Fredrik Lauritzen, Gøran Paulsen, Truls Raastad, Linda Hildegard Bergersen and
Simen Gylterud Owe
J Appl Physiol 107:1923-1934, 2009. First published Oct 1, 2009; doi:10.1152/japplphysiol.00148.2009
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J Appl Physiol 107: 1923–1934, 2009.
First published October 1, 2009; doi:10.1152/japplphysiol.00148.2009.
Gross ultrastructural changes and necrotic fiber segments in elbow flexor
muscles after maximal voluntary eccentric action in humans
Fredrik Lauritzen,1,2 Gøran Paulsen,2 Truls Raastad,2 Linda Hildegard Bergersen,1
and Simen Gylterud Owe1
1
Department of Anatomy and Centre for Molecular Biology and Neuroscience, Institute of Basic Medical Sciences, University
of Oslo, and 2Norwegian School of Sports Sciences, Oslo, Norway
Submitted 11 February 2009; accepted in final form 28 September 2009
muscle damage; streaming; eccentric exercise; myofibrillar disruptions; electron microscopy
skeletal muscle activated and
stretched beyond the optimal length for the generation of force
was originally proposed by Sir Bernard Katz in 1939. Later, in
the 1960s and 1970s, distinct ultrastructural changes in the
myofibrillar structure were observed in patients with various
skeletal muscle dystrophies (19, 20, 70). Finally, similar ultrastructural changes were also observed after in vivo human
experiments using eccentric muscle actions (29, 31, 55). Dur-
THE OCCURRENCE OF DAMAGE IN
Address for reprint requests and other correspondence: S. G. Owe, Dept. of
Anatomy and Centre for Molecular Biology and Neuroscience, Institute of
Basic Medical Sciences, Univ. of Oslo, P.O. Box 1105 Blindern, N-0317 Oslo,
Norway (e-mail: s.g.owe@medisin.uio.no).
http://www. jap.org
ing the last 25 years, factors related to skeletal muscle damage
after physical exercise have been investigated in a number of
studies. Particularly, eccentric muscle actions (i.e., lengthening
contractions) have proven to cause more damage than isometric or concentric action (3, 49, 55). Damage has been measured
directly using histological methods, and indirectly by relying
on changes in skeletal muscle force, plasma creatine kinase
(CK) levels, and self-reported skeletal muscle soreness as
damage markers. Among these markers, change in forcegenerating capacity is considered to be the most reliable (74),
although indirect methods can never be used as indisputable
evidence (27). Consequently, indirect markers of muscle damage should ideally always be supported by direct measures
(22). Nevertheless, in human studies, only minuscule parts of
the experimental muscle can be studied by obtaining biopsies
(⬃100 mg); hence markers that reflect overall muscle damage
can yield valuable information. Among histological methods,
electron microscopic analyses are preferable, since damage can
be distinguished from other structural changes known to occur,
i.e., separate myofibrillar damage from misalignment. Furthermore, structural features can be directly visualized by electron
microscopy, independent of the use of markers.
As a locus for eccentric muscle action-induced damage, the
majority of morphological studies conclude that the Z disk is
the most vulnerable structure (e.g., Ref. 26). Alteration of the
Z disk includes streaming and disintegration, first described as
a central core disease by Engel et al. (20) and Gonatas et al.
(32) as irregular Z disks with a widened and zigzagged appearance. More severe lesions are associated with patches of dense
material located in the midst of sarcomeres. Alterations in
Z-disk morphology have been reported after both electrically
activated eccentric muscle actions in animals (43) and humans
(17), and after voluntary eccentric muscle actions in humans
(23, 31, 33). Other ultrastructural myofibrillar alterations reported after eccentric muscle actions include disorganized
myofilaments, hypercontracted and/or overstretched sarcomeres, and myofibrils out of register (29).
A problem when comparing studies is the lack of a common
method to quantify muscle damage. Hence, based on the
histological data presented, comparing the outcome of different
damage protocols has been difficult. For instance, in studies
where muscle damage has been quantified as myofibrillar
disruptions, more extensive damage is reported than in studies
where only random Z-disk disruptions are quantified (17, 31,
33). However, because different exercise protocols are used in
these studies, deciding whether the discrepancy is a result of
differences in the analytical approach or a result of different
exercise protocols is not feasible. Whether the ultrastructural
changes demonstrated after in vivo human experiments apply-
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1923
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Lauritzen F, Paulsen G, Raastad T, Bergersen LH, Owe SG.
Gross ultrastructural changes and necrotic fiber segments in elbow
flexor muscles after maximal voluntary eccentric action in humans.
J Appl Physiol 107: 1923–1934, 2009. First published October 1,
2009; doi:10.1152/japplphysiol.00148.2009.—Eccentric muscle actions are associated with ultrastructural changes. The severity and
types of change depend on the nature of the stimulation protocol, and on
the method for assessing such changes, and can be regarded as a
continuum from mild changes to pathological-like changes. Most
studies describing more severe changes have been performed on
animals and only a few in humans, some using electrical stimuli.
Hence, a debate has emerged on whether voluntary actions are
associated with the pathological-like end of the continuum. The aim of
this study was to determine whether severe muscle damage, i.e.,
extensive ultrastructural changes, is confined to animal studies and
studies on humans using electrical stimuli. Second, because there is no
generally approved method to quantify the degree of muscle damage,
we compared two published methods, analyzing the Z disks or
sarcomeres, as well as novel analyses of pathological-like changes. A
group of untrained subjects performed 70 voluntary maximal eccentric muscle actions using the elbow flexors. On the basis of large
reductions in maximal force-generating capacity (on average, ⫺62 ⫾
3% immediately after exercise, and ⫺35 ⫾ 6% 9 days later), five
subjects were selected for further analysis. Biopsies were taken from
m. biceps brachii in both the exercised and nonexercised arm. In
exercised muscle, more disrupted (13 ⫾ 4 vs. 3 ⫾ 3%) and destroyed
(15 ⫾ 6 vs. 0%) Z disks were found compared with nonexercised
muscle. A significant proportion of exercised myofibers had focal
(85 ⫾ 5 vs. 11 ⫾ 7%), moderate (65 ⫾ 7 vs. 11 ⫾ 6%), and extreme
(38 ⫾ 9 vs. 0%) myofibrillar disruptions. Hypercontracted myofibrils,
autophagic vacuoles, granular areas, central nuclei, and necrotic fiber
segments were found to various degrees. The present study demonstrates that the more severe end of the continuum of ultrastructural
changes occurs in humans after voluntary exercise when maximal
eccentric muscle actions are involved.
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ULTRASTRUCTURAL CHANGES AFTER VOLUNTARY ECCENTRIC MUSCLE ACTIONS
ing voluntary muscle actions actually are not signs of damage,
but rather remodeling of the myofibrillar structure (i.e., adaptation, Refs. 47, 77), has also been discussed. The lack of
damage in some studies could be due to the use of only mild
stimuli. In this study, the aim was to thoroughly investigate
muscle ultrastructure after extreme voluntary stimuli, causing
very large reductions in maximum force. Hence, we address
whether muscle damage rather than remodeling occurs with
exercise. Two previously published methods for quantitative
assessment of damages at the ultrastructural level were compared. We also qualitatively and quantitatively report novel
observations of a number of pathological-like changes possibly
associated with maximal voluntary exercise.
MATERIALS AND METHODS
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Subjects. Five healthy students (3 women and 2 men, 25 ⫾ 2 yr,
169 ⫾ 9 cm, 66 ⫾ 4 kg; means ⫾ SD) voluntarily participated in the
study. All subjects signed a consent document after being informed
about the possible risks of conducting unaccustomed, maximal eccentric muscle actions. The subjects were physically active, but none was
engaged in regular heavy strength training of the elbow flexors 6 mo
before the study. Subjects were instructed not to take any form of
medications or supplements. The study complied with the standards
set by the Declaration of Helsinki and was approved by the Regional
Ethics Committee of Southern Norway. The 5 subjects were chosen
from a total of 33 subjects who performed voluntary eccentric muscle
actions based on a ⬎50% reduction in force-generating capacity. In a
study where data from all 33 subjects were published (59), the 5
subjects included here are referred to as high responders.
Experimental design. Changes in force-generating capacity were
monitored 9 days after two identical bouts of unilateral, voluntary
maximal eccentric muscle actions. Test of muscle function was
followed by blood sampling. The dominant or nondominant arm was
randomly chosen to perform the exercise protocol. The same arms
were exercised at both occasions, and the nonexercised arm functioned as control for all tests and measurements. The subjects were
familiarized with the strength tests before day 1 of the experiment.
Baseline values were obtained immediately before exercise. Biopsies
were taken at a total of six different time points; at 1 h, 48 h, 4 days,
7 days, 21 days, and 23 days after exercise, from both control and
exercised skeletal muscle. However, biopsies were only taken at three
time points from each subject. Since our study presents 5 high
responders selected from a total of 33 subjects, the number on each
biopsy time point was low. Consequently, biopsies were pooled into
three groups for further statistical analysis: early (1 ⫹ 48 h), middle
(4 ⫹ 7 days) and late (21 ⫹ 23 days). The three biopsy time points of
each subject were matched with these groups, so that all five subjects
had one biopsy time point in each temporal group.
Eccentric muscle actions. The subjects performed 70 unilateral,
maximal isokinetic eccentric muscle actions (30°/s) with the elbow
flexors applying a REV 9000 dynamometer (Technogym, Gambettola,
Italy). The subjects were positioned in the chair and fasten with belts
over the hip, chest, and shoulder, and the upper arm was stabilized by
a cushion. Thus the shoulder joint was kept in a slightly flexed
position (30 –35° from the vertical axis) and prevented from moving
during the elbow exercise. The work consisted of 14 sets of 5
repetitions with 30-s rest in between sets. The range of motion in the
elbow joint was 40 –175° (180° equals full extension of the elbow
joint), and the forearm (elbow) was kept in a supinated position during
the extension. At the completion of each eccentric muscle action, the
arm was immediately returned passively to the starting position. The
exercise bout lasted for ⬃15 min.
Skeletal muscle function. The force-generating capacity was measured as voluntary maximal isometric torque at 90° in the elbow joint.
Preceding the test, subjects warmed up on an arm ergometer (Loda,
Groningen, The Netherlands) for 3 min at 30 –50 W, followed by four
submaximal repetitions of concentric contractions in the dynamometer. Thereafter, subjects performed two maximal isometric repetitions,
lasting 5 s, separated by 30-s rest. Peak isometric torque was registered and used for further analyses.
Blood sampling and CK activity. Blood was drawn from an antecubital vein into a 10-ml serum vacutainer tube. After coagulating for
30 – 45 min at room temperature (⬃20°C), the blood was centrifuged
at 2,700 g for 10 min at 4°C. Serum was immediately pipetted into
Eppendorf tubes and stored at ⫺80°C until analysis. CK was analyzed
with an automated chemistry analyzer (Modular P, Hitachi HighTechnologies, Tokyo, Japan), with analytic coefficient of variation
being ⬍5%. Values are given as units of enzyme activity per liter of
serum (U/l).
Muscle soreness. Muscle soreness [delayed onset muscle soreness
(DOMS)] was rated on a visual analog scale, where 0 represented “not
sore at all” and 100 mm “extremely sore”. DOMS was assessed in m.
biceps brachii and m. brachialis after the subjects had stretched,
contracted, and palpated the elbow flexors.
Skeletal muscle biopsy procedure. A 6-mm Pelomi needle (Albertsund, Denmark) with manual suction was used to obtain tissue
samples (100 –150 mg) from the midsection (central) of m. biceps
brachii. Subjects lay in supine position, while the procedure was done
under local anesthesia (Xylocain adrenaline, 10 mg/ml ⫹ 5 ␮g/ml;
AstraZeneca). For the three biopsies, each needle insertion was placed
1–2 cm lateral or medial to the previous insertion to avoid affected
tissue from earlier biopsies. Visible blood contamination and adipose
and connective tissue were carefully removed in saline. A smaller
tissue section with intact unidirectional fibers (⬃10 mg) was dissected, dried on filter paper, and immersed in fixative (4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M sodium phosphate
buffer pH 7.4) and stored at ⫹4°C until analysis.
Tissue embedding. Fixed samples were embedded with the use of
freeze substitution, as described by Bergersen et al. (5). Briefly, the
specimens were cryoprotected by immersion in increasing concentrations of glycerol (10, 20, and 30%) in phosphate buffer for 30 min at
each step, and overnight at 4°C in the 30% solution. Then samples
were plunged into liquid propane cooled to ⫺170°C by liquid nitrogen
in a Universal Cryofixation System KF80 (Reichert-Jung, Vienna,
Austria). The samples were then immersed in a solution of 0.5%
uranylacetate in anhydrous methanol overnight at ⫺90°C in a cryosubstitution unit (AFS, Reichert). The following day, temperature was
raised stepwise in 4°C increments per hour from ⫺90 to ⫺45°C.
Samples were washed multiple times with anhydrous methanol to
remove residual water and uranylacetate and infiltrated with Lowicryl
HM20 resin at ⫺45°C, with a stepwise increase in the ratio of resin to
methanol (1:2, 1:1, and 2:1 to pure Lowicryl). Resin polymerization
was catalyzed by ultraviolet light at a wavelength of 360 nm for 2
days at ⫺45°C, followed by 1 day at room temperature (⬃20°C). The
skeletal muscle tissue samples were longitudinally oriented before
ultrathin sections (80 nm) were cut with a diamond knife (ultra 45°,
Diatome) on a Reichert-Jung ultramicrotome (Reichert ultracut S,
Vienna, Austria) and mounted on nickel grids (200 mesh TB, Electron
Microscopic Sciences) using an adhesive pen (Electron Microscopy
Sciences). Ultrathin sections were contrasted in uranyl acetate (5% in
40% ethanol for 90 s) and lead citrate (0.3% for 90 s) before they were
analyzed in a Tecnai 12 FEI transmission electron microscope.
Electron microscopic analysis. Changes in myofibrillar ultrastructure were analyzed by two different methods: 1) method modified
from Gibala et al. (31); and 2) from Crameri et al. (17).
In brief, using the method from the Gibala study, ⬃10 fibers with
a length of more than 3 grid holes (365 ␮m) from each sample were
scanned for sarcomeres with nonlinear Z lines and disorganized
myofilaments, using ⫻1,700 to ⫻2,550 original magnification. A fiber
was considered to be disrupted if it contained any apparent disturbances in the normal myofibrillar banding pattern (see Fig. 2, A and
B). Fibers were classified as having focal damages when 1–2 sarco-
ULTRASTRUCTURAL CHANGES AFTER VOLUNTARY ECCENTRIC MUSCLE ACTIONS
specimens had been analyzed, the number of subjects with each of
these findings was summarized, as well as the percentage of fibers that
had the given finding among the affected specimens.
Statistics. Paired Student’s t-tests were used on data, where values
from all time points were compared (see Fig. 3), and results are
presented as means ⫾ SE. Bivariate relationships were examined with
the Pearson’s product-moment correlation coefficient test. When comparing values that had been subdivided into time points, nonparametric tests were used because of the low number of subjects. To compare
the numbers of affected subjects, ␹2 tests were used. To compare the
percentage of affected fibers in specimens between exercise and
control, Mann-Whitney U-tests were used. For comparison of repeated measurements, Friedman tests with Dunn posttests were used.
Results from data treated as nonparametric are presented as median ⫾
minimum/maximum. Statistical significance was set at P ⱕ 0.05.
RESULTS
Skeletal muscle function. Force-generating capacity during
maximal isometric elbow actions was reduced by 62 ⫾ 3%
immediately after exercise (P ⬍ 0.01; Fig. 1). The recovery
was slow, and force-generating capacity was still reduced by
35 ⫾ 6% 9 days later (P ⬍ 0.01), and by 24 ⫾ 6% (P ⬍ 0.01)
21 days after exercise. In control skeletal muscles, peak isometric torque was reduced 2–7% after exercise, and it was
significantly different from preexercise values 1 and 4 days
after exercise. Compared with the exercised arm, the reductions in force-generating capacity in the control arm were
minor at all time points (P ⬍ 0.01).
Muscle soreness and serum CK activity. Muscle soreness
peaked 2 and 3 days after exercise, with values of 56 ⫾ 12 and
55 ⫾ 11, respectively (P ⬍ 0.01). Serum CK activity increased
exponentially from 129 ⫾ 23 U/l preexercise to a peak of
9,698 ⫾ 4,270 U/l 4 days after exercise (P ⬍ 0.05) before it
declined to 3,653 ⫾ 1,582 U/l 1 wk after exercise.
Muscle damage. Muscle damage, as described in Gibala et al.
(31) (Fig. 2, A and B, as myofibrillar disruptions) and in Crameri
et al. (17) (Fig. 2, C–E, as Z-disk disruptions), was identified in
the biopsies taken after the exercise protocol. When quantified, the
proportion of disruptions (Fig. 3, A and B) was higher in the
exercised muscle compared with the control muscle, independent of quantification method used (P ⬍ 0.01 in both analyses).
However, large differences in amount and severity of disruptions were registered between the two methods (Fig. 3C). For
further analysis, the quantitative ultrastructural data from all
time points after exercise were pooled into either exercised or
unexercised control skeletal muscle (Fig. 3).
Fig. 1. Mean reduction in peak isometric torque at
different time points in skeletal muscle after 70 eccentric muscle actions. Values are change in percentage ⫾
SE of preexercise value; n ⫽ 5 subjects. }, Control
muscle; ■, exercised muscle. 70 Eccentric muscle actions using the arm flexors were performed on day 0 and
on day 21. Note that peak isometric torque in exercised
muscle was still not recovered 3 wk after exercise.
Greater reductions in peak isometric force were found in
exercised muscle compared with control muscle at all
time points after exercise (P ⬍ 0.01). Shaded boxes
indicate time points for the assessment of skeletal muscle biopsies.
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meres in parallel and/or in series were disrupted, moderate when 3–9
sarcomeres in parallel and/or in series were disrupted, and extreme if
ⱖ10 sarcomeres in parallel and/or in series had changes in morphology. Fibers with five or more centrally placed skeletal muscle fiber
nuclei and extensive structural changes were termed degenerative/
regenerative. In addition, fibers with ⬎10 focal disruptions were
termed moderate, and fibers with ⬎10 moderate disruptions were
termed extreme.
With the method adapted from Crameri et al. (17), 10 random
pictures were taken in the electron microscope with a ⫻2,550 original
magnification from each biopsy (the “search” function in the electron
microscope was used to randomly choose 10 different locations within
each sample). The numbers of total Z disks in each picture were
estimated by multiplying the number of Z disks along the x- and
y-axis, and then the Z disks were classified as intact (normal straight
Z disk), disrupted (Z disks still present but showing morphological
changes from the intact Z disks), or destroyed (Z disks no longer
recognizable as an intact structure) (see Fig. 2, C–E). In addition to
these criteria from Crameri et al. (17), an overlay with a central
100-nm-wide zone and two lateral zones of 100-nm width was
constructed above each picture to more precisely evaluate the straightness of each Z disk and reduce observer bias (Z-disk lateral deviations
from the central zone on one or both sides ⫽ disrupted; lateral
deviations of ⬎100 nm from the central zone or no clear lateral
boundary of the Z disk ⫽ destroyed). Additionally, using the same
pictures, mitochondrial area, the area occupied by mitochondria in
each electron micrograph, was measured and averaged for control and
exercised muscle. During all analyses, the sample identity was concealed from the investigator until all quantifications were completed.
Identification of other intracellular alterations. The following structural alterations were identified according to the literature: hypercontraction and contraction clumps (19, 58), granular sarcoplasm and
leukocytes (19), autophagic vacuoles (21), central nuclei (38), and
rounded nuclei as a sign of abnormal myonuclear shape.
The following criteria were used for quantification. Hypercontraction was recognized when the parallel I-bands of the Z disks were no
longer discernable and subdivided into two categories, covering areas
of more than two sarcomeres, but ⬍100 ␮m2, and covering areas of
⬎100 ␮m2. Granules were recognized as light stained, small circular
objects, and granular areas were quantified when having a width of
⬎5 ␮m to separate granular areas from more common intermyofibrillar granules. Autophagic vacuoles were defined as light stained, round
objects with membrane-like interior structures, and only those with a
diameter of ⬎0.5 ␮m were considered. To separate these findings
from more commonly appearing vacuoles, a cutoff of ⬎10 vacuoles
per 100 ␮m2 were chosen. Myonuclei interior to at least one myofibril
from the cell membrane were quantified as internal. Nuclei with a
length-to-width ratio of ⬍1.5 were quantified as rounded. When
foreign cells were present inside of the muscle cell membrane, the
fiber was termed as having one or more necrotic segments. When all
1925
1926
ULTRASTRUCTURAL CHANGES AFTER VOLUNTARY ECCENTRIC MUSCLE ACTIONS
Myofibrillar disruptions. This method was adapted from
Gibala et al. (31). An average of 9.8 ⫾ 0.2 fibers were
investigated from each biopsy, yielding a total of 148 fibers in
both groups, and a mean fiber length of 525 ⫾ 61 and 647 ⫾
37 ␮m from exercised and control skeletal muscle, respectively. Control muscles were primarily intact, but 11 ⫾ 7 and
11 ⫾ 6% of all of the fibers contained focal or moderate
disruptions (Fig. 3A). In contrast, in exercised muscle, 85 ⫾
5% of all fibers contained focal disruptions, and 65 ⫾ 7%
moderate disruptions (P ⬍ 0.01). In the exercised muscle only,
extreme disruptions (38 ⫾ 9%, P ⬍ 0.01) were found. The
reduction in force-generating capacity at 1 and 48 h after
exercise (pooled) correlated with the percentage of fibers with
moderate (r ⫽ ⫺0.93) and extreme (r ⫽ ⫺0.94) myofibrillar
disruptions (P ⬍ 0.05).
Z-disk disruptions. This method was adapted from Crameri
et al. (17). Ten randomly taken electron micrographs were
investigated from each subject. A mean of 3,446 ⫾ 264 and
3,881 ⫾ 226 Z disks from each subject were analyzed in
exercised and control skeletal muscle, respectively. In exercised skeletal muscle, 72 ⫾ 10% of the Z disks were intact,
compared with 96 ⫾ 3% in control skeletal muscle (P ⬍ 0.01)
(Fig. 3B). More Z disks categorized as disrupted (13 ⫾ 4 vs.
3 ⫾ 3%) and destroyed (15 ⫾ 6 vs. 0.4 ⫾ 0.2%) were found
in exercised skeletal muscle compared with control skeletal
muscle (P ⬍ 0.01). The reductions of isometric torque 1 and
J Appl Physiol • VOL
48 h after exercise correlated with the level of disrupted (r ⫽
⫺0.90) and destroyed (r ⫽ ⫺0.92) Z disks (P ⬍ 0.05). The two
different approaches to quantify muscle damage were correlated (Fig. 3C) when Z disks categorized as disrupted or
destroyed were compared with the level of fibers with extreme
myofibrillar disruption (r ⫽ 0.82, P ⬍ 0.01 and 0.7, P ⬍ 0.05,
respectively).
Mitochondrial density. A 32.5% reduction in average mitochondrial area was observed in the electron micrographs used
for Z-disk disruption analysis when exercised muscle from the
early biopsy time point (1⫹48 h after exercise) was compared
with control skeletal muscle (1.33 and 1.97 ␮m2 respectively,
P ⬍ 0.05).
Hypercontractions. At all time points and in both exercised
and control muscles, hypercontractions were observed (Fig. 4).
In control specimens, two subjects had small hypercontractions
of ⬍100 ␮m2 at early, 9% (6 –33%) of the fibers, and middle,
13% (8 –18%) of the fibers, time points. In one of these
subjects, 10% of the fibers also had contractions at the late time
point. In exercised muscles, three subjects had small hypercontractions at the early time point, 11% (6 –36%) of the fibers,
and two subjects at the middle, 17% (9 –25%) of fibers, and
late, 33% (8 –58%) of the fibers. In only one subject were small
hypercontractions found in all exercised muscle specimens.
Large hypercontractions of ⬎100 ␮m2 were found in three
controls, 18% (6 –33%) of the fibers, and in all five exercised
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Fig. 2. Sample electron micrographs of exercised skeletal muscle illustrating six categories of muscular disruption. A: first, based on the ultrastructural skeletal
muscle damage quantification criteria adopted from Gibala et al. (31), focal (*) and moderate (**) disruption were identified and are shown [X ⫽ 6,000, scale
bar (sb) ⫽ 2 ␮m]. B: an extreme disruption is shown (X ⫽ 4,200, sb ⫽ 5 ␮m). C–E: second, electron micrographs of exercised skeletal muscle illustrating three
categories of Z-disk morphology, according to ultrastructural skeletal muscle-damage quantification criteria adopted from Crameri et al. (17), are shown.
C: example shown of a Z disk (Z), A-band (a), I-band (I), M-line (M), and a mitochondria (m) have been labeled in a sarcomere with intact Z-disk. Also shown
are disrupted (D) and destroyed (E) Z disks. In addition to the original criteria in Crameri et al., objective values for the width of Z-disks were chosen to reduce
observer bias (lateral deviations of ⬎25 nm from a 100-nm-wide zone constructed above the Z disk ⫽ disrupted; lateral deviations of ⬎100 nm from Z-disk
zone, or no clear lateral boundary of the Z disk ⫽ destroyed). X ⫽ 26,500, sb ⫽ 0.5 ␮m.
ULTRASTRUCTURAL CHANGES AFTER VOLUNTARY ECCENTRIC MUSCLE ACTIONS
muscles, 18% (10 –56%) of the fibers at the early time point,
whereas, at the middle time point, four control specimens, 18%
(8 –50%) of the fibers, and three exercised specimens, 58%
(11–100%) of the fibers, were affected. At the late time point,
large hypercontractions were found in one control (10% of the
fibers) and four exercised muscles [30% (6 –100%) of the
fibers]. Notably, all maximum values for exercised muscles
were higher than corresponding controls, although the median
did not differ significantly. The number of subjects that had
J Appl Physiol • VOL
small or large hypercontractions did not differ significantly
between groups.
Granular areas. At the early time point, granular areas were
found in all five control subjects, 13% (7–18%) of the fibers, and
in two exercised muscles, 8% (6 –10%) of the fibers (Fig. 5). At
the middle time point, only two subjects had granular areas
affecting 33% (11–55%) and 23% (8 –39%) in control and
exercised muscle, respectively. At the late time point, granular
areas were found in one control (11% of the fibers) and in one
exercised muscle (13% of the fibers).
Autophagic vacuoles. Early, autophagic vacuoles were
found in exercised muscle from all five subjects and in four
subjects at the middle and late time point, affecting 22%
(9 – 63%), 40% (9 – 85%), and 17% (7–18%) of the fibers at the
early, middle, and late time points, respectively (Fig. 6). By
comparison, one control muscle with 13% of the fibers affected
was found at the early time point, and three subjects, 18%
(8 –22%)of the fibers affected, and one subject with 10% of the
fibers affected were found at the middle, and late time points,
respectively. Significantly more exercised than control muscle
specimens were affected at the early time point.
Internal myonuclei. Three, two, and none of the control
specimens had internal nuclei in 18% (10 –20%), 13% (13–
14%), and none of the fibers at the three time points, respectively (Fig. 7, A–C). Observations in exercised muscles differed in that two, four, and four subjects had 10%, 24%
(11–36%), and 25% (0 – 60%) of the fibers at early, middle, and
late time points, respectively. There are significantly more
specimens with internal nuclei among the exercised than the
controls at the late time point.
Rounded myonuclei. In only one control muscle, at the
early time point, were rounded nuclei found in 19% of the
fibers. Similar findings appeared to be more common in
exercised muscles and were found in three, two, and three
subjects at the early, middle, and late time points, and in
10% (9 –13%), 16% (8 –23%), and 12% (8 –27%) of the
fibers, respectively (Fig. 7, D–F).
Necrotic segments. Three exercised muscle specimens were
confirmed to have necrotic segments, one at the middle and
two at the late time point, with the two most severely affected
specimens being from the same subject. The percentage of
affected fibers was 33 and 34% (12–56%) (Fig. 8).
DISCUSSION
The aim of this study was to challenge the view that severe
muscle damage, i.e., extensive ultrastructural changes seen
together with an inflammatory reaction and segmental necrosis,
is confined to animal studies or studies using electrical stimulation. Exercised skeletal muscles were examined, in regard to
both the magnitude and the types of damage, after 70 voluntary
maximal eccentric muscle actions, using the elbow flexors and
a large range of motion. Claims that only minor muscle
damage or no damage at all after maximal voluntary eccentric
muscle actions occurs in lower limbs in humans (17) are not
supported by the present findings of ultrastructural disruptions
and necrotic segments of muscle in the fibers from the upper
body when using high responders. Neither are claims that
ultrastructural changes are rather signs of remodeling and
adaptations (77). Rather, in the most extreme cases, the findings were similar to those reported in patients with skeletal
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Fig. 3. Quantitative assessment of muscle damage. A: mean percentage of
skeletal muscle fibers with different degree of myofibrillar disruption, according to the skeletal muscle-damage quantification method adopted from Gibala
et al. (31), in control (open bars) and exercised skeletal muscle (solid bars).
More fibers with focal, moderate, and extreme myofibrillar disruptions were
found in exercised skeletal muscle compared with control skeletal muscle
(#P ⬍ 0.01). B: mean percentage of Z disks categorized as disrupted and
destroyed, according to the skeletal muscle-damage quantification method
adopted from Crameri et al. (17) in control (open bars) and exercised skeletal
muscle (solid bars). In exercised skeletal muscle, more Z disks were categorized as disrupted and destroyed compared with control skeletal muscle (#P ⬍
0.01). C: comparison between the two methods of directly accessing ultrastructural skeletal muscle damage. Percentage of fibers with extreme myofibrillar disruptions correlated with percent of Z disks categorized as disrupted
(r ⫽ 0.82, P ⬍ 0.01).
1927
1928
ULTRASTRUCTURAL CHANGES AFTER VOLUNTARY ECCENTRIC MUSCLE ACTIONS
muscle dystrophies (19, 20) and to studies using animal
models and electrical stimulation to initiate eccentric muscle
actions (43).
Muscle damage and changes in force-generating capacity.
One could claim that the first prerequisite to discuss whether
muscle damage occur in humans after voluntary eccentric
muscle actions is a thorough evaluation of the exercise protocol
and of the exercise-induced changes in force-generating capacity in investigations of muscle damage. Generally, we argue
that the authors who have spoken against the existence of
muscle damage in humans have used exercise protocols that
have to be considered as “mild”. In fact, several of the studies
report reductions in force-generating capacity that were either
small/moderate (⬍30%; Refs. 17, 48) and/or the protocol did
not include maximal/near maximal eccentric muscle actions
over a large range of motion; e.g., Yu et al. (76, 77) and Malm
et al. (48). Moreover, the use of DOMS to indicate muscle
damage (e.g., Refs. 76, 77) has no support in the literature (56,
73). We found only moderate muscle soreness associated with
a high degree of muscle damage. In studies in which muscle
damage has been claimed, the changes in force-generating
capacity have generally been large (11, 34; T. Raastad, unpubJ Appl Physiol • VOL
lished observations). In line with these studies, we found a 62%
reduction in force-generating capacity, which was even larger
than reported in the classical studies using similar methods (16,
35, 53). Most likely, this was due to the selection of five
subjects from a larger group based on their reduction in
force-generating capacity. Actually, our subjects responded
similar to so-called “high-responder subjects” reported on in
recently published studies (10, 60, 65). Hence, long-lasting
reductions in force-generating capacity is well established as a
marker for muscle damage, and the assumption that changes in
force-generating capacity reflect the degree of muscle damage
evaluated at the ultrastructural level (22, 31; T. Raastad, S. G.
Owe, G. Paulsen, D. Enns, K. Overgaard, R. M. Crameri, S.
Kiil, A. N. Belcastro, L. H. Bergersen, J. Hallen, unpublished
observations) is supported by the present study. Care was taken
to fulfill known criteria to induce muscle damage: 1) a large
range of motion (13, 22, 40, 54, 68); and 2) high force in each
muscle action (6, 12, 46, 71). In support of criteria 1, eccentric
muscle actions with a large range of motion by direct comparison yields a more pronounced reduction in peak isometric
torque than using a small range of motion in human subjects
(57). Second, Black et al. (6) have recently verified that force
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Fig. 4. Two degrees of hypercontraction in exercised skeletal muscle fibers. A: small hypercontraction: a number of myofibrils show hypercontracted sarcomeres
(h) underneath the plasma membrane (p), with swollen Z disks and invisible I-bands, covering an area of ⬍100 ␮m2 (time t ⫽ 48 h after exercise, X ⫽ 4,200,
sb ⫽ 5 ␮m). B: histogram of the number of subjects in whom small hypercontractions were found at the three time points. C: box plot showing median, maximum,
and minimum percentages of fibers in which small hypercontractions are found. D: large hypercontraction. Noticeably, there is also clumping of myofibrils
(contraction clumps). Z disks are not aligned or perpendicular to the plasma membrane. Overstretched areas are present between contraction clumps (t ⫽ 7 days
after exercise, X ⫽ 1,700, sb ⫽ 10 ␮m). E: histogram of the number of subjects in whom large hypercontractions were found at the three time points. F: box
plot showing median, maximum, and minimum percentages of fibers in which large hypercontractions are found.
ULTRASTRUCTURAL CHANGES AFTER VOLUNTARY ECCENTRIC MUSCLE ACTIONS
1929
Fig. 5. Granular areas of ⬎5-␮m width. A: granular material beneath the basal lamina and a vacuole possibly undergoing exo/endocytotic activity (*). The
sublaminal area is filled with granular material and is partly encircled by hypercontracted fibers and autophagic vacuoles (t ⫽ 48 h after exercise, X ⫽ 4,200,
sb ⫽ 5 ␮m). B: histogram of the number of subjects in whom granular areas were found at the three time points. C: box plot showing median, maximum, and
minimum percentages of fibers in which granular areas are found.
respectively). At this time point, ⬎10 biopsies had been taken
from the same skeletal muscle.
Direct comparison of two quantitative methods. A considerable amount of publications have addressed the issue of
ultrastructural changes after exercise (23, 24, 28 –30; e.g., Ref.
33), but comparisons of morphological outcome after different
damage protocols are difficult, because there is no unison
method on how to quantitatively assess the degree of muscle
damage. We have compared two approaches (17, 31) on the
same samples. Although a correlation was found between
methods, the method quantifying myofibrillar disruptions (31)
yielded much higher values than the Z-disk disruption method
(17). A reason for concern about using the Z-disk disruption
method is that areas with disruption are normally intermingled
with intact areas. Hence, a random sampling as in the Z-disk
disruption method can be expected to overlook some of the
damaged areas by chance. Looking into how big an area is
being analyzed, 10 pictures, each ⬃30 ⫻ 40 ␮m in size, are
taken randomly, representing a total of ⬃12,000 ␮m2 of total
surface area analyzed in each specimen. The whole surface
area of the same specimen is ⬃1–2,000,000 ␮m2; thus the
Fig. 6. Areas with more than 10 autophagic vacuoles per 100 ␮m2. A: representative example of autophagic vacuoles in an area with disrupted and
hypercontracted sarcomeres, Z-disk streaming, and disorganized myofilaments (t ⫽ 7 days after exercise, X ⫽ 8,200, sb ⫽ 2 ␮m). B: histogram of the number
of subjects in whom autophagic vacuoles were found at the three time points (*P ⬍ 0.05). C: box plot showing median, maximum, and minimum percentages
of fibers in which autophagic vacuoles are found.
J Appl Physiol • VOL
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generation per se dictates the muscle damage outcome in
humans. The biopsy sampling procedure or central fatigue
seems to have little effect on the force-generating capacity, as
evidenced by the small reduction seen in control skeletal
muscles.
Furthermore, because of the small changes seen in the
control muscle when quantifying muscle damage, changes in
ultrastructure could not be caused only by the needle biopsy
procedure, as has previously been proposed (63). Possible
negative effects of the anesthetics were omitted by the used
Xylocain with adrenaline injected in the skeletal muscle fascia
and by taking biopsies 2–3 cm from the area of injection.
Successive biopsies were taken 1–2 cm laterally or medially
from the initial sample in both control and exercised arms. In
other human studies, successive biopsies from the same skeletal muscles are often taken days and weeks after the initial
exercise protocol, and mechanical force is exerted on the
muscle, potentially inflicting morphological damage. However,
in a study by Staron et al. (66), two biopsies were taken every
week, in 8 wk. Still, degenerative changes were not found in
the control arm before the 7th and 9th wk (1.7 and 4.9%,
1930
ULTRASTRUCTURAL CHANGES AFTER VOLUNTARY ECCENTRIC MUSCLE ACTIONS
randomly chosen areas constitute only 0.6 –1.2% of the specimen. The specimen itself already represents a limited selection: 10 fibers of a total of ⬃250,000 fibers in the biceps
brachii muscle (36) or 500,000 fibers in the vastus lateralis
muscle (39). Hence, delimitating the analyzed area by a factor
of 100 could be detrimental, taking the heterogeneity of muscle
fibers in consideration. When compared, myofibrillar disruptions were always associated with Z-disk disruptions, but
Z-disk disruptions could be seen without myofibrillar disruptions. Hence, Z-disk disruption could represent a milder ultrastructural change than myofibrillar focal disruptions, whereas
destroyed Z disks are observed in areas together with myofibrillar disruptions. Z-disk streaming is known to be a common
structural alteration in healthy, young adults (51).
Severity of muscle damage can be compared between studies using the same methods of quantification. In the study by
Gibala et al. (31), disruption in the biceps brachii muscle was
evident in 3% of the baseline fibers, 33% of the fibers subjected
to concentric muscle actions, and 82% of the fibers subjected to
eccentric muscle actions. In the present study, 11 and 85% of
the fibers were classified with disruptions in the control and
exercised arm, respectively. Of all fibers analyzed, extreme
disruptions were evident in 41% immediately after and 50%
48 h after eccentric muscle actions in the Gibala study, compared with 38% at all time points combined in the present
J Appl Physiol • VOL
study. How submaximal eccentric exercise performed in the
Gibala et al. (31) study can induce a similar amount of extreme
disruptions as a maximal eccentric exercise protocol is unclear,
because the force developed during each action seems to be
critical for the resulting muscle damage (71). Fibers with
disruption after the concentric work were relatively numerous,
considering the small changes in force-generating capacity
found and compared with other studies (18, 55).
Crameri et al. (17) found that ⬃30% of all Z disks analyzed
could be classified as disrupted 5 h after voluntary exercise
with eccentric muscle actions, and the number of disrupted Z
disks decreased rapidly, to ⬃15% 24 h after exercise. Interestingly, the findings in the electrically stimulated leg were
opposite as the number of disrupted Z disks slowly increased to
an amount of 30% 4 days after the exercise. When looking at
the more severe alterations, destroyed Z disks, none was found
in the leg performing voluntary exercise, whereas, in the
electrically stimulated leg, an increase peaking at ⬃22% of all
Z disks was found 1 day after exercise. In comparison, we
found an average of 13% of disrupted Z disks (all time points),
not very different from what Crameri et al. (17) found after
their voluntary exercise protocol. This similarity was unexpected, given that Crameri et al. registered ⬃16 and ⬃24%
reduction of force-generating capacity immediately and 24 h
after voluntary exercise, respectively, and that full recovery
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Fig. 7. Exercised skeletal muscles with extensive damage and internally placed or rounded myonuclei. A: two central myonuclei in a muscle fiber with several
moderate disruptions as classified in Fig. 2. B: histogram of the number of subjects in whom internal myonuclei were found at the three time points (*P ⬍ 0.05).
C: box plot showing median, maximum, and minimum percentages of fibers in which internal myonuclei are found. D: two rounded myonuclei (n) in a muscle
fiber where striation is nearly abolished. E: histogram of the number of subjects in whom rounded myonuclei were found at the three time points. F: box plot
showing median, maximum, and minimum percentages of fibers in which rounded myonuclei are found. A and B: t ⫽ 7 days after exercise, X ⫽ 1,700, sb ⫽
10 ␮m.
ULTRASTRUCTURAL CHANGES AFTER VOLUNTARY ECCENTRIC MUSCLE ACTIONS
1931
was reported 48 h after exercise, whereas, 3 wk after exercise,
our high-responder subjects still showed impairment in forcegeneration capacity of ⬃24%. However, in our studies, 15% of
the Z disks in the exercised arm were classified as destroyed
compared with none in the voluntary exercise and 22% in the
electrical stimulation exercise performed by Crameri et al.
(18). Hence, using the same methods for analysis but a more
severe voluntary exercise protocol, the results obtained from
the present study are most similar to the electrically stimulated
fibers reported in the former study. Caution should be made
when comparing due to our selection of subjects and due to the
very limited sample area that is analyzed using this method.
Pathological-like changes. In addition to Z-disk streaming
and integration, first described in central core disease, and
myofibrillar disruptions, early linked to eccentric muscle actions, several other features previously linked to disease and
animal models were observed. First, hypercontraction, as described by Lotz and Engel (44), to various degrees, is shown in
Fig. 4. These analyses are prone to artifacts caused by the
biopsy procedures (9), as evidenced in the frequent findings in
control specimens. Albeit not statistically significant, large
hypercontractions appeared to progress over time in the exercised muscle and to decrease in the controls. Exercised muscles
systematically showed the most severe cases at all time points.
The appearance of autophagic vacuoles has been reported
after strenuous, long-lasting running in mice (64) and is deJ Appl Physiol • VOL
scribed in the inflammatory muscle disease, inclusion body
myositis (37). Central nuclei are not normally present in
healthy, young subjects (52). Significantly more subjects were
found to have densities of vacuoles above cutoff at the early
time point in the exercised muscle (Fig. 6), and, although not
statistically significant, the upper values recorded in exercised
muscle were up to threefold higher than in the controls. This is
in line with animal studies (2) and similar to findings from the
mutant mdx mouse (69). The percentage of exercised muscle
fibers that displayed internal nuclei appeared to increase at the
middle and late time points, whereas the percentage of affected
fibers was low in all control samples.
When analyzing for rounded myonuclei, some nuclei are
expected to be rendered round by chance as an effect of the
sectioning technique. However, rounded nuclei were infrequently encountered and only seen in one control sample.
Possibly the cutoff at a ratio of 1.5 could have contributed to
keep the numbers low. Rounded nuclei were seen in approximately one-half of the exercised muscle samples in relatively
few fibers. Necrotic segments were also encountered and were
exclusively found in exercised muscle. Of the two subjects
affected, one had necrotic segments in around one-half of the
fibers analyzed. From light and electron microscopic studies
(25), necrotic segments can be seen repeatedly over the length
of a fiber; hence there is a risk of sampling areas that are either
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Fig. 8. Immune cell infiltration in exercised skeletal muscle. A: extravascular erythrocytes (arrows) and immune cells in the intercellular space between two
myofibers 7 days after exercise. B: immune cells resembling macrophages (one circled in red) underneath the basal lamina 23 days after exercise. These and other
cells were numerous in the necrotic segments identified (A and B: X ⫽ 1,700, sb ⫽ 10 ␮m). C: higher magnification of the border between an immune cell and
intact sarcomeres (t ⫽ 7 days after exercise, X ⫽ 4,200, sb ⫽ 5 ␮m). D: histogram of the number of subjects in whom necrotic segments were found at the three
time points. E: box plot showing median, maximum, and minimum percentages of fibers in which necrotic segments are found.
1932
ULTRASTRUCTURAL CHANGES AFTER VOLUNTARY ECCENTRIC MUSCLE ACTIONS
J Appl Physiol • VOL
etal muscle fibers. While such small lesions do not necessarily
affect the skeletal muscle’s ability to generate and transmit
force, larger lesions likely will. In the present study, we often
observed fibers with extreme damage; in fact, 65 and 55% of
the exercised skeletal muscle fibers contained this degree of
disruption 0 –2 and 5–7 days after exercise, respectively. Often
the lesions covered the whole fiber diameter and extended
several micrometers longitudinally. In addition, eccentric muscle actions may result in loss of intermediate filaments (4, 25,
42), and loss of intermediate filaments have been observed to
correlate with reduction in force-generating capacity (41).
Consequently, if we assume that the same myofibers have both
myofibrillar disruptions and disruptions of intermediate filaments affecting lateral force transmission, the resulting reduction in force-generating capacity should reflect the number of
affected fibers.
Ultrastructural findings after eccentric muscle actions have
been suggested to be primarily remodeling and not damage.
However, based on the intrinsic differences in exercise protocols, this does not exclude the possibility that what we and
others observe is preferentially damage, and not remodeling
(e.g., Ref. 31). Importantly, the initial component of recovery
in force-generating capacity is faster than can be explained by
repair of sarcomeres; hence damage is not the single cause of
reduced force-generating capacity. Rather, disturbances seen in
the myofibrillar ultrastructure are one manifestation of damage
to force-generating and force-transmitting structures, which
also include other components, such as extracellular structures
(67). Alternatively, damage to the excitation-contraction coupling has been regarded as the primary cause of the reduced
force-generating capacity after eccentric muscle actions (72),
while others have proposed that excitation-contraction coupling dysfunction is secondary after disruption of sarcomeres
(61). Indicative of such dysfunction, we observed a significant
low-frequency fatigue (20:100 Hz ratio) (data not shown),
repeatedly reported in previous studies (15).
Conclusions. The present study shows that maximal voluntary
eccentric muscle actions can result in considerable myofibrillar
and Z-disk damage, as well as pathological-like findings, including necrotic fiber segments. The severity of damage apparently
depends on individual differences, on the exercise protocol in use
(exercise protocols that include high-force eccentric muscle actions and large range of motion induce more severe damage), and
on the choice of methods to quantify damage. Importantly, in
view of the findings presented, the substantial amount of data
previously reported using animal models should still be taken into
account in the understanding of human response to unaccustomed
eccentric muscle actions.
GRANTS
The authors at the Department of Anatomy (University of Oslo, Norway)
received financial support from the Norwegian Research Council.
DISCLOSURES
No conflicts of interest are declared by the author(s).
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