J Appl Physiol 103: 926–931, 2007.
First published June 21, 2007; doi:10.1152/japplphysiol.01422.2006.
Calpain-3 is autolyzed and hence activated in human skeletal muscle 24 h
following a single bout of eccentric exercise
Robyn M. Murphy,1 Craig A. Goodman,2 Michael J. McKenna,2 Jason Bennie,2 Murray Leikis,3 and
Graham D. Lamb1
1
Department of Zoology, La Trobe University, Victoria; 2Muscle, Ions and Exercise Group, Centre for Ageing, Rehabilitation,
Exercise and Sport, School of Human Movement, Recreation and Performance, Victoria University, Melbourne; and
3
Department of Nephrology, Royal Melbourne Hospital and Western Hospital, Melbourne, Australia
Submitted 14 December 2006; accepted in final form 15 June 2007
2⫹
CALPAINS ARE NONLYSOSOMAL, Ca -activated cysteine proteases. Skeletal muscle fibres contain not only the ubiquitous
calpains, ␮-calpain and m-calpain (also referred to as calpain-1
and calpain-2), but also a muscle-specific calpain known as
calpain-3 or p94 (32), for which the mRNA expression is
10-fold higher than for the other calpains in muscle (34). There
is some understanding of the physiological functions of the
ubiquitous calpains, which among other things appear to be
involved in initial proteolytic dismantling of the sarcomere that
occurs before the major degradation of proteins via ubiquitinproteasomal pathways (6, 15, 16). In contrast, the role of
calpain-3 remains an enigma. Complete absence of calpain-3
from skeletal muscle and/or the inability of calpain-3 to undergo autolysis (from its full-length 94-kDa form to 60-, 58-,
and 56/55-kDa forms) results in limb-girdle muscular dystrophy type 2A in humans (10, 31, 33). Also, calpain-3 needs to
be bound to the large elastic sarcomeric protein, titin, to
maintain its integrity. Mutations in the titin gene adjacent to the
calpain-3 binding site, which inhibit calpain-3 binding there,
result in tibial muscular dystrophy (17). Somewhat surprisingly, overexpression of this protease has no noticeable effect
on muscle function (35). Furthermore, there was contention for
many years about whether or not the activation of calpain-3 in
muscle is Ca2⫹ dependent, because it appeared to autolyze
even in the absence of Ca2⫹. It is now clear, however, that
activation of calpain-3 is indeed Ca2⫹ dependent and that the
earlier findings were due to the particularly high sensitivity of
the process to very low levels of Ca2⫹ (8, 9, 13, 26, 28). One
recent suggestion is that calpain-3 normally plays an important
role in sarcomere maintenance in mature muscle and that its
absence or loss of proteolytic activity results in degeneration
and death of muscle cells (9, 21). However, there is no
evidence to date as to precisely when or why calpain-3 is
normally activated. Given that autolysis of calpain-3 is necessary for its normal function as a protease (30), it is important
to know what conditions result in its autolysis. Studies to date
have found that calpain-3 remains in its full-length form and
thus inactive in healthy human muscle (8, 26) at rest as well as
after typical concentric exercise (26), and no studies have
reported a physiological intervention or circumstances that
results in calpain-3 autolysis (9).
Our laboratory has found recently that neither calpain-3 nor
␮-calpain were autolyzed in human skeletal muscle following
an “all-out” sprint exercise bout or endurance cycling in trained
subjects (26). Unless it is particularly excessive, such exercise
is not normally deleterious to the muscles, and muscle performance typically recovers within an hour. In contrast, eccentric
exercise, where muscles are stretched while contracting, such
as occurs in downhill walking, can result in muscle weakness
lasting 24 h or more (14, 18, 36). Following such exercise there
is often overt damage of the sarcomeric structure, typically
involving Z-disk streaming and marked widening of the I band
(2, 29). Although many fibers may show some damage (2, 14),
usually only a comparatively small percentage of the total fiber
volume is affected (2, 18), and the muscle weakness is due in
large part to a reduction in excitation-induced Ca2⫹ release
from the sarcoplasmic reticulum (5, 18). Significantly, it has
been observed in mouse fibers that the resting intracellular
Ca2⫹ concentration ([Ca2⫹]) remains elevated for hours (5, 18)
and even days (22) following eccentric contraction. A common
marker of muscle damage is serum creatine kinase (CK), which
leaks from muscles if the surface membrane is damaged,
something that might occur during the exercise itself or sub-
Address for reprint requests and other correspondence: R. Murphy, Dept. of
Zoology, La Trobe Univ., Victoria 3086, Australia (e-mail: r.murphy@latrobe.
edu.au).
The costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
␮-calpain; proteolysis; autolysis; muscle damage
926
8750-7587/07 $8.00 Copyright © 2007 the American Physiological Society
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Murphy RM, Goodman CA, McKenna MJ, Bennie J, Leikis M,
Lamb GD. Calpain-3 is autolyzed and hence activated in human
skeletal muscle 24 h following a single bout of eccentric exercise.
J Appl Physiol 103: 926–931, 2007. First published June 21, 2007;
doi:10.1152/japplphysiol.01422.2006.—The function and normal regulation of calpain-3, a muscle-specific Ca2⫹-dependent protease, is
uncertain, although its absence leads to limb-girdle muscular dystrophy type 2A. This study examined the effect of eccentric exercise on
calpain-3 autolytic activation, because such exercise is known to
damage sarcomeric structures and to trigger adaptive changes that
help prevent such damage on subsequent exercise. Six healthy human
subjects performed a 30-min bout of one-legged, eccentric, knee
extensor exercise. Torque measurements, vastus lateralis muscle biopsies, and venous blood samples were taken before and up to 7 days
following the exercise. Peak isometric muscle torque was depressed
immediately and at 3 h postexercise and recovered by 24 h, and serum
creatine kinase concentration peaked at 24 h postexercise. The amount
of autolyzed calpain-3 was unchanged immediately and 3 h after
exercise, but increased markedly (from ⬃16% to ⬃35% of total) 24 h
after the exercise, and returned to preexercise levels within 7 days. In
contrast, the eccentric exercise produced little autolytic activation of
the ubiquitous Ca2⫹-activated protease, ␮-calpain. Eccentric exercise
is the first physiological circumstance shown to result in calpain-3
activation in vivo.
CALPAINS AND ECCENTRIC EXERCISE IN HUMANS
METHODS
Subjects. Six healthy subjects, comprising 3 men and 3 women,
participated in this study (age 26.3 ⫾ 8.1 yr, height 173.7 ⫾ 17 cm,
mass 76.3 ⫾ 14.9 kg, mean ⫾ SD). All subjects were physically
active, and none had in the previous 6 mo participated in resistance
training or had recent trauma to the knee joint. Subjects were informed
of all test procedures and associated risks before giving written
informed consent. All experimental protocols were approved by the
Victoria University Human Research Ethics Committee. Inclusion of
both women and men is unlikely to have influenced the results,
because sex does not affect either the relative changes in muscle force
or the amount of muscle damage (seen as Z-disk streaming) observed
following eccentric exercise (36).
Overview of testing. Subjects underwent an eccentric exercise bout
to induce damage of the knee extensor muscles. A vastus lateralis
muscle biopsy was taken for measurement of calpain autolysis at five
time points: preexercise (pre), immediately postexercise (post), and at
3 h, 24 h, and 7 days postexercise. Peak isometric torque of the knee
extensors was measured on 6 different days to initially determine
variability and then deterioration in muscle function following eccentric exercise. Peak torque was measured on 3 separate days preceding
the eccentric exercise bout, which comprised an initial familiarization
test and repeat tests on the next two visits to determine variability,
enabling interpretation of the subsequent postexercise and recovery
data. On the day of eccentric exercise, peak isometric torque was
measured pre, immediately post, and at 3 h after eccentric exercise;
subsequent measurements occurred at 24 h and 7 days after eccentric
exercise. Venous blood samples were taken to determine increases in
serum CK as a marker of muscle damage, at the times corresponding
to biopsy sampling.
Maximal eccentric knee extension and isometric muscle strength
test. Both the eccentric exercise bout and measurements of maximal
isometric torque of the knee extensors were performed on an isokinetic dynamometer (Cybex Norm 770, Henley Health Care). The
dynamometer was calibrated for angle, torque and velocity immediately before use, and all data were corrected for gravity. Subjects were
strapped to the dynamometer chair across the hips and chest to restrict
J Appl Physiol • VOL
upper body movement, and they were strapped across the thigh to
stabilize the active leg. During the initial test, each subject’s positions
were recorded and were replicated during each subsequent isokinetic
test session. The dynamometer’s axis of rotation was aligned with the
anatomic axis of rotation of the knee, while the lever arm was
extended with the pad covering the distal shin. All measurements were
conducted on the right leg, as all subjects were right-hand dominant.
A real-time visual display of torque and strong verbal support were
provided to encourage subjects to exert maximal force during each
contraction.
Each trial of maximal isometric knee extensor torque consisted of
a standard warm-up (except the immediately postexercise measure)
and three maximal 5-s contractions, at a knee joint angle of 45°, with
a 30-s recovery between contractions.
The eccentric exercise test was based on previous studies that
induced muscle damage of the knee extensors (3, 7, 24, 25). Briefly,
subjects performed 300 repetitions of maximal eccentric knee extension, consisting of 10 sets of 30 repetitions at 30°/s. Each set was
separated by a 1-min rest period. The subject’s leg was initially fully
extended. At commencement of the eccentric bout, the subject was
instructed to maximally resist the downward movement of the lever
arm, through the full range of motion. When the leg was fully flexed,
the subject was then instructed to relax the leg, while the investigator
returned the lever arm (leg) to a fully extended position. This procedure ensured that the mode of muscle contraction was purely eccentric. All torque data are expressed relative to the preexercise value for
each individual.
Muscle biopsies. A muscle biopsy was taken at pre, post, and at 3 h,
24 h, and 7 days after eccentric exercise. Following injection of a local
anesthetic (1% Xylocaine) into the skin and subcutaneous tissue, a
small incision was made and biopsies were taken from the midportion
of the vastus lateralis muscle of the right leg. Subjects were supine for
the pre and 3 h, 24 h, and 7 days after biopsies, and they were seated
for the immediately after eccentric exercise biopsy. Each biopsy was
taken by the same experienced medical practitioner from separate
incisions, at a constant depth. Samples were immediately blotted on
filter paper and then frozen in liquid N2 and stored at ⫺80°C until
analyzed for calpains.
The biopsy immediately preceded the torque measurements.
General. All chemicals were obtained from Sigma (St. Louis, MO)
unless otherwise stated.
Muscle homogenate preparation. Skeletal muscle samples [9 ⫾ 5
(SD) mg] were homogenized (10:1 wt/vol) in 0.4 M Tris 䡠 Cl, pH 6.8,
and 25 mM EGTA ([Ca2⫹] ⬍ 10 nM), following which SDS was
added to a final concentration of 4% and homogenates were incubated
at 4°C for 20 – 40 min. Samples were spun (3,000 g, 5 min), and the
supernatant added (2:1 vol/vol) to SDS loading buffer (0.125 M
Tris 䡠 HCl, 10% glycerol, 4% SDS, 4 M urea, 10% mercaptoethanol,
and 0.001% bromophenol blue, pH 6.8). Samples were heated to 95°C
for 4 min and stored at ⫺20°C until analyzed by Western blotting.
Western blotting of ␮-calpain and calpain-3 in muscle homogenates. Similar volumes of muscle protein present in the supernatant as
described above, were separated on an 8% SDS-PAGE gel and
transferred to nitrocellulose. Membranes were exposed to either
mouse anti-␮-calpain (1 in 1,000; Sigma monoclonal, clone 15C10) or
mouse anti-calpain-3 (1 in 200; Novocastra monoclonal 12A2, Newcastle, UK), following which goat anti-mouse horseradish peroxidase
(1 in 20,000; Bio-Rad, Hercules, CA) was added to the membranes.
Bands were visualized using West Pico chemiluminescent substrate
(Pierce, Rockford, IL), and densitometry was performed using Quantity One software (Bio-Rad). ␮-calpain is visualized as an 80-kDa
protein that autolyzes to proteins of 78 and 76 kDa (4). Calpain-3 is
observed as a 94-kDa protein, which autolyzes to proteins of ⬃60, 58,
and 56 kDa when activated (8, 20, 33, 34, 37), with there frequently
being only two of these proteolytic bands (58 and 56 kDa) apparent in
human muscle (10, 26). For both ␮-calpain and calpain-3, the Western
blot data for each subject and each time point were quantified by
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sequently owing to activation of lipases triggered by the raised
cytoplasmic [Ca2⫹] (1). It is also well known that even a single
bout of eccentric exercise can be enough to result in adaptations within the muscle fibers that reduce the extent of damage to a subsequent bout of eccentric exercise. One of these
adaptive mechanisms is thought to be an increase in muscle
fiber length by addition of extra sarcomeres (23). Given the
suggested role of calpain-3 in sarcomeric remodeling (21), it
seems quite possible that calpain-3 would be activated by
eccentric contraction.
Eccentric exercise has been previously shown to result in
reduced levels of mRNA for calpain-3 and ␮-calpain 1 day
after the exercise (11), although the functional significance of
this is unclear. To date there has been no examination of the
effects of eccentric exercise on calpain-3 autolysis and activation. Such examination seems warranted given that calpain
activation is highly Ca2⫹ dependent, and there is a prolonged
rise in intracellular [Ca2⫹] following eccentric exercise, at least
in murine muscle. We hypothesized that calpain-3 and/or
␮-calpain would be autolyzed, and hence activated, following
eccentric exercise. Here we show that eccentric exercise in
humans results in appreciable autolysis of calpain-3; this is the
first physiological circumstance shown to result in calpain-3
activation in vivo. Significantly, the autolysis of calpain-3
primarily occurs not during the exercise itself but 24 h afterward.
927
928
CALPAINS AND ECCENTRIC EXERCISE IN HUMANS
RESULTS
Torque and [CK] after the eccentric exercise. As expected,
peak isometric torque was decreased following the eccentric
exercise (Fig. 1). Immediately after exercise, relative torque
was 76 ⫾ 8% (P ⬍ 0.05) of the preexercise levels and was
84 ⫾ 7% (P ⬍ 0.05) at 3 h after exercise. At 24 h and 7 days
after eccentric exercise, peak isometric torque did not differ
from preexercise levels. Before exercise, [CK] was 133 ⫾ 37
U/l, and the value at 3 h postexercise (235 ⫾ 25 U/l; P ⬍ 0.05)
was significantly greater than before exercise, immediately
postexercise, and 7 days postexercise (Fig. 2). Serum [CK]
peaked at 24 h after exercise (339 ⫾ 49 U/l; P ⬍ 0.05 greater
than all other time points). By 7 days after the exercise bout,
serum [CK] had returned to the “pre” exercise levels.
Amount of calpain-3 and its autolysis. Figure 3, A and B,
shows representative Western blots for calpain-3 for two subjects. Before the exercise (pre), calpain-3 existed in the muscle
Fig. 1. Effect of eccentric exercise on the peak isometric torque response
immediately after (post), and 3 h, 24 h, and 7 days following the initial exercise
bout. Torque data are expressed as a percentage of that produced before
exercise (pre). Values are means ⫾ SE; n ⫽ 6 subjects. *P ⬍ 0.05, different
from pre, 24 h, and 7 days (1-way ANOVA with repeated measures, NewmanKeuls post hoc analyses).
J Appl Physiol • VOL
Fig. 2. Effect of eccentric exercise on serum creatine kinase immediately after
(post), and 3 h, 24 h, and 7 days following the exercise bout (pre). Values are
means ⫾ SE; n ⫽ 6 subjects. *P ⬍ 0.05, different from pre, post, and 7 days;
#P ⬍ 0.05, greater than all other times (1-way ANOVA with repeated
measures, Newman-Keuls post hoc analyses).
of the subjects predominantly as a 94-kDa protein (top band),
which requires autolysis (to the 58- and 56-kDa forms) to be
proteolytically active (13, 30). Figure 3C shows the proportion
of calpain-3 that was autolyzed (i.e., amount in 58- and 56-kDa
forms compared with sum of all calpain-3 bands) at each time
after exercise expressed relative to that present before exercise
in the same subject. On average, the proportion of calpain-3
that was autolyzed was increased more than threefold at 24 h
after the eccentric exercise bout compared with that present
either before exercise (pre) or 3 h and 7 days after exercise
(Fig. 3C). To indicate how much of the total muscle calpain-3
was autolyzed on average, the percentage of calpain-3 in
autolyzed forms was expressed as a percentage of the total
calpain-3 present in the same muscle sample, without normalizing to the preexercise levels in the subject. Preexercise 16 ⫾
2% of the calpain-3 was autolyzed, and this amount increased
substantially 24 h after exercise to 35 ⫾ 12%.
Amount of ␮-calpain and its autolysis. The effects of the
eccentric exercise on ␮-calpain were also investigated. Figure
4, A and B, shows representative Western blots for ␮-calpain
for the same two subjects as shown for calpain-3 in Fig. 3, A
and B. ␮-calpain exists as an 80-kDa protein (top band) and
once activated autolyzes to 78- and 76-kDa bands. Analysis of
the pooled data (Fig. 4C) showed no significant difference in
the amount of autolysis of ␮-calpain across the trial, irrespective of whether or not the values were normalized to the levels
present in each subject before exercise. However, there did
appear to be noticeable autolysis in some individual cases, such
as in the immediate postexercise sample shown in Fig. 4B.
There was no correlation between the percentage of autolyzed
␮-calpain and the percentage of autolyzed calpain-3 across the
different subjects and time points (linear regression analysis,
r2 ⫽ 0.114, P ⫽ 0.16).
Extent of autolysis of calpain-3 and ␮-calpain in preexercise samples. Finally, the small level of ␮-calpain autolysis
observed in the subjects here in preexercise state (pre samples,
9 ⫾ 3% of the total ␮-calpain) was not significantly different
from that found previously (26) in a group of healthy, active
men before exercise (10 ⫾ 2%, n ⫽ 11; P ⬎ 0.05, 2-tailed,
unpaired t-test). The amount of autolyzed calpain-3 in the
preexercise samples in the present study (16 ⫾ 2%) was
slightly greater than in that previous study (9 ⫾ 1%, n ⫽ 11;
P ⬍ 0.05, 2-tailed, unpaired t-test). It should be noted that this
slightly higher baseline level of calpain-3 autolysis would, if
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expressing the density of the bands of the autolyzed products relative
to the total density of all the bands for that calpain (i.e., autolyzed and
unautolyzed) in the sample. This indicated what proportion of the
given calpain was autolyzed in that particular sample, irrespective of
any minor differences in protein loading.
Biopsy material was not available for one subject at both the pre
and immediately postexercise time points, and so no protein data are
presented for this subject. For another subject, there was no sample
immediately postexercise, so data presented are for n ⫽ 5 for pre, 3-h,
24-h and 7-day samples and n ⫽ 4 for immediately postexercise
samples.
CK. Serum CK concentration ([CK]) was determined pre, post, and
3 h, 24 h, and 7 days after eccentric exercise. Arterialized blood was
sampled from a dorsal hand vein for the pre and post samples, with all
subsequent samples obtained from an antecubital vein. Each sample
was placed into a plain evacuated test tube, and blood allowed to
coagulate for 30 min at room temperature and then centrifuged at
1,500 g for 10 min. The serum layer was removed and frozen at
⫺20°C until analyzed in duplicate for [CK] using an Olympus GmbH
AU1000 analyzer (Olympus Diagnostics, Clare). The normal reference range of [CK] using this method is 45–130 U/l.
Statistics. Data are expressed as means ⫾ SE, unless otherwise
indicated. Data were analyzed using a one-way ANOVA with repeated measures. When the ANOVA revealed a significant effect,
Newman-Keuls post hoc analyses were performed. Pre and post
samples were compared using a Student’s t-test (paired, 2-tailed). All
analyses were performed using Prism V 4.01. A probability value of
P ⬍ 0.05 was deemed to indicate significant difference.
CALPAINS AND ECCENTRIC EXERCISE IN HUMANS
929
anything, be expected to have detracted from, rather than
exaggerated, the relative changes in calpain-3 autolysis observed here after exercise (Fig. 3C).
DISCUSSION
In this study, we report the first example of physiological
circumstances that result in calpain-3 autolysis. We have
shown that a single bout of eccentric exercise resulted in
calpain-3 autolysis and hence in its activation. At 24 h postexercise ⬃35% of the total calpain-3 present in the muscle was in
the autolyzed state. Given that 1) the total proportion of
sarcomeres showing noticeable disruption after eccentric exercise is relatively small (⬍10%) (2, 18) (which is also likely the
case here because subjects were able to produce near-maximal
Fig. 4. Effect of eccentric exercise on the extent of
␮-calpain autolysis. A and B: Western blots of ␮-calpain
for same 2 subjects as in Fig. 3. For each Western blot, the
Coomassie blue stain of the SDS-PAGE gel showing
MHC following transfer indicates the relative loading of
the samples. ␮-calpain exists predominantly in its unautolyzed 80-kDa form (top band) at most time points, with
only a little autolysis to 78- and 76-kDa isoforms at any
time point. C: percentage of total ␮-calpain in autolyzed
forms (n ⫽ 5 subjects, except n ⫽ 4 for “post” as
indicated). Values are means ⫾ SE.
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Fig. 3. Effect of eccentric exercise on the extent of
calpain-3 autolysis. A and B: Western blots of calpain-3 for 2 subjects. For each Western blot, the
Coomassie blue stain of the SDS-PAGE gel showing myosin heavy chain (MHC) following transfer
indicates the relative loading of the samples. The
94-kDa calpain-3 (top band) can be seen autolyzed
to 58- and 56-kDa bands at various time points.
C: amount of 58- and 56-kDa calpain-3 (i.e., in
autolyzed forms) expressed as a proportion of the
total calpain-3 present at each time, normalized to
the proportion of autolyzed calpain-3 present before
exercise in the same subject (“pre” time sample)
(see METHODS) (n ⫽ 5 subjects except n ⫽ 4 at post).
Values are means ⫾ SE. 7d, 7 days. *P ⬍ 0.05
different from all other time points (1-way ANOVA
with repeated-measures, Newman-Keuls post hoc
analyses).
930
CALPAINS AND ECCENTRIC EXERCISE IN HUMANS
J Appl Physiol • VOL
this was not enough to reach significance levels; in any case it
was far smaller than the amount of autolysis present 24 h after
the exercise. Serum [CK] increased significantly in the 3 h
following the exercise and was maximal at the 24 h time point
(Fig. 2), which could be due to both acute damage to the
muscle and subsequent membrane damage caused by raised
cytoplasmic [Ca2⫹] (1). Thus the prolonged rise in resting
intracellular [Ca2⫹] following eccentric exercise may be responsible for both calpain-3 autolysis and CK loss.
We did not examine whether the other ubiquitous calpain,
m-calpain, was autolyzed during or following the exercise,
although given the potential role of m-calpain in muscle
regeneration (15), a future study should examine its autolysis
and activity following eccentric exercise, particularly given
that a twofold increase in the mRNA levels for m-calpain has
been observed a day after eccentric exercise (11).
Finally, on a speculative note, we suggest that the rise in
calpain-3 autolysis seen 24 h after the eccentric exercise is
reflecting an important role of calpain-3 in sarcomeric repair
and remodeling following the eccentric exercise. This would
be consistent with previous suggestions, based on studies with
calpain-3 knockout mice, that calpain-3 plays a vital role in
regulating and remodeling sarcomeric structure in mature muscle (9, 21). Furthermore, one of the unique features of eccentric
exercise experienced by individuals unaccustomed to the exercise protocol is that the muscle undergoes changes that help
prevent damage on a subsequent similar exercise bout. Thus it
is possible that calpain-3 may be involved not only in the repair
and reassembly of the original sarcomeric structure following
damage by eccentric contractions, but also in any subsequent
adaptive changes, such as adding extra sarcomeres to lengthen
the myofibrils (23, 29). Given that calpain-3 contains a putative
nuclear translocation domain (32) and it has been found localized to the nucleus (34), it may have a role not only as a
sarcomeric protease but also as a signaling molecule (31).
The present study showed increased calpain-3 autolysis in a
cohort of healthy individuals, who likely possessed the normal
adaptive mechanisms to eccentric exercise. Although not examined in the present study, we expect that if limb-girdle
muscular dystrophy type 2A patients had performed the same
eccentric exercise they would have experienced a similar level
of acute damage to their muscles as normal individuals [as is
the case with calpain-3 knockout and normal mice; (12)], but
their muscle fibers may not be able to subsequently undergo the
normal repair mechanisms because of the absence of functional
calpain-3 (21). Consequently, we speculate that the progressive
dystrophic changes that occur in limb-girdle muscular dystrophy type 2A patients over their life (and also in calpain-3
knockout mice) may be at least in part due to inadequate repair
of the cumulative minor damage that likely arises from the
mild eccentric muscle contractions that are a normal component of daily activity, particularly because this effect would be
compounded if the muscles also fail to show the normal
adaptation to eccentric exercise. This could be explored in
future experiments examining the long-term effects and adjustments that occur in muscles of normal and calpain-3 knockout
mice following eccentric exercise.
ACKNOWLEDGMENTS
We thank all subjects for their efforts.
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torque within 24 h after the exercise), and 2) all of the
calpain-3 within a muscle fiber is normally tightly bound (27)
[most likely to titin (19, 33)], the high proportion of calpain-3
autolysis suggests that the autolysis was a widespread process
within the muscle fibers and that a substantial amount of
autolysis must have taken place in many and perhaps in all of
the fibers in the exercised muscles and was not limited solely
to the localized regions of overt damage. We note that there are
both type I and type II muscle fiber types in human vastus
lateralis samples, and the findings here do not reveal whether
calpain-3 activation occurred in a fiber type-dependent manner.
Of note, increased calpain-3 autolysis was apparent 24 h
after the eccentric exercise protocol, but it was not detectable
either immediately after or 3 h after the exercise. This makes it
unlikely that the peak in calpain-3 autolysis was caused directly by the exercise itself or by the acute effects associated
with it, such as the repeated rises in cytoplasmic [Ca2⫹]
eliciting the muscle contractions. Nevertheless, as explained
below, it is quite likely that this autolysis of calpain-3 is caused
by raised cytoplasmic [Ca2⫹] but that it primarily occurs in
response to a very prolonged rise in resting [Ca2⫹] rather than
to the acute rises during muscular activity.
Overall, ␮-calpain showed a different response to the exercise protocol compared with calpain-3, there being no significant change in the proportion of autolyzed ␮-calpain at any
time postexercise. Interestingly though, in two subjects there
did appear to be an increased amount of autolyzed ␮-calpain
immediately postexercise (e.g., Fig. 4B), possibly indicating
that the eccentric exercise caused some ␮-calpain activation in
those particular subjects. It could be that those two subjects
were slightly different in their training status or lifestyle
compared with the rest of the group or that their muscles were
more sensitive to the nature of the eccentric exercise regime.
There was no significant correlation between the level of
calpain-3 autolysis and that of ␮-calpain across the subjects
and time course of the study. This indicates that the factor(s)
leading to the increased level of calpain-3 autolysis at 24 h
were relatively specific for calpain-3 compared with ␮-calpain.
One plausible scenario is that the autolysis of calpain-3 is due
to a prolonged rise in the resting [Ca2⫹] in the muscle fibers
following the eccentric contractions. Such a rise has been
observed in mouse skeletal muscle after eccentric contractions,
with the resting [Ca2⫹] being increased from its normal level of
⬃100 nM to ⬃250 nM for more than 24 h (22). At submicromolar levels of Ca2⫹ there is virtually no autolysis or activation
of ␮-calpain (15, 26, 27), but calpain-3 does show autolysis
and activation (13, 26), although the process proceeds extremely slowly at such low Ca2⫹ levels and can take up to 24 h
for much of the calpain-3 to become autolyzed (13). This
slowness in the Ca2⫹-dependent autolysis of calpain-3 is also
apparent at higher [Ca2⫹], because it was found that exposure
to 2.5 ␮M Ca2⫹ caused a small amount of autolysis of
␮-calpain within 1 min but there was no detectable autolysis of
calpain-3 unless the period of exposure was increased (26).
This latter finding also can account for the fact that the
eccentric exercise regime here appeared to cause proportionately more autolysis of ␮-calpain than of calpain-3 during the
actual course of the exercise period. However, in view of the
variability in the measurements and the relatively small number of subjects examined, it is possible that there was some
level of calpain-3 autolysis occurring during the exercise, but
CALPAINS AND ECCENTRIC EXERCISE IN HUMANS
Present addresses: M. Leikis, Wellington Hospital, Dept. of Renal
Medicine, Private Bag 7902, Wellington 6001, New Zealand; J. Bennie,
School of Exercise and Nutrition Sciences, Deakin University, Melbourne
3125, Australia.
GRANTS
R. M. Murphy is supported by National Health and Medical Research
Council (NHMRC) Fellowship 380842. This study was in part supported by
NHMRC Grants 256603 and 433034.
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