Increased oxidative capacity does not protect skeletal
muscle fibers from eccentric contraction-induced injury
T. J. PATEL,1 D. CUIZON,1 O. MATHIEU-COSTELLO,1 J. FRIDÉN,2 AND R. L. LIEBER1
of Orthopaedics, Medicine, and Bioengineering, Biomedical Sciences Graduate Group,
University of California and Veterans Affairs Medical Centers, San Diego, California 92161;
and 2Department of Hand Surgery, Göteborg University, Göteborg, Sweden S-412
1Departments
Patel, T. J., D. Cuizon, O. Mathieu-Costello, J. Fridén,
and R. L. Lieber. Increased oxidative capacity does not
protect skeletal muscle fibers from eccentric contractioninduced injury. Am. J. Physiol. 274 (Regulatory Integrative
Comp. Physiol. 43): R1300–R1308, 1998.—Isometric electrical stimulation was delivered to rabbit dorsiflexor muscles at
10 Hz for 1 s on and 1 s off over 30 min, 5 days/wk for 3 wk to
induce an increase in muscle oxidative capacity. Stimulationtrained muscles as well as untrained muscles were then
subjected to a 30-min eccentric exercise bout to test whether
increased oxidative capacity provided a protective effect
against muscle injury. Electrical stimulation resulted in
significant training of both the extensor digitorum longus
(EDL) and tibialis anterior (TA) muscles, with EDL citrate
synthase (CS) activity increasing an average of 67% (P ,
0.0001) and TA CS activity increasing by 27% (P , 0.05). For
all parameters measured, the magnitude of change was much
greater for EDL than for TA muscle. Dorsiflexor fatigability
decreased significantly during the 3-wk training period (P ,
0.0001), whereas the EDL and TA individually showed strong
decreasing trends in fatigability after training. TA and EDL
capillary density measured histomorphometrically increased
from 839 6 56 to 1,026 6 71 mm22 (P 5 0.07) and from 589 6
37 to 792 6 66 mm22 (P , 0.05), respectively. TA and EDL
capillary-to-fiber ratio increased from 1.32 6 0.10 to 1.55 6
0.16 (P . 0.2) and 1.08 6 0.07 to 1.36 6 0.14 (P . 0.1),
respectively. Type 2A fiber type percentage increased after
stimulation training by 68% (P , 0.0001) for the EDL and by
32% (P . 0.1) for the TA at the expense of type 2D fibers.
Despite the large training effect for the EDL and the modest
training effect for the TA, no differences were observed
between stimulation-trained and untrained groups for maximum dorsiflexion torque (P . 0.3) or maximum tetanic
tension (P . 0.3) after eccentric contraction-induced injury.
Additionally, no significant correlation was observed between
CS activity and maximum tetanic tension after eccentric
contraction-induced injury for either muscle (P . 0.2). Thus
we conclude that increasing muscle oxidative capacity by
isometric electrical stimulation training did not protect muscle
against eccentric contraction-induced injury.
muscle metabolism; fiber type; muscle mechanics
NUMEROUS INVESTIGATIONS DEMONSTRATE that skeletal
muscle injury and soreness result from the forced
lengthening of activated muscle [i.e., eccentric contraction (EC)]. Experimental models involving both humans (6, 9) and animals (1, 21) have identified a
number of factors that affect the initial injury itself as
well as subsequent tissue deterioration. For example,
mechanical stress (38) and strain (14) have both been
shown to be strong predictors of the magnitude of force
loss after EC. After the initial injury, inflammation
causes further tissue deterioration (4, 5, 22).
R1300
EC-induced injury is also fiber type specific. For
example, using the rat downhill running model, preferential damage of the deep vastus intermedius muscle
fibers was shown (35) and was interpreted as indicating
preferential activation of these fibers. In eccentric
exercise of human quadriceps muscles, Fridén and
co-workers (10) demonstrated a greater incidence of
myofibrillar disruption to the ultrastructurally identified type 2B fibers compared with either type 2A or type
1 fibers. They also interpreted the decreased isokinetic
torque observed at high angular velocities as indicating
selective type 2B fiber damage. Lieber and Fridén (13)
demonstrated selective damage of histochemically identified type fast glycolytic (FG) fibers in the rabbit
tibialis anterior (TA) muscle after 30 min of cyclic EC.
On the basis of this observation, they hypothesized that
the low oxidative capacity of FG fibers predisposed
them to injury either due to rigor cross-bridge formation or to initiation of degradative cellular events
associated with loss of cellular adenylate charge (cytosolic [ATP]/[ADP] and [Pi]) that may serve as a stimulus for lactate production or may activate cellular
degradative processes during and after cyclic EC. The
appeal of this hypothesis was that it provided a metabolic basis for the observed protective effect of eccentric
training before eccentric exercise (9). However, this
hypothesis has not been tested. It is possible that the
eccentric action itself causes cellular changes independent of cellular metabolism, such as increased sarcomere number (23) or increased cytoskeletal strength
(2), that may be protective against EC-induced muscle
injury. Thus the purpose of this study was to increase
muscle oxidative capacity to test the hypothesis that
muscle damage resulting from EC is a function of
oxidative capacity. Brief versions of this work have
been presented (25, 26).
METHODS
Animal care. TA and extensor digitorum longus (EDL)
muscles from New Zealand White rabbits (n 5 43, mass 5
3.11 6 0.04 kg) were used in this study. Animal care adhered
to the Guide for the Care and Use of Laboratory Animals and
was approved by the University of California, San Diego, and
Veterans Affairs Committees on Animal Subjects. After terminal experiments, all animals were euthanized by intracardiac
perfusion of glutaraldehyde fixative.
Experimental design. Experimental animals were divided
into the following four groups: control (C, n 5 17), consisting
of normal animals age and weight matched to the experimental animals; stimulation-trained only animals (S 1 C, n 5 9)
that received 3 wk of isometric electrical stimulation training;
EC-only animals (EC, n 5 8) that received only a single
30-min bout of eccentric exercise, and stimulation-trained
0363-6119/98 $5.00 Copyright r 1998 the American Physiological Society
OXIDATIVE CAPACITY DOES NOT AFFECT MUSCLE INJURY
plus EC animals (S 1 EC, n 5 9) that received 3 wk of
isometric electrical stimulation training followed by a single
bout of noninvasive eccentric exercise (described below). The
3-wk electrical stimulation period was chosen based on pilot
studies demonstrating that this treatment duration was
sufficient to increase muscle oxidative capacity but not so
great as to result in fiber size decrease or fast-to-slow fiber
type transformation that results from chronic stimulation
(30). This permitted testing the effect of increased oxidative
capacity alone on muscle injury. Isoflurane gaseous anesthesia was used for induction and maintenance of anesthesia to
permit rapid recovery after daily stimulation treatment.
In vivo noninvasive EC treatment. Noninvasive EC was
produced as previously described (16). Briefly, rabbits were
induced with and then maintained on isoflurane anesthesia
(,2% at 1 l/min). Heart and respiratory rates were monitored
manually throughout testing.
A dual-mode servomotor (model 6400; Aurora Scientific)
with an adjustable foot plate attached to the motor arm
measured dorsiflexion torque during muscle activation. For
all noninvasive mechanical testing, rabbits’ hindlimbs were
stimulated supramaximally with electrodes placed perpendicular to the peroneal nerve as the ankle was moved from
,100° to 70° over a 400-ms period (stretched) and then
returned to the starting position (shortened). The stretching
movement during peroneal nerve activation induced eccentric exercise of TA and EDL muscles. Muscles were not
activated during shortening. This stimulation and applied
deformation pattern was repeated every 2 s for 30 min,
resulting in 900 cyclic ECs.
In vivo noninvasive isometric stimulation training treatment. For noninvasive isometric stimulation training treatment alone, anesthesia was induced as described in In vivo
noninvasive EC treatment, and electrical stimulation of the
right and left peroneal nerves was elicited at 10 Hz and cycled
for 1 s on and 1 s off over a 30-min stimulation training
treatment period. Each foot, secured to a foot plate connected
to a torque motor, was maintained at tibiotarsal and femoraltibial joint angles of 100° and 90°, respectively, during
stimulation training treatment. Dorsiflexion torque was continuously monitored throughout stimulation training treatment. In addition, for each day of stimulation training
treatment, fatigue index of the dorsiflexors as a whole was
calculated as the ratio of dorsiflexion torque at 2 min to
maximum torque (which occurred during the first 30 s). This
permitted analysis of the change in fatigability of the dorsiflexors over the 3-wk stimulation training period. Dorsiflexor
fatigue index was also calculated as the ratio of torque at 2
min to initial torque (data not shown), and no qualitative or
statistical difference was observed compared with dorsiflexor
fatigue index, calculated as the ratio of torque at 2 min to
maximal torque, as described above. Fatigue index was also
calculated for isolated TA and EDL muscles during in situ
contractile testing as described by Burke et al. (3). Individual
muscle fatigue index was calculated as the ratio of force at 2
min to the initial force after a 2-min isometric stimulation
bout at 40 Hz and train duration of 330 ms. Both right and left
dorsiflexors in each animal were stimulation trained. This
permitted analysis of frozen as well as perfused tissue at the
end of the study (see In situ muscle contractile testing). After
each stimulation training treatment, animals recovered on a
heating pad and were monitored until they were fully awake
and resuming normal activity.
Joint torque testing. For joint torque determination, both
twitch and tetanic contractions were measured. Stimulation
frequencies of 5, 10, 15, 20, 40, 60, 80, 100, and 200 Hz, with a
train duration of 1,000 ms (for frequencies ,80 Hz) or 800 ms
R1301
(for frequencies .80 Hz) were used to generate the torquefrequency relationship. All torque records demonstrated a
clear maximum. Maximum tetanic torque was defined as the
peak of the force-frequency relationship, which occurred at
either 100 or 200 Hz for all rabbits tested.
In situ muscle contractile testing. Three days after the final
stimulation treatment of each experimental group, rabbits
were anesthetized and the TA and EDL tendons were isolated
and clamped to separate motors (models 360 and 6400;
Aurora Scientific). On the basis of previous studies, the
greatest loss of force occurs 3 days after eccentric exercise
(16). Thus we chose this time period to provide the greatest
sensitivity to the treatment effects. Testing methods used
were as described previously (15). Briefly, an incision was
made from the ankle to mid-thigh and the peroneal nerve was
located. The leg was immobilized using Steinmann pins at the
distal femur and mid-tibia that were secured in a rigid
fixture. Distal TA and EDL tendons were clamped and
aligned with their respective motor measurement axes. A
small cuff electrode was placed around the nerve. Muscle
temperature was maintained at 37°C using radiant heat,
mineral oil, and a servo-temperature controller (model 73A;
Yellow Springs Instruments, Yellow Springs, OH). TA and
EDL muscle lengths were adjusted until twitch tension was
maximal (Lo ), and contractile properties were measured with
supramaximal stimulation during twitch and tetanic contractions. Stimulation frequencies were the same as those used in
the noninvasive procedure.
After contractile testing and removal of muscles on one side
for freezing for immunohistochemical and biochemical analysis, contralateral muscles were perfusion fixed in situ for
stereological analysis (19). The entire vasculature was perfused by direct cannulation of the left ventricle, with the right
atrium cut open to permit outflow. Perfusion consisted of an
initial saline wash to remove blood cells (11.06 g NaCl/l; 350
mosM; 20,000 USP units heparin/l) followed by 6.25% glutaraldehyde solution in 0.1 M sodium cacodylate buffer (total
fixative osmolarity 1,100 mosM; pH 5 7.4). All perfusions
were performed at a nonpulsatile pressure of 120 mmHg. To
insure adequate perfusion, the vasodilator prazosin was
administered at a dose of 0.33 mg/kg the night before the
terminal experiment. After 10 min of perfusion with the
glutaraldehyde fixative, the TA and EDL were excised and the
midbelly (1.5 3 0.5 3 0.4 cm) was cut into thin longitudinal
strips, stored in glutaraldehyde fixative, and processed for
electron microscopy as described previously (19). The nonperfusion-fixed muscles were oriented on cork, frozen in
isopentane cooled by liquid nitrogen (2159°C), and stored at
280°C for processing.
Tissue analysis was performed on TA and EDL muscles
from each group, but these muscles were not always the
identical muscles from which contractile data were obtained
(46 of 52 muscles tested were also subjected to tissue analysis).
Immunohistochemistry. Serial transverse sections of TA
and EDL (n 5 30 muscles) were sectioned at 220°C and
mounted onto glass slides. Sections were stained with antibodies against various myosin heavy chain (MHC) isoforms. The
following antibodies (provided by Prof. S. Schiaffino, Padua,
Italy, and generated for use on rat muscle) were used: F8,
reactive with slow MHC; BF13, reactive with all type 2 MHC;
BF35, reactive with all MHC except the 2X MHC; and SC71,
reactive with 2A MHC. On the basis of the observation that,
unlike other muscles in rabbit, TA and EDL muscles do not
have a protein band that migrates in the 2B position (11),
muscle fibers were classified as type 1, type 2A, or type 2D,
the only three fibers present in normal rabbit TA and EDL.
R1302
OXIDATIVE CAPACITY DOES NOT AFFECT MUSCLE INJURY
Furthermore, pilot experiments demonstrated that muscle
fibers typed on the basis of immunohistochemistry were
similar to those fibers classified histochemically (27) with
myofibrillar ATPase and oxidative and glycolytic enzyme
activity. Individual fibers were typed from examination of
serial sections stained with all antibodies. Fiber areas were
measured using Image1 analysis program (Universal Imaging, West Chester, PA) interfaced to a Nikon Microphot light
microscope (Nikon, New York, NY). Fibers (n 5 119 6 8 fibers
from each EDL muscle and n 5 140 6 8 fibers from each TA
muscle) were sampled within frozen sections based on the
systematic sampling principles suggested by Weibel (40).
Stereological methods. Mitochondrial and capillary parameters were measured. They were estimated by light (capillary) and electron microscopy (mitochondria) using procedures described previously (19, 20). Briefly, 1-µm-thick
transverse and longitudinal sections were cut on an LKB
Ultratome III, stained with 0.1% aqueous toluidine blue
solution, and examined by light microscopy. Ultrathin transverse sections (50–70 nm) were contrasted with uranyl
acetate and bismuth subnitrate (33). Electron micrographs
for morphometry were taken on 70-mm films with a Zeiss 10
electron microscope.
Eight randomly chosen blocks were cut into four transverse
sections (angle between each section and fiber axis, a 5 0°)
and four longitudinal sections (a 5 90°) from each of five TA
and EDL muscles from S 1 EC and EC groups. Capillary
density [QA(0)] 5i.e., number per fiber cross-sectional area
[a(f )]6 was estimated by point counting on 1-µm-thick transverse sections examined at magnification 3400. On average,
35 6 2 (SE) fields were examined per sample, yielding ,1,120
fiber profiles for each muscle. We measured a(f ) with an
image analyzer (Videometric 150, American Innovision). On
average, 136 6 8 (SE) fibers were measured per sample. They
were randomly selected by systematic sampling of the same
transverse sections used to estimate QA(0). Sarcomere length
was measured in each longitudinal section (average of 10
measurements of groups of consecutive sarcomeres systematically sampled over the entire area of each section examined at
magnification 31,000). We then normalized a(f ) and QA(0) to
a sarcomere length of 2.1 µm, as previously described (20), to
remove the effect of variable fiber shortening between samples.
Capillary-to-fiber ratio [NN(c,f )] was computed as the
product of QA(0) and a(f ). Capillary surface per fiber volume,
SV(c,f ), was measured by intersection counting on vertical
sections with a cycloid grid as described previously (20). A
total of 60 6 3 (SE) fields randomly selected by systematic
sampling on the same longitudinal sections used to measure
sarcomere length were examined at a magnification of 3400.
Volume density of mitochondria per volume of muscle fiber,
[VV(mit,f )] was estimated by standard point counting at a
final magnification of 349,000. Twenty micrographs were
obtained by systematic sampling in one ultrathin section
from each block (80 micrographs per sample). Contact prints
of the electron micrograph films were projected onto a 144point square grid using a microfilm reader (Documator DL 2;
Jenoptic, Jena).
CS assay. Citrate synthase (CS) activity was measured to
estimate whole muscle oxidative capacity as one estimate of
the magnitude of stimulation training. Transverse tissue
blocks, 1–1.5 mm thick, (n 5 120 blocks from 60 muscles;
mass 5 48 6 1 mg) were homogenized, and CS activity was
measured spectrophotometrically by placing the homogenate
in a medium containing (in mM): 1 5,5-dithiobis-(2-nitrobenzoic acid), 3 acetyl CoA, and 100 Tris buffer at pH 8.0. The
reaction was initiated using oxaloacetic acid as the substrate,
and absorbance was measured every 30 s for 4 min. The slope
of the absorbance-versus-time relationship, determined by
linear regression, yielded reaction rate. Two separate crosssectional blocks of the entire muscle midbelly were obtained
from each muscle to insure that CS activity measurements
were representative of the entire cross section. Betweensample variation was typically ,5%.
SDH assay. Succinate dehydrogenase (SDH) activity was
measured in frozen sections to estimate individual muscle
fiber oxidative capacity. Ten-micrometer-thick transverse sections from TA (n 5 7 muscles from EC group and n 5 8
muscles from S 1 EC group) and EDL (n 5 7 muscles from EC
group and n 5 8 muscles from S 1 EC group) were mounted
on microscope slides and placed on a motor-driven microscope
stage. This permitted single fiber SDH activity measurement
from fibers in three different regions of the tissue section
under computer control. Briefly, SDH activity was measured
densitometrically by incubating the tissue section with a
medium containing (in mM): 1.5 nitroblue tetrazolium, 5
EDTA, 59 succinic acid, 0.75 sodium azide, 30 methylphenylmethlyl sulfate, and 100 phosphate buffer adjusted to
pH 7.6 and by recording fiber optical density every 2 min for
14 min, which was well within the linear portion of the
reaction (18). SDH activity (OD/min) of individual fibers (n 5
1,783 fibers from 15 EDL muscles, n 5 2,100 fibers from 15 TA
muscles) was calculated from the slope of the linear regression of optical density-versus-time relationship. Individual
fibers were then immunohistochemically classified into fiber
types using four serial sections and the MHC monoclonal
antibodies described in Immunohistochemistry.
Statistical analysis. Peak dorsiflexion torque versus day
was analyzed by one-way analysis of variance (ANOVA) with
repeated measures (15 different days). To determine whether
fatigue index showed a significant increase over the experimental period, fatigue index versus day was analyzed using
linear regression (15 different days). Fatigue indexes for
isolated TA and EDL in EC and S 1 EC groups were analyzed
by one-way ANOVA. CS activity was analyzed by two-way
ANOVA, using stimulation training (or not) and EC injury (or
not) as grouping variables. Morphometric parameters obtained from groups EC and S 1 EC were compared using the
unpaired Student’s t-test. For most parameters, one-way
ANOVA was used to compare across EC, S 1 EC, C, and S 1 C
groups followed by multiple paired comparisons using Fisher’s protected least-significant differences test. Coefficient of
variation percentages were arcsine transformed before oneway ANOVA. All results are expressed as means 6 SE. Level
of statistical significance was set to P , 0.05, and statistical
power was calculated using standard equations (36) when
results were not significant.
RESULTS
Time course of torque change over treatment period.
Maximum dorsiflexion torque for each treatment session remained constant throughout the 3-wk stimulation training treatment period (Fig. 1A). This confirmed, as planned, that the magnitude of the
stimulation treatment was not so great as to cause
muscle fiber atrophy as is observed secondary to the
chronic stimulation paradigms (17, 32, 34), and substrate utilization was not so great as to cause declining
torque over the treatment period as a result of metabolic fatigue. No torque increase was observed either,
demonstrating that isometric stimulation training did
not cause muscle hypertrophy. Dorsiflexor fatigue index measurements of all rabbits, calculated from each
OXIDATIVE CAPACITY DOES NOT AFFECT MUSCLE INJURY
Fig. 1. A: peak dorsiflexion torque throughout the 3-wk stimulation
training treatment period for all stimulation-trained (5) rabbits (n 5
18). No significant difference between time periods was observed (P .
0.2). B: mean dorsiflexor fatigue index measurements over the 3-wk
stimulation training period in all stimulation-trained rabbits. Dorsiflexor endurance capacity increased throughout the stimulation
training period (P , 0.0001).
treatment session, increased significantly over the 3-wk
stimulation training treatment (P , 0.0001), demonstrating that stimulation training reduced whole dorsiflexor fatigability (Fig. 1B). In addition, fatigue index
measurements from isolated TA and EDL muscles
showed strong increasing trends. TA fatigue index
increased insignificantly from 0.34 6 0.10 in the EC
group to 0.51 6 0.12 in the S 1 EC group (P . 0.3),
whereas EDL fatigue index increased from 0.21 6 0.06
in the EC group to 0.46 6 0.13 in the S 1 EC group (P .
0.1). The statistical power of these comparisons was
50% for the TA and 80% for the EDL. This stimulation
paradigm should thus be viewed as a method for
altering muscle metabolism, as indicated by fatigue
index measurements, independent of changes in maximum muscle force generation capacity. One-way ANOVA
with repeated measures revealed no significant peak
torque change (P . 0.2) over the 3-wk stimulation
training period and no significant difference between
S 1 C and S 1 EC groups (P . 0.2).
Changes in CS activity. CS activity showed a significant main effect of stimulation training for both the TA
R1303
(P , 0.05) and EDL (P , 0.0001) but no significant
main effect of injury for either muscle (P . 0.4) and no
significant interaction (P . 0.4). Despite the significant
main effect of stimulation training, paired comparisons
between C and S 1 C and between EC and S 1 EC
groups for TA muscles were not significantly different
(P . 0.07). After stimulation training, TA CS activity
increased from 22.1 6 1.6 µmol · g tissue21 · min21 in the
C group to 25.5 6 2.2 µmol · g tissue21 · min21 in the S 1
C group (P . 0.2) and from 19.6 6 3.3 µmol · g21 · min21
in the EC group to 26.8 6 2.0 µmol · g21 · min21 in the
S 1 EC group (P . 0.07). In contrast, EDL CS activity
increased significantly from 15.4 6 0.7 µmol · g21 · min21
in the C group to 26.9 6 1.6 µmol · g21 · min21 in the S 1
C group (P , 0.0005) and from 15.7 6 2.0
µmol · g21 · min21 in the EC group to 25.2 6 1.7
µmol · g21 · min21 in the S 1 EC group (P , 0.005). The
magnitude of CS activity increase was much greater for
the EDL compared with the TA because control TA CS
activity was significantly greater (P , 0.005) compared
with control EDL CS activity. This difference in CS
activity between TA and EDL is consistent with previously published values (30). The stimulation training
paradigm thus significantly increased the oxidative
capacity of both the TA and EDL, although the magnitude of the effect was much greater for the EDL
(increasing from an average of 15.6 6 1.2
µmol · g21 · min21 to 26.1 6 1.1 µmol · g21 · min21 or 67%)
compared with the TA (increasing from an average of
20.6 6 2.0 µmol·g21 ·min21 to 26.1 6 1.5 µmol·g21 ·min21
or 27%).
Changes in muscle fiber enzymatic properties. Isometric electrical stimulation training over the 3-wk period
also affected individual muscle fiber oxidative capacity
and fiber type distribution with no effect on muscle
fiber size. No significant change was observed in TA or
EDL muscle a(f ), as measured on perfusion-fixed specimens (Table 1, P . 0.7) or from frozen sections used for
quantitative SDH measurements (data not shown).
Although the stimulation training paradigm was not
sufficient to induce the fast-to-slow transformation
observed in chronic stimulation models, it did induce a
significant shift of fast twitch fiber subtypes in TA and
EDL. Fiber type percentages were compared between
EC (n 5 7) and S 1 EC (n 5 8) groups in both the TA
and EDL. In TA, type 2A fiber type percentage increased insignificantly from 39.5 6 3.9 to 52.2 6 7.0%
(P . 0.1, Table 2) at the expense of type 2D fibers,
which decreased from 51.9 6 4.4 to 34.1 6 5.5% (P ,
0.05, Table 2). Similarly, in EDL, type 2A fiber type
percentage increased significantly from 31.9 6 2.4 to
53.6 6 2.7% (P , 0.0001) at the expense of type 2D
fibers, which decreased from 58.1 6 4.1 to 23.7 6 4.5%
(P , 0.0001, Table 2). Differences in type 1 fiber type
percentage were not significant in either TA (4.2 6 1.4
to 2.2 6 0.5%; P . 0.1) or EDL (2.3 6 0.6 to 1.3 6 0.4%;
P . 0.1) after stimulation training. Fibers coexpressing
type 2A and 2D MHC isoforms significantly increased
from 3.5 6 1.0 to 10.1 6 2.1% in the TA (P , 0.05) and
from 7.3 6 2.9 to 21.4 6 4.5% in the EDL (P , 0.05)
after stimulation training. We interpret 2A-2D coexpres-
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OXIDATIVE CAPACITY DOES NOT AFFECT MUSCLE INJURY
Table 1. Muscle morphometric parameters
Tibialis Anterior
Parameter
S 1 EC
EC
Extensor Digitorum
Longus
S 1 EC
EC
Sarcomere
length, µm
2.06 6 0.08 2.02 6 0.06 2.29 6 0.06 2.18 6 0.02
Fiber crosssectional
area, µm2
1,552 6 197 1,582 6 97 1,763 6 227 1,831 6 36
Capillary den22
sity, mm
1,026 6 71
839 6 56
792 6 66
589 6 37*
Capillary-tofiber ratio
1.55 6 0.16 1.32 6 0.10 1.36 6 0.14 1.08 6 0.07
Capillary surface per
fiber
volume,
cm21
149 6 12
114 6 7*
153 6 14
118 6 18
Mitochondrial
volume density, %
4.8 6 0.6
4.0 6 0.4
4.2 6 0.6
2.9 6 0.3
Values represent means 6 SE; n 5 5 animals per group. Fiber
cross-sectional area and capillary density are at sarcomere length 5
2.1 µm. * Significant difference between eccentric contraction only
(EC) and stimulation 1 EC (S 1 EC) animals (P , 0.05).
sion as representing type 2D fibers transforming to
type 2A but not completely transformed by the end of
the stimulation training treatment period. In both
muscles, consistent with the increased oxidative capacity as measured by CS activity, TA and EDL increased
expression of the MHC isoform (type 2A) associated
with higher oxidative capacity. Unfortunately, this fiber
subtype transformation confounded our ability to measure fiber type-specific oxidative capacity changes due
to stimulation training. This was because many of the
type 2A fibers measured after stimulation training
were actually transformed or transforming type 2D
fibers. Because type 2D fiber oxidative activity is
normally less than one-half that of type 2A fibers (Fig.
2, A and C), even if these fibers increased oxidative
capacity, as they express type 2A MHC, they still might
not achieve the average oxidative capacity of normal
type 2A fibers. This was not the case for type 2D fibers;
Table 2. Muscle fiber type percentages
Tibialis
Anterior, %
Fiber
Type
1
2A
2D
2A/2D
Extensor Digitorum
Longus, %
S 1 EC
EC
S 1 EC
EC
2.2 6 .5
(3 6 1)
52.2 6 7.0
(68 6 6)
34.1 6 5.5
(49 6 9)
10.1 6 2.1
(14 6 3)
4.2 6 1.4
(6 6 2)
39.5 6 3.9
(55 6 4)
51.9 6 4.4*
(77 6 10)
3.5 6 1.0*
(5 6 1)
1.3 6 .4
(2 6 1)
53.6 6 2.7
(60 6 8)
23.7 6 4.5
(24 6 3)
21.4 6 4.5
(26 6 7)
2.3 6 .6
(6 6 2)
31.9 6 2.4*
(41 6 5)
58.1 6 4.1*
(75 6 9)
7.3 6 2.9*
(9 6 3)
Values represent means 6 SE; n 5 7 animals for EC group, and n 5
8 animals for the S 1 EC group. Values in parentheses represent
average fiber counts per muscle of each fiber type. Fibers expressing
.2 myosin heavy-chain isoforms are not presented in this table
because they represent ,2% of total fiber population in both tibialis
anterior and extensor digitorum longus. Thus values may not add to
100%. * Significant difference between S 1 EC and EC (P , 0.05).
all fibers observed in the S 1 EC group were type 2D
fibers that had not transformed. A significant increase
in SDH activity in the TA was observed with type 2D
fibers increasing from 0.012 6 0.001 OD/min to 0.016 6
0.001 OD/min (P , 0.01) and type 2D fibers in the EDL
increasing insignificantly from 0.011 6 0.001 to 0.013 6
0.001 OD/min (P 5 0.06). In addition, all fiber types had
a more homogeneous oxidative capacity and size after
stimulation training (Fig. 2, B and D). For example,
both TA and EDL decreased fiber size coefficient of
variation after stimulation training (TA, from 39.6 6
2.0 to 32.5 6 1.1%, P , 0.0001; EDL, from 40.8 6 2.5 to
37.2 6 1.8%, P . 0.2) and also decreased SDH activity
coefficient of variation (TA, from 37.5 6 1.9 to 22.4 6
2.4%, P , 0.0001; EDL, from 49.2 6 4.3 to 24.6 6 1.4%,
P , 0.0001).
Torque achieved during eccentric exercise. It was
important to ensure that the eccentric exercise bout
intensity was comparable between S 1 EC and EC
groups. We were concerned that, if isometric stimulation training caused muscle fatigue, lower torques
would be obtained during eccentric exercise, resulting
in less muscle injury, and would thus provide the
appearance that electrical stimulation training actually protected the muscle from injury when, in fact, the
S 1 EC muscles would simply have received a lessintensive eccentric exercise bout. Thus torque was
monitored during EC as previously described (15).
Peak eccentric torque of the S 1 EC group slightly
exceeded that of the EC group during the first few
minutes of eccentric exercise (0.78 6 0.11 Nm for the
S 1 EC group compared with 0.61 6 0.06 Nm for the EC
group, P . 0.2; Fig. 3, inset), but over the entire 30-min
period, the summed, integrated eccentric impulse produced by the S 1 EC group was 6.9% less than the EC
group (55,017 6 6,384 vs. 59,141 6 7,741 Nm · min, P .
0.6; Fig. 3).
Microcirculatory alterations after stimulation training. Isometric electrical stimulation training increased
the surface area of the muscle capillary bed in both the
TA and EDL (Table 1). The most conspicuous changes
were a 22% increase for the TA (P 5 0.07) and a 34%
significant increase for the EDL QA(0) (P , 0.05)
(normalized to a sarcomere length of 2.1 µm) and a 17
and 26% increase in NN(c,f ) for the TA (P . 0.2) and
EDL (P . 0.1), respectively. However, SV(c,f ) ratio
increased by ,30% in both muscles, a difference not
significant for the EDL (P 5 0.07) and significant for
the TA (P , 0.05). This, in conjunction with the
observation that fiber size did not change significantly
(P . 0.8), suggests a significant increase in oxygendelivering ability by the microcirculation to the muscle
fibers. Mitochondrial volume density did not increase
significantly for the EDL (P 5 0.07) and, interestingly,
did not increase for the TA (P . 0.3) even though an
increase in oxygen delivery capabilities was seen.
Torque and tension changes after eccentric exercise.
Isometric dorsiflexion torque 3 days after the eccentric
exercise was not significantly different between S 1 EC
and EC groups at any stimulation frequency (P . 0.07;
Fig. 4A). The magnitude of the torque decrease was
OXIDATIVE CAPACITY DOES NOT AFFECT MUSCLE INJURY
R1305
Fig. 2. Single-fiber oxidative capacity
vs. fiber size for each fiber type from 1
representative extensor digitorum longus (EDL) and tibialis anterior (TA)
muscle from each group. r, Type 1; k,
type 2A; l, type 2D fibers. A: EDL
muscle fibers from group eccentric contraction (EC) (n 5 148 fibers). B: EDL
muscle fibers from group S 1 EC (n 5
121 fibers). C: TA muscle fibers from
group EC (n 5 165 fibers). D: TA muscle
fibers from group S 1 EC (n 5 132
fibers). In both EDL and TA, singlefiber oxidative capacity was more homogeneous after stimulation training. For
clarity, type 2A/2D fibers are not shown.
Data from other 26 muscles showed
similar changes. SDH, succinate dehydrogenase.
comparable to that which we previously reported (16)
using the same model. Despite this result, because a
greater stimulation training effect was observed in
EDL than in TA, as indicated by CS activity, we were
concerned that a protective effect in only one muscle
might be masked by measuring both muscles acting
together during ankle torque production. In normal
rabbit dorsiflexors, based on physiological cross-sectional area and moment arm (12), the EDL generates
about twice the torque compared with the TA. Thus
Fig. 3. Torque achieved during 30-min eccentric exercise treatment
in S 1 EC (r, n 5 9) and EC (s, n 5 8). Inset shows initial 3 min of
eccentric exercise treatment when S 1 EC group generated higher
torques than EC. For clarity, standard error bars are provided only
for the final 2 data points in each graph.
Fig. 4. A: dorsiflexion torque measured 3 days after eccentric exercise in S 1 EC (r, n 5 9) and EC (s, n 5 8). B: TA and EDL maximum
tetanic tension measured 3 days after eccentric exercise in S 1 EC
(filled bars) and EC (open bars). Hatched bars represent mean 6 SE
of normal TA or EDL muscle.
R1306
OXIDATIVE CAPACITY DOES NOT AFFECT MUSCLE INJURY
torque measurements are biased to represent EDL
properties. We thus measured the isolated maximum
tetanic tension of each muscle to determine whether
muscle oxidative capacity affected injury magnitude.
No significant difference between S 1 EC and EC
groups was observed in injury magnitude (as measured
by decreased tetanic tension) for either the TA or the
EDL (P . 0.3, Fig. 4B) despite the differential isometric
stimulation training effect on both muscles. Because
such a wide range in oxidative capacities from EC and
S 1 EC groups was observed, we were concerned that
any protective effect of stimulation training might be
masked by the heterogeneity of the CS activity within
groups. We regressed muscle maximum tetanic tension
on CS activity (Fig. 5) and found no significant correlation between these two variables for either the TA (P .
0.8, r2 5 0.003) or EDL (P . 0.1, r2 5 0.13). Thus
increasing muscle oxidative capacity with isometric
stimulation training did not protect muscle from ECinduced injury.
DISCUSSION
The purpose of this study was to determine whether,
after increasing muscle oxidative capacity, skeletal
muscle would be protected from EC-induced injury. The
study’s premise was the observation of selective damage to type FG muscle fibers in rabbits (13) and type 2B
muscle fibers in humans (10), which, assuming that
they are analogous to the type 2D fibers measured in
this study, have an oxidative capacity that is less than
one-half of type 2A or type 1 fibers. Three weeks of
stimulation training treatment had no effect on muscle
fiber size (Table 1) and increased the percentage of type
2A fibers (from ,35 to ,50%, Table 2) at the expense of
2D fibers (which decreased from ,50 to ,30%, Table 2)
in both TA and EDL muscles. The oxygen-metabolizing
capability of the tissue was also greatly increased as
indicated by a 67 and 27% increase in EDL and TA CS
activity, respectively, the 22% increase in TA (P 5 0.07)
Fig. 5. Relationship between citrate synthase activity (abscissa) and
muscle maximum tetanic tension in TA (circles, n 5 16) and EDL
(squares, n 5 16) muscles. Open symbols represent EC group, and
filled symbols represent S 1 EC group. No significant correlation was
observed between oxidative capacity and maximum tetanic tension
for either the TA (P . 0.8, r2 5 0.003) or EDL (P . 0.1, r2 5 0.13).
and 34% increase in EDL QA(0) (P , 0.05), and the 17%
increase in TA (P . 0.2) and 26% increase in EDL
NN(c,f ) (P . 0.1) (Table 1). Statistical power for many of
the morphometric parameters was only 50% for the TA
and 80% for the EDL, a reflection of low sample size for
these measures. Fatigue index measurements also
indicated a metabolic adaptation of the dorsiflexors.
Dorsiflexor fatigue index over the 3-wk stimulation
training period increased by 30% (P , 0.0001) from the
initial to final day of stimulation training (Fig. 1B).
Furthermore, individual muscle fatigue indexes indicated a 50% (P . 0.30) increase for the TA and a 119%
(P . 0.10) increase for the EDL. Although these increases in the individual muscles were not statistically
significant, both muscles reduced their fatigability, and
the differential effect seen on the two muscles at the
functional level was consistent in both direction and
magnitude with those seen at the biochemical level.
Despite all of the stimulation training effects observed,
no difference in injury magnitude between EC and S 1
EC groups was observed for either the TA or EDL. This
conclusion is strengthened by the observation that,
although stimulation training preferentially affected
the EDL compared with the TA, the magnitude of
injury between the EC and S 1 EC groups for these two
muscles was the same. Thus we conclude that increased muscle oxidative capacity alone did not protect
muscle fibers from EC-induced injury.
This lack of a protective effect was not simply due to
incomplete activation of stimulation-trained muscles
during eccentric exercise, because torque measured for
S 1 EC muscles was not significantly different from EC
muscles during eccentric exercise. Peak dorsiflexion
torque measured three days after eccentric injury was
not different between EC and S 1 EC groups (Fig. 4A).
These torque measurements were representative of
changes to both the TA and EDL at the individual
muscle level, because no difference in maximum tetanic
tension was observed between these groups (Fig. 4B). It
is theoretically possible that no torque difference would
be observed between groups and yet, at the muscle
force level, differential effects could be observed that
would lead to a different experimental conclusion.
However, we would predict a greater protective effect of
increased oxidative capacity on the EDL, because the
control EDL had a CS activity only about one-half that
of the TA. Thus, in terms of CS activity and mitochondrial density, the EDL received the greater stimulation
training effect and yet this effect was not protective
(Fig. 4B).
Obviously, this study tested only oxidative capacity
as a possible basis for the differential injury to type 2D
compared with type 2A or type 1 muscle fibers. Thus
the structural or functional basis for selective type 2D
and FG muscle fiber damage remains unknown. One
feature of these fibers besides their low oxidative
capacity that may predispose them to injury is their
unusually large size. For example, in the rabbit EDL,
type 2D fibers have a cross-sectional area (4,000 µm2 )
that is ,41% greater than either type 1 or type 2A
fibers. It is possible that this large size renders the
OXIDATIVE CAPACITY DOES NOT AFFECT MUSCLE INJURY
fibers vulnerable to injury. If one assumes that myofibrillar force must ultimately be transmitted through
the fiber surface to the extracellular matrix, then larger
fibers, with their decreased surface-to-volume ratios,
would have a greater transmembrane stress compared
with smaller fibers. Indeed, this argument was put
forth by Petrof and colleagues (29), who reported
increased disruption of the larger fibers of the mdx
mouse EDL muscle compared with the mouse diaphragm. These authors used both maximum muscle
tension as well as the exclusion of low-molecularweight dye as indexes of fiber disruption. In the context
of this experiment, it might be interesting to continue
the stimulation training paradigm to the point where
muscle fiber size begins to decrease (31) to see if fiber
size alone can predispose a muscle to fiber injury.
In contrast to mechanical or metabolic differences
between fiber types, other mechanisms could explain
selective injury. Warren and colleagues (39) recently
explained selective fast twitch fiber injury on the basis
that fast twitch fibers are used less often than slow
twitch fibers, and thus are more likely to be injured
upon repetitive activation. In support of this hypothesis, they demonstrated that the normal EDL was more
injured than the normal soleus; however, the differential injury observed between the two muscles was
markedly diminished when both muscles were subjected to 14 days of hindlimb suspension. Unfortunately, neither muscle fiber size, fiber type, nor oxidative capacity were measured in this study, and thus the
basis for their observation remains unknown.
Another mechanism that may cause selective FG
fiber type damage can be hypothesized based on the
injury model presented by Morgan (23). He suggested
that muscle fiber damage occurs when sarcomeres are
forced to lengthen onto their descending limb of the
length-tension curve. Because in our previous studies
(13, 14, 16) initial muscle length was set to Lo and
subsequently stretched 25% beyond Lo, it is likely that
muscles were stretched onto their descending limb. For
a selective FG fiber type damage to occur, according to
Morgan’s hypothesis, FG fibers must be stretched to a
greater extent than other fiber types. This may be the
case if there is a systematic difference between motor
unit fiber lengths as reported in anatomical studies (8,
24) and may be inferred from biomechanical measurement of motor unit stiffness (28). Not only may fiber
lengths be different between different motor unit types,
it is also possible that optimal sarcomere length may
vary between motor unit types.
Finally, a potential mechanism involving loss of
calcium homeostasis cannot be excluded. Although
others have postulated that EC may result in membrane damage, leading to increased intracellular calcium (7), it is also possible that fiber type-specific
differences in calpain or calpistatin may result in fiber
type-specific damage secondary to EC. Recent studies
demonstrated that µ-calpain is activated during the
necrotic phase of mdx mouse dystrophy necrosis and
that activation ceases upon degeneration and/or regeneration (37). Thus fiber type-specific changes in calcium
R1307
homeostasis may not only be involved in the protective
mechanism but also in the adaptive mechanism that
renders muscle fiber less vulnerable to EC-induced
muscle damage after eccentric training. Further studies are required to resolve this issue at the cellular,
biochemical, and biomechanical levels.
We thank Dr. David Pierotti and Taby Ahsan for technical assistance, Dr. Jennifer Fujimoto for advice regarding isoflurane anesthesia, and Professor Stefano Schiaffino, Padua, Italy, for MHC antibodies.
This work was supported by National Institute of Arthritis and
Musculoskeletal and Skin Diseases Grant AR-40050 and National
Heart, Lung, and Blood Institute Grant 5 PO1 HL-17731, Veterans
Affairs, and the Swedish Medical Research Council.
Address for reprint requests: R. L. Lieber, Dept. of Orthopaedics
(9151), UC San Diego School of Medicine and Veterans Affairs
Medical Center, 3350 La Jolla Village Drive, San Diego, CA 92161.
Received 3 September 1997; accepted in final form 23 January 1998.
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