armstrong. Redistribution of cell membrane probes following contraction-induced injury of mouse soleus muscle. Cell Tissue Res. 282: 311-320, 1995. |
| 32. | Westerlind, K. C., W. C. Byrnes, and R. S. Mazzeo. A comparison of the oxygen drift in downhill vs. level running. J. Appl. Physiol. 72: 796-800, 1992. |
| 33. | Widrick, J. J., D. L. Costill, G. K. McConell, D. E. Anderson, D. R. Pearson, and J. J. Zachwieja. Time course of glycogen accumulation after eccentric exercise. J. Appl. Physiol. 72: 1999-2004, 1992. |
| 34. | Wrogemann, K., and S. D. J. Pena. Mitochondrial calcium overload: a general mechanism for cell-necrosis in muscle diseases. Lancet 1: 672-674, 1976. |
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Fdt,
µmol /(N · m · s)
