J Appl Physiol 106: 804–812, 2009.
First published January 8, 2009; doi:10.1152/japplphysiol.90872.2008.
Endurance exercise activates matrix metalloproteinases in human
skeletal muscle
Eric Rullman,1 Jessica Norrbom,1,2 Anna Strömberg,1 Dick Wågsäter,3 Helene Rundqvist,2 Tara Haas,4
and Thomas Gustafsson1
1
Division of Clinical Physiology, Department of Laboratory Medicine, Karolinska University Hospital, Karolinska Institute,
Stockholm; 2Department of Physiology and Pharmacology, Karolinska Institute, Stockholm; 3Department of Medicine,
Atherosclerosis Research Unit, King Gustav V Research Institute, Karolinska Institute, Stockholm, Sweden; and 4School
of Kinesiology and Health Sciences, York University, Toronto, Ontario, Canada
Submitted 27 June 2008; accepted in final form 7 January 2009
CHANGES IN THE FUNCTIONAL and morphological characteristics
of skeletal muscle occur under various conditions and in
response to a wide range of pathological and physiological
stimuli (26). Most, if not all, such processes require a remodeling of the extracellular matrix (ECM), by degradation and
regeneration. In fact, the vast majority of genes upregulated in
response to exercise training are related to ECM (31).
Matrix metalloproteinases (MMPs) compromise a diverse
family of proteases that degrade a similarly large variety of
proteins. Each MMP is targeted to a specific variety of ECM
proteins. Expression of various MMPs is found in most
tissues and has been demonstrated to play essential roles
during growth, development, and angiogenesis (2, 5, 9).
MMP-2 and MMP-9 are both collagenases with a special
ability to degrade collagen type IV, the most prevalent
protein in skeletal muscle basal lamina. Despite their similarity in substrate specificity, the general patterns of expression and transcriptional regulation of MMP-2 and MMP-9
are very different; they also seem to be involved in partly
different biological process (4, 9, 14, 34). The MMP-2 gene
lacks a TATA box, is constituently expressed in most
tissues, and is induced only modestly by growth factors and
cytokines. MMP-2, activated by MMP-14, is considered to
cleave the basal lamina and ECM during angiogenesis and
thus makes a path where endothelial sprouts can migrate and
form new capillaries (9, 13). MMP-2 has furthermore been
implicated as important for successful regeneration of experimentally damaged muscle fibres in the mouse (19). In
contrast, MMP-9 shows none or a very modest basal expression, but its promoter region contains a variety of
response elements sensitive to a wide range of growth
factors and cytokines (33). MMP-9 has been proposed to
affect general collagen turnover in skeletal muscle and in
vitro models of disease (7, 9, 28). It might also to be a
regulator of growth factor bioavailability via proteolytic
release (8, 21). Thus, despite their common substrate specificity, MMP-2 and MMP-9 are principally different in terms
of transcriptional regulation, and they have been associated
with different aspects of tissue remodeling (4, 9, 14, 34).
Physical exercise is a complex process constituting several factors that could affect the expression levels of MMP-2
and MMP-9 in skeletal muscle, such as local ischemia and
increases in muscle stretching; in shear stress; and in the
wall tension of blood vessels (2, 24, 28). Despite the
established importance of ECM remodeling in the skeletal
muscle during adaptation to exercise and the central role of
MMPs in these processes, MMP levels in exercising skeletal
muscle have been largely overlooked. One exception is a
report in which MMP activity was demonstrated to be
crucial for exercise-induced skeletal muscle angiogenesis in
the rat (14). MMP-2 and MMP-14 increased with chronic
electrical stimulation and inhibition of MMP activity completely abolished angiogenesis. Furthermore, expression and
exercise-induced changes of MMP levels in human skeletal
muscle have to our knowledge only one been explored in
Address for reprint requests and other correspondence: E. Rullman, Div. of
Clinical Physiology C1-88, Karolinska Univ. Hospital, Huddinge 141 86
Stockholm, Sweden (e-mail: [email protected]).
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.
extracellular matrix; remodeling
804
8750-7587/09 $8.00 Copyright © 2009 the American Physiological Society
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Rullman E, Norrbom J, Strömberg A, Wågsäter D,
Rundqvist H, Haas T, Gustafsson T. Endurance exercise activates matrix metalloproteinases in human skeletal muscle. J Appl
Physiol 106: 804 –812, 2009. First published January 8, 2009;
doi:10.1152/japplphysiol.90872.2008.—In the present study, the effect of exercise training on the expression and activity of matrix metalloproteinases (MMPs) in the human skeletal muscle was investigated.
Ten subjects exercised one leg for 45 min with restricted blood flow and
then exercised the other leg at the same absolute workload with unrestricted blood flow. The exercises were conducted four times per week for
5 wk. Biopsies were taken from the vastus lateralis muscles of both legs
at rest before the training period, after 10 days and 5 wk of training, and
2 h after the first exercise bout for analysis of MMP and tissue inhibitor
of metalloproteinase-1 (TIMP-1) mRNA, enzyme activity, and protein
expression. Levels of MMP-2, MMP-14, and TIMP-1 mRNA in muscle
tissue increased after 10 days of training regardless of blood flow
condition. MMP-2 mRNA level in laser-dissected myofibers and MMP-2
activity in whole muscle increased with training. The level of MMP-9
mRNA and activity increased after the first bout of exercise. Although
MMP-9 mRNA levels appeared to be very low, the activity of MMP-9
after a single bout of exercise was similar to that of MMP-2 after 10 days
of exercise. MMP-2 and MMP-9 protein was both present throughout the
extracellular matrix of the muscle, both around fibers and capillaries, but
MMP-2 was also present within the skeletal muscle fibers. These results
show that MMPs are activated in skeletal muscle in nonpathological
conditions such as voluntary exercise. The expression and time pattern
indicate differences between the MMPs in regards of production sites as
well as in the regulating mechanism.
MMP IN HUMAN SKELETAL MUSCLE WITH EXERCISE
one earlier study. This study demonstrated that a single bout
of exercise induced expression of MMP-9 mRNA as well as
increased MMP-9 activity (29).
In the present study, we explore the effects of exercise
training with similar mechanical load but with differences in
exercise-induced metabolic alteration on the levels, temporal
patterns, and activity of MMP in human skeletal muscle tissue.
We report that 1) members of the MMP family are present in
the skeletal muscle of healthy humans; 2) MMP-9 and MMP-2
are induced by exercise, both by pre- and posttranslational
mechanisms, but with a different time pattern; and 3) inhibiting
factor tissue inhibitor of metalloproteinase-1 (TIMP-1) is induced in parallel. The present findings encourages further
studies to establish both the regulatory mechanisms of transcriptional upregulation and the activation as well as the
biological significance of the MMP family in the adaptation of
the skeletal muscle to exercise.
METHODS
Ten healthy male subjects were included in the study. Their mean
(range) age, height, and weight were 24 (20 –27) yr, 181 (173–190)
cm, and 75 (63–90) kg, respectively, and their mean (range) maximal
oxygen uptake (V̇O2max) was 51 (43– 64) ml 䡠 kg⫺1 䡠 min⫺1. Welltrained subjects (V̇O2max ⬎65 ml 䡠 kg⫺1 䡠 min⫺1) were excluded to
maximize the subjects’ training responses.
Experimental Model
Exercise in the supine position was performed with the dynamic
constant-load knee-extension exercise for 45 min at 60 revolutions/
min. A method introduced by Eiken and Bjurstedt (3) and thoroughly
described in a recent publication (12) was used to restrict blood flow
during exercise. Briefly, the subjects performed 45 min of one-legged
exercise with a 20% restriction in blood flow at their highest tolerable
workload. This was immediately followed by exercise with the opposite leg without blood flow restriction and at the same workload as
the first leg. Hereby both legs exercised with the same mechanical
load, but the blood flow restriction leads to functional ischemia and an
increase in metabolic stress (10) and it is associated with enhanced
expression of several growth factors (1, 10, 12) compared with
exercise at the same absolute workload with unrestricted blood flow.
The subjects performed one-legged exercise four times per week
during a 5-wk period. Subjects exercised one leg for 45 min under
restricted blood flow (R) condition and then the other leg under
normal, unrestricted (UR) condition. The workload for each exercise
bouts was measured, and the total workload performed for each week
was calculated.
Muscle biopsies were obtained using the percutaneous needle
biopsy technique from the vastus lateralis muscle of both legs on three
occasions: before and 2 h after the first exercise bout, after 10 days of
training, and after 5 wk of training; the latter two biopsies were
obtained 24 h after the previous bout of exercise. All biopsy samples
were frozen within 10 –15 s in liquid nitrogen and stored at ⫺80°C
until further analysis. The study was approved by the Ethics Committee of Karolinska Institutet. Written informed consent of all subjects was obtained.
RNA Extraction and Reverse Transcription
Total RNA was prepared by the acid phenol method and quantified
spectrophotometrically by absorbance at 260 nm. The integrity of total
RNA was controlled on a 1% agarose gel electrophoresis. Two
micrograms of RNA were reverse transcribed by Superscript reverse
transcriptase (Life Technologies) using random hexamer primers
(Roche Diagnostics) in a total volume of 20 ␮l.
Real-Time PCR to Quantify mRNA. Detection of mRNA was
performed on an ABI-PRISM7700 Sequence Detector (PerkinElmer Applied Biosystems). Primer and probe for MMP-2, MMP-9,
Fig. 1. A: illustration of a representative zymogram. MMP, matrix metalloproteinases; Pre, sample obtained before exercise; Post, sample obtained 2 h after the first exercise bout; 10 days and
5 weeks, sample obtained after 10 days and 5 wk
of exercise training, respectively; R and UR, sample obtained from the leg exercised with restricted
and unrestricted blood flow respectively. B: comparison of MMP-9 mRNA related to GAPDH
mRNA (left) and 18S rRNA (right). Values are
means ⫾ SE presented as fold change.
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Subjects
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MMP IN HUMAN SKELETAL MUSCLE WITH EXERCISE
RNA degradation, RNAase inhibitor was added to the PBS solutions
(50 U/100 l; SUPERase, Ambion). The sections were dehydrated in a
graded series of 75, 95, and 2 ⫻ 100% ethanol for 1 min each,
followed by a 5-min immersion in pure xylene and air drying for 20
min. The laser capture and microdissection was performed using the
P.A.L.M. system (Carl Zeiss microimaging). From muscle tissue
sections, larger areas of muscle tissue covering all cell types and
individual muscle fibers were dissected from the same section. After
capturing the sum of 100 muscle fibers and subsequently an area of
tissue of the same size as the fibers from each section, the caps were
inserted into 0.5-ml microcentrifuge tubes filled with 50 ␮l of extraction buffer (PicoPure RNA isolation kit, Arcturus Engineering). After
incubation in 42°C for 30 min, the extraction buffer was collected by
a brief spin (800 g for 2 min), the caps were removed, and the
microcentrifuge tubes were stored in ⫺80°C. Total RNA was extracted from the cell lysate using the PicoPure RNA isolation kit
according to the manufacturer’s protocol. The isolated RNA was
stored frozen in ⫺80°C until use. Isolated RNA were reverse transcribed by Superscript reverse transcriptase (Life Technologies,
Solna, Sweden) using random hexamer primers (Roche Diagnostics)
in a total volume of 30 ␮l. MMP-2 and 18S were analyzed as
described above.
Laser Capture Microdissection
Zymography was performed in seven of the subjects. Zymography
supplies were purchased from Invitrogen. MMP-2, pro-MMP-9, and
active MMP-9 distribution was determined from muscle biopsy homogenate before and 2 h after the first exercise bout, after 10 days of
training, and after 5 wk of training. A detailed description of the
protocol used has been published previously (29). Briefly, 25 ␮g of
protein homogenized in phosphate-buffer were diluted into 2⫻ Trisglycine SDS sample buffer and electrophoretically separated under
nonreducing conditions. Proteins were incubated in renaturing buffer
Fig. 2. MMP-2 mRNA and activity in human vastus
lateralis muscle before and after a single bout of exercise (A) and over 5 wk of training (B). Pre, biopsy taken
before the first exercise bout; 10 days, biopsy taken
after 10 days of exercise training; 5 w, biopsy obtained
after 5 wk of training. Values are means ⫾ SE presented as fold change (mRNA n ⫽ 10 subjects; protein
n ⫽ 7 subjects). Interaction in the ANOVA is reported
in the figure, and the symbol in the figure denotes the
located difference found in the post hoc test performed
following the ANOVA analysis. *Significant differences between before exercise and 10 days in both
conditions, P ⬍ 0.05.
A
A
Muscle fibers were dissected from a subset (n ⫽ 3) of the subjects,
before and after 10 days of training, and mRNA of MMP-2 was
measured. Frozen biopsy samples were embedded in OCT (TissueTek, Sakura, Finetek) and kept frozen at ⫺80°C until further analysis.
Cross sections (8 ␮m) of biopsy samples were cut at ⫺20°C, placed
on glass slides, and immediately put in cold acetone for 5 min for
fixation, followed by washing in PBS for 3 ⫻ 15 min. To prevent
Protein Extraction and Zymography
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MMP-14 and TIMP-1 were ordered as assay on demand (Hs00234422_m1,
Hs00234579_m1, Hs00237119_m1, Hs00171558_m1 Perkin-Elmer Applied Biosystems). Selected as endogenous control to correct for
potential variations in RNA loading was 18S rRNA (4310893E,
Perkin-Elmer Applied Biosystems). ␤-Actin increases in response to
repeated exercise, especially during restricted blood flow, as indicated
by an increased amount relative to 18S levels, and thereby ␤-actin was
unsuitable as a housekeeping gene. There was a tendency to an
interaction (condition ⫻ exercise) of MMP-9 mRNA after a single
bout of exercise. To reduce the risk of a type II error due to possible
random variability in 18S, an ad hoc analysis of this case using GADH
as housekeeping gene was performed. The result was, however,
similar regardless of the choice of housekeeping gene (see Fig. 1B).
The mix for 18S rRNA was prepared according to the manufacturer’s
recommendation and run at a 1:2,000 (corresponding to 0.25 ng RNA)
dilution in separate wells. The cDNA for MMP-2, MMP-14, TIMP-1,
and GAPDH was diluted 1:100 (corresponding to 5 ng RNA) and
MMP-9 1:5 (corresponding to 100 ng RNA) due to low levels of
MMP-9 mRNA. A more detailed description of the procedure is
available in a recent publication (12). The basal resting relative
distribution of MMP-2 and -9 was performed for each individual; the
change in threshold cycle (⌬CT) value was obtained by subtracting
CT values from MMP-2 with CT values from MMP-9. The relative
expression of was then calculated by 2⫺⌬CT.
MMP IN HUMAN SKELETAL MUSCLE WITH EXERCISE
for 30 min at room temperature. The gels were incubated overnight at
37°C in developing buffer. After 1 h staining with Coomassie blue and
destaining for 2 days with 10% acetic acid and 40% methanol in
water, gelatinase activity was evident by clear bands against a dark
blue background. Quantification of the bands was performed using
digital camera Fujifilm LAS-1000 and densitometry software Fujifilm
Image gauge version 3.46.
Immunohistochemistry
noResearch) diluted 1:500 was added in the double-staining protocols.
As a negative control, the primary antibodies were excluded from the
protocol.
Statistics
The data were analyzed using logarithmic-transformed ratios for
mRNA and logarithmic-transformed densitometric values for zymographies. A two-way ANOVA for repeated measures was used to
evaluate the effects of training (before, 10 days, and 5 wk) in the two
exercise conditions (UR and R) on basal mRNA and protein content.
Differences were considered significant at P ⬍ 0.05. Planned comparison was used (i.e., post hoc test) to locate differences corresponding to significant interactions or when no interaction was found to
locate differences corresponding to significant main effects in the
ANOVA models. Due to high variance, a nonparametrical analysis
(Wilcoxon matched-pair test) was performed on the MMP-9 data in
addition to the ANOVA. In the figures, samples are expressed relative
to the corresponding trial control sample (preexercise), which was set
at 1. Data are presented as means ⫾ SE unless otherwise stated.
RESULTS
Training Response
The average cumulative workload (4 bouts) during each of
the 5 training wk were 1,975, 2,461, 2,803, 3,157 and 3,350 W,
respectively, corresponding to a 1.3-, 1.5-, 1.8-, and 2.0-fold
increase in workload over the last 4 wk compared with the first
week.
Fig. 3. MMP-9 mRNA (A) and total protein (B)
(active ⫹ pro-MMP) in human vastus lateralis
muscle before and after a single bout of exercise
(A) and over 5 wk of training (B). Pre, biopsy taken
before the first exercise bout; 10 d, biopsy taken
after 10 days of exercise training; 5 w denote
biopsy obtained after 5 wk of training. Subjects
with contraction threshold values over 35 in the Pre
sample are excluded from the figure (n ⫽ 2), and 2
outliers are marked with x in the figure. Values are
means ⫾ SE presented as fold change (mRNA n ⫽
10 subjects, protein n ⫽ 7 subjects). Interaction in
the ANOVA is reported in the figure, and the
symbols in the figure denote the located difference
found in the post hoc test performed following the
ANOVA analysis. ‡Significant differences between
before and postexercise in both conditions, P ⬍
0.05. ‡‡Significant differences between before exercise and 10 days in both conditions using nonparametric statistics, P ⬍ 0.05.
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Frozen biopsy samples were embedded in OCT (Tissue-Tek,
Sakura Finetek) and kept frozen at ⫺80°C until further analysis. Cross
sections (5 ␮m) of biopsy samples were cut at ⫺21°C, placed on glass
slides. Sections for MMP-9 staining were put in 4% formaldehyde,
and sections for MMP-2 staining were put in 99% ethanol for 10 min
for fixation in room temperature. After washing in PBS for 3 ⫻ 5 min,
the sections were incubated with PBS containing 3% BSA and 0.1%
Triton X-100 (Dako) for 30 min at room temperature. A rabbit
anti-human MMP-9 antibody (Sigma-Aldrich) diluted 1:500 in PBS
with 1% BSA and 0.3% Triton X-100 was applied to the sections and
incubated at 37°C for 60 min, and subsequently the sections were
incubated overnight at 4°C with a rabbit anti-human MMP-2 antibody
(Sigma-Aldrich) diluted 1:250 in PBS with 1% BSA. Following
washing, the sections were incubated for 60 min with a cyanine-3conjugated secondary antibody donkey anti-rabbit (Jackson ImmunoResearch) diluted 1:500 in 1% BSA at 37°C for 60 min. The
sections were mounted with Vectashield (Vector Laboratories). Double staining was achieved by coincubation of the primary antibody
together with sarcolemmal marker, a mouse anti-human caveolin-3
antibody (Santa Cruz Biotechnology) diluted 1:500 or an endothelial
cell marker mouse anti-human CD31 (Dako) diluted 1:100. A FITCconjugated donkey anti-mouse secondary antibody (Jackson Immu-
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MMP IN HUMAN SKELETAL MUSCLE WITH EXERCISE
mRNA
Laser Capture Microdissection
MMP-2. MMP-2 mRNA expression did not change in response to a single bout of exercise regardless of exercise
condition. The basal level of MMP-2 mRNA was higher after
10 days of training (effect of training, P ⬍ 0.001) and remained
elevated after 5 wk of training, independent of training condition (no interaction between training and condition). MMP-2
data are presented in Fig. 2.
MMP-9. There was an increase in MMP-9 transcript after a
single bout of exercise regardless of exercise condition.
MMP-9 mRNA were also increased compared with preexercise
levels after 10 days of exercise training if tested with a
nonparametric test (P ⬍ 0.05), but because of the high variance
this increase did not reach significance using an ANOVA (P ⫽
0.16). MMP-9 data are presented in Fig. 3.
MMP-14. MMP-14 mRNA expression did not change in
response to a single bout of exercise regardless of exercise
condition. The basal level of MMP-14 mRNA was higher after
10 days of training (effect of training, P ⬍ 0.01) and remained
elevated after 5 wk of training, independent of training condition (no interaction between training and condition). MMP-14
data are presented in Fig. 4.
TIMP-1. There was a tendency to an increase, albeit insignificant (effect of exercise, P ⫽ 0.059), of TIMP-1 mRNA in
response to a single bout of exercise regardless of exercise
condition. The basal level of TIMP-1 mRNA was higher after
10 days of training (effect of training, P ⬍ 0.05) and remained
elevated after 5 wk of training, independent of training condition (no interaction between training and condition). TIMP-1
data are presented in Fig. 4.
MMP-2 transcript was detectable in all samples taken before
exercise. With exercise training, the MMP-2/18S ratio was
increased in all subjects (n ⫽ 3) both in isolated muscle fibers
and dissected areas covering all cell types, including cells such
as endothelial cells. Median fold change was 4.5 in laser
capture-microdissected muscle fibers. MMP-9 mRNA levels
were to low to be detected in any of the laser capturemicrodissected samples. A photograph of a microdissected
muscle fiber is presented in Fig. 5.
Zymography
Immunohistochemistry
MMP-9 staining was evident in the extracellular matrix
around muscle fibers and a colocalization with CD31-positive
Fig. 4. MMP-14 and tissue inhibitor of metalloproteinase-1 (TIMP-1) mRNA in human vastus lateralis muscle before and after a single bout of exercise
(A) and over 5 wk of training (B). Pre, biopsy taken
before the first exercise bout; 10 d, biopsy taken
after 10 days of exercise training; 5 w, biopsy
obtained after 5 wk of training. Values are means ⫾
SE presented as fold change (n ⫽ 10 subjects).
Interaction in the ANOVA is reported in the figure,
and the symbol in the figures denotes the located
difference found in the post hoc test performed
following the ANOVA analysis. *Significant differences between before exercise and 10 days in both
conditions, P ⬍ 0.05.
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MMP-2. MMP-2 protein was unchanged after a single bout
of exercise in both exercise conditions. Basal level of MMP-2
activity was higher after 10 days of training (effect of training,
P ⬍ 0.05) and remained elevated after 5 wk of training,
independent of training condition (no interaction between
training and condition). MMP-2 data is presented in Fig. 2, and
a representative zymogram is presented in Fig. 1.
Total MMP-9. There was an increase in total MMP-9 (proMMP-9 ⫹ active MMP-9) level after a single bout of exercise
regardless of exercise condition (main effect of exercise) (P ⬍
0.05). There was no change in total MMP-9 with training.
MMP-9 data are presented in Fig. 3, and a representative
zymogram is presented in Fig. 2.
MMP IN HUMAN SKELETAL MUSCLE WITH EXERCISE
809
DISCUSSION
areas, indicating high abundance around capillaries. No
MMP-9 immunoreactivity was observed within skeletal muscle
fibers. For MMP-9 immunohistochemistry, see Fig. 6. MMP-2
staining was more scattered. It was a clear staining around
muscle fibers, but the staining seemed to be more intense
adjacent to capillaries. In addition, MMP-2 immunoreactivity
was apparent also within the skeletal muscle fibers. See Fig. 7
for MMP-2 immunohistochemistry.
Fig. 6. Immunohistochemical staining of MMP-9.
MMP-9 (red) (A), ⫹ sarcolemmal marker caveolin-3
(green) (B), MMP-9 (red) ⫹ endothelial cell marker
CD31 (green) (C), and negative control (D). All
images are from human skeletal muscle sections
before exercise. B and C are counterstained with
4,6-diamidino-2-phenylindole (blue). The distributions of the staining indicate MMP-9 to be distributed in the extracellular matrix (solid arrows) around
capillaries and muscle fibers but with no expression
within skeletal muscle fibers.
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Fig. 5. Laser capture-microdissected muscle fiber. The circle indicates a
muscle fiber that has been microdissected out for mRNA analysis. Altogether,
100 fibers were isolated from each section. Fibers were microdissected with a
clear margin to the cell boundaries to avoid contamination from adjacent cells.
Arrows and numbers come from laser-capture software.
Skeletal muscle remodeling involves the degradation and
synthesis of intracellular and extracellular proteins. In this
study, we investigated the expression of selected MMPs in
response to exercise training in humans.
Our results show that MMP-2 is activated in repeatedly
exercised skeletal muscle and that factors that activate and a
factor that inhibit MMPs are induced simultaneously. These
observations are novel for human skeletal muscle, but they are
supported by studies on exercise and the human tendon and by
electrical stimulation of an animal model (14, 15, 20). The
absolute number of transcripts of MMP-9 was much lower than
that of MMP-2, but it increased after a single exercise bout.
Thus the expression patterns of MMP-2, MMP-14, TIMP-1,
and MMP-9 indicate that these factors are regulated by different stimuli or through dissimilar mechanisms during exercise.
This is consistent with differences in the cis-element composition of the MMP-9, MMP-2, and MMP-14 promoters (33).
TIMP-1 mRNA increased alongside the increase in MMP-2
and MMP-14, which is consistent with a previous report on
exercise in the rat (15). Thus these studies collectively suggest
a concurrent initiation of both activation and inhibition of the
MMP-family by exercise, and they encourage further exploration of whether type and mode of exercise influence these
processes differently.
A recent study suggested that changes in skeletal muscle
MMP-9 mRNA are associated with an inflammatory response
to the biopsy procedure (15). To test this, we measured MMP-9
mRNA in muscle biopsies of eight subjects taken from the
same leg at 2-h intervals in the absence of exercise. The
MMP-9 mRNA level did not change with time (P ⫽ 0.3, data
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MMP IN HUMAN SKELETAL MUSCLE WITH EXERCISE
Fig. 7. Immunohistochemical staining of MMP-2
(A), MMP-2 (red) ⫹ sarcolemmal marker caveolin-3
(green) (B), MMP-2 (red) ⫹ endothelial cell marker
CD31 (green) (C), and negative control (D). All
images are from human skeletal muscle sections
before exercise. B and C are counterstained with
4,6-diamidino-2-phenylindole (blue). The distributions of the staining indicate MMP-2 to be distributed in the extracellular matrix around muscle fibers
(solid arrows) as well as capillaries (open arrows)
and also within skeletal muscle fibers.
J Appl Physiol • VOL
cated that MMP-2 was present in capillaries, which is consistent with a report showing that rat skeletal muscle endothelial
cells express MMP-2 (22). However, MMP-2 expression was
evident around but also within skeletal muscle fibers indicating
that skeletal muscle fibers contribute to the overall activity of
MMP-2. This is further supported by the observation of
MMP-2 mRNA in laser-dissected muscle fibers. RT-PCR data
indicate that MMP-9 mRNA abundance in human skeletal
muscle is very low and thus presumably also the translation
and secretion of this factor. Immunohistochemistry data reveal
that MMP-2 and MMP-9 protein are present in resting skeletal
muscle, but lack of zymographic activity at rest imply that both
are in an inactive state and become activated with physical
exercise. Because the activity levels were similar for the two
factors, it seems likely that MMP-9 accumulates over time in
the ECM of the skeletal muscle. Thus the expression and time
pattern support differences in production site as well as regulating mechanisms.
Although earlier reports showed that MMP-9 is induced in
ischemic muscles, and that the promoter region of MMP-9 is
responsive to many cellular signals and growth factors associated with metabolic stress (6, 18, 33), we were unable to
confirm our hypothesis that expression of MMP-9 in exercised
muscle is higher when blood flow is restricted than when it is
unrestricted. However, in a similar study involving exercise
with restricted blood flow, our laboratory observed that phosphorylation of p38 increased after acute exercise, and that
restriction of blood flow had no further effect. p38 regulates
MMP-9 transcription in various cell lines, including C2C12
cells (25, 30). This indirect evidence constitutes further proof
against metabolic stress as a major regulatory stimulus responsible for the exercise-induced increase in MMP-9 observed in
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not shown), indicating that changes in MMP-9 mRNA level
cannot be attributed solely to biopsy procedures. The gelatinolytic activity of MMP-9, which after a single bout of exercise
was similar to that of MMP-2 after 10 days of exercise, is
further evidence of the biological relevance of MMP-9.
MMP-9 mRNA was elevated compared with preexercise levels
after 10 days of exercise training if tested with a nonparametric
test, but due to the high variance this increase did not reach
significance using an ANOVA (P ⫽ 0.16). In the human
tendon, MMP-9 activity can be detected up to 3 days after a
single bout of exercise (20), which suggests that it has a
relatively long half-life, and that the effect of exercise on
MMP-9 level is cumulative, which is consistent with our
results.
The expression of MMP-2 and MMP-9 protein was confirmed by immunohistochemistry of samples of resting skeletal
muscle from which no previous biopsies had been obtained,
which minimized any possible biopsy effects. MMP-9 was
clearly localized in the ECM, around muscle fibers and in
capillaries. Double staining with CD31 and caveolin 3 showed
that MMP-9 was present throughout the extracellular matrix of
the muscle, i.e., around capillaries and muscle fibers. The
absence of MMP-9 immunoreactivity within skeletal muscle
fibers, and absence of MMP-9 transcripts within laser-dissected myofibers, indicate that skeletal muscle fiber is not the
primary source of MMP-9 expression in human skeletal muscle
tissue. The widespread prevalence of MMP-9 in the ECM
supports previous reports showing that MMP-9 is secreted by
several cell types in skeletal muscle tissue, including satellite
and endothelial cells (11, 19). The distribution of MMP-2
staining was scattered, and it had an extra- and an intracellular
appearance. Double staining with CD31 and caveolin-3 indi-
MMP IN HUMAN SKELETAL MUSCLE WITH EXERCISE
GRANTS
This work was supported by the Swedish Heart-Lung Foundation, the
Swedish Research Council, the Swedish National Center for Research in
Sports, the Swedish Society of Medicine, Swedish Society for Medical Research, the Magn. Bergvall Foundation and the Swedish Foundation for
International Cooperation in Research and Higher Education (STINT).
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the present study. As was observed for MMP-9, restricted
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In conclusion, members of the MMP family are present in
the skeletal muscles of healthy humans. MMP-9 is induced
by a single bout of exercise, presumably by posttranslational
activation and an increase in mRNA. In contrast, MMP-2,
MMP-14, and TIMP-1 levels increase after 10 days of
exercise training. Blood flow restriction to the exercising
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MMP-2 and MMP-9. Further studies are needed to elucidate
mechanisms responsible for transcriptional upregulation and
activation of the MMP family and to determine the biological significance of MMPs for the adaptation of the skeletal
muscle to exercise.
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