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1 Neuromuscular Research Center, Department of Biology of Physical Activity, 4 Department of Health Sciences, University of Jyväskylä, 40351 Jyväskylä, 3 LIKES Research Center for Sport and Health Sciences, 40700 Jyväskylä, 5 Department of Sports Medicine, Deaconess Institute of Oulu, 90100 Oulu, Finland; 2 Sports Medicine Research Unit, Bispebjerg Hospital, 2400 Copenhagen NV, and Copenhagen Muscle Research Centre, Rigshospitalet, 2100 Copenhagen Ø, Denmark
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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This experiment
tested the hypothesis that running-induced damage to rat skeletal
muscle causes changes in synthesis and degradation of basement membrane
type IV collagen and to proteins regulating its degradation. Samples
from soleus muscle and red and white parts of quadriceps femoris muscle
(MQF) were collected 6 h or 1, 2, 4, or 7 days after downhill
running. Increased muscle 
-glucuronidase activity indicated greater
muscle damage in the red part of MQF than in the white part of
MQF or soleus. In the red part of MQF, type IV collagen expression was
upregulated at the pretranslational level and the protein concentration
decreased, whereas matrix metalloproteinase-2 (MMP-2), a protein that
degrades type IV collagen, and tissue inhibitor of metalloproteinase-2
(TIMP-2), a protein that inhibits degradation, were increased in
parallel both at mRNA and protein levels. Type IV collagen mRNA level
increased in the white part of MQF and soleus muscle. The protein
concentration increased in the white part of MQF and was unchanged in
soleus muscle. MMP-2 and TIMP-2 changed only slightly in the white part of MQF and soleus muscle. The changes seem to depend on the severity of
myofiber injury and thus probably reflect reorganization of basement
membrane compounds.
matrix metalloproteinase; tissue inhibitor of metalloproteinase; damage; collagen degradation
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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TYPE IV COLLAGEN IS
THE MAIN component of basement membranes and is thought to have a
critical role in the cellular arrangement within tissue, e.g., by
ensuring mechanical stability in skeletal muscle fibers
(10). This is interesting as it is only a small part of
the total extracellular matrix (49). It has previously been demonstrated that lifetime endurance training can increase type IV
collagen content in rat skeletal muscle (27), and type IV
collagen steady-state mRNA levels were found to elevate rapidly after
an acute, strenuous exercise bout (17). It is, however, unknown what mechanisms are behind these changes, and whether a single
bout of intense exercise will affect type IV collagen degradation and
its concentration. Proteolytic degradation of specific extracellular
matrix compounds, which occurs during physiological and pathological
processes, is initiated by the family of zinc-dependent proteases
called matrix metalloproteinases (MMPs) (33).
MMP-2 (6) and MMP-9 (32) are
believed to break down native type IV collagen. The actual activities
of MMPs are under complex pre- and posttranslational regulation
including activation of proMMPs to MMPs by enzymatic cleavage and
inhibition by tissue inhibitors of matrix metalloproteinases (TIMPs)
(14). TIMP-1 and TIMP-2 are capable of inhibiting the
activities of all known MMPs in vitro with different binding affinities
(12) and can form complexes with proMMP-9
(13) and proMMP-2 (12, 24), respectively. The
expression of MMP-2 and MMP-9 has previously been investigated in
drug-induced skeletal muscle damage in normal and mutant mdx mice, the murine model of Duchenne muscular dystrophy
(22). We know of no studies that describe the
expression of TIMP-1 and TIMP-2 in rat skeletal muscle. On this basis,
the goal of the present experiment was to test the hypothesis that
intense, eccentric treadmill running causes changes in synthesis and
degradation of basement membrane type IV collagen and to proteins
regulating its degradation in rat skeletal muscle. Soleus and
quadriceps femoris (MQF) muscles were investigated, and muscle
-glucuronidase activity was used as an indicator of muscle injury
(41).
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Animals and exercise. Ten-week-old female Wistar rats (n = 50, body wt 190 ± 15 g), supplied by the Laboratory Animal Centre of the University of Jyväskylä (Jyväskylä, Finland), were used in this study. The animals were housed under constant temperature (22°C), humidity (40%), and light-dark (light between 6:00 AM and 6:00 PM) conditions in groups of four or five per cage (Scanbur, Køge, Denmark) with free access to pelleted chow (R36, Labfor, Stockholm, Sweden) and tap water. Treatment of the animals was in accordance with the European Convention for the Protection of the Vertebrate Animals Used for Experimental and Other Scientific Purposes and was approved by the Ethical Committee for Animal Care and Use at LIKES Research Center.
Eight rats were used as sedentary controls, and 42 rats were made to run on a motor-driven treadmill with
13.5° downhill tracks at a
speed of 17 m/min for total of 130 min. This exercise model has
previously been used to elicit muscle damage (17). During
the exercise, 5-min running bouts (26 altogether) were separated by
2-min rest periods. The animals were adapted to treadmill running
during the first 5 min at a lower speed. After running, the rats were
randomly assigned to five groups (8 or 9 each). Six hours or 1, 2, 4, or 7 days after the exercise, the animals were placed under anesthesia
and killed by decapitation.
Tissue preparation.
Soleus muscle and the red (the deep, red portions of the lateralis,
intermedius, and medialis muscles) and the white (the superficial,
white portion of lateralis muscle) parts of quadriceps femoris muscle
(MQF) were rapidly excised. Samples for light microscopy were
cut from the midbelly regions of soleus muscle and MQF, mounted on a
specimen holder with Optimal Cutting Temperature (OCT) compound (Miles,
Elkhart, IN), and frozen in isopentane cooled in liquid nitrogen. Tissue for total RNA isolation and protein
measurements was frozen in liquid nitrogen. All samples were stored at
70°C until further analysis.
20°C overnight to precipitate RNA. Sedimentation at
10,000 g for 20 min at 4°C was again performed, and the
resulting RNA pellet was dissolved in 0.3 ml denaturing solution and
precipitated with 0.3 ml isopropanol at 20°C overnight. After
centrifugation (10,000 g for 10 min at 4°C), the RNA
pellet was resuspended in 75% ethanol, centrifuged, vacuum-dried, and
dissolved in 50 µl diethyl pyrocarbonate (DEPC)-treated water. The
concentration and purity of each sample were evaluated
spectrophotometrically using absorbances at 260 and 280 nm.
For enzyme activity measurements and concentration of type IV collagen,
the frozen muscle samples from left hindlimbs were homogenized with an
Ultra-Turrax homogenizer in two 7-s bursts at 4°C in a cold solution
containing 0.1 M NaCl, 0.1% (wt/vol) Triton X-100, 0.1 M glycine, and
0.02 M Tris · HCl, pH adjusted to 7.4. The homogenates
(6-10% wt/vol) were centrifuged at 12,000 g for 20 min
at 4°C, and the supernatants were taken for assay of the enzyme
activities. Pellets were used for analysis of type IV collagen. The
muscle pellets were suspended in 0.2 M ammonium bicarbonate and
digested first with collagenase (Worthington Biochemical, Lakewood, NJ)
for 20 h at room temperature, followed by trypsin (Sigma, St.
Louis, MO) for another 20 h at room temperature (21). Enzyme reaction was stopped by trypsin inhibitor (Sigma)
(21). The samples were centrifuged at 15,000 g
for 30 min at 4°C. Supernatants were collected for type IV collagen
concentration measurements.
Northern and slot-blot analysis.
For Northern blotting, 10 µg total RNA was separated in 1%
agarose-formaldehyde gels under denaturating conditions and transferred onto a nylon membrane (Schleicher and Schuell, Dassel, Germany) by 3-h
downward capillary transfer in 10× SSC buffer, pH 7.0. For slot blots,
10 µg total RNA was incubated in a buffer containing formaldehyde for
15 min at 68°C and then applied to a nylon membrane by using a vacuum
filtration manifold (Minifold II; Scheicher and Schuell). All filters
were put in 0.05 M NaOH solution for 5 min and rinsed in 2× SSC
solution to bind the RNA to the membranes. The cDNA probes were labeled
by commercial random primer labeling kit (Amersham Pharmacia Biotech,
Uppsala, Sweden) with 
-[32P]dCTP according to the
manufacturer's manual. The membranes were prehybridized in a
solution containing 50% formamide (vol/vol), 5× SSC, 5× Denhardt's,
10% (wt/vol) dextran sulfate, 50 mM sodium phosphate (pH 6.8), 100 µg/ml salmon sperm DNA, and 1% SDS (wt/vol) for 2-3 h at
42°C, after which the radioactive probe was added and then hybridized
for 24 h at 42°C. After hybridization, the membranes were washed
and exposed to Kodak X-Omat or Agfa Curix film at 70°C. For the
quantification and comparison of relative amounts of mRNA, signal
intensities of the bands were scanned by densitometry (Personal
Densitometry SI, Molecular Dynamics, Sunnyvale, CA). The values of
integrated optical density were used for further analysis. Equal
amounts of RNA in each slot or lane were confirmed using a 24-mer
oligonucleotide (5'-ACG-GTA-TCT-GAT-CGT-CTT-CGA-ACC-3') for 18S rRNA
(4). Blots were hybridized with a 2.8-kb EcoRI insert in pBR322 plasmid of mouse 72-kDa gelatinase (MMP-2)
(37), a 1.8-kb EcoRI-HindIII insert
in pBluescript II plasmid of mouse 92-kDa gelatinase (MMP-9)
(38), a 0.6-kb EcoRV insert in pBluescript II
plasmid of mouse TIMP-1 (9), a 1.7-kb EcoRI
insert in pBluescript II plasmid of mouse TIMP-2 (42), a
1.8-kb EcoRI-HindIII in pBluescript II plasmid of
mouse 1(IV) collagen (29), a 2.2-kb insert in pRGR5
plasmid of rat pro1(III) collagen (11), and a 0.3-kb insert in pBluescript II plasmid of mouse pro1(I) collagen
(31). The recombinant plasmids were kindly provided by Dr.
K. Tryggvason (Department of Medical Biochemistry and Biophysics,
Karolinska Institute, Stockholm, Sweden), Dr. M. Kurkinen (Wayne State
University School of Medicine, Center for Molecular Medicine and
Genetics, Detroit, MI), and Dr. E. Vuorio (Department of Molecular
Biology, University of Turku, Turku, Finland).
-Glucuronidase activity assay.
-Glucuronidase activity was assayed in essence according to Barrett
(1a). Briefly, 450 µl 0.1 M acetate buffer, pH
4.2, was added to 50 µl supernatant from muscle homogenate and
incubated 5 min at 37°C. Then 250 µl substrate (5 mM
p-nitrophenyl--D-glucuronide, Sigma) was
added and incubated 18 h at 37°C. After overnight incubation 1.5 ml 0.1 M glycine buffer (ice cold), pH 10, was added. The tubes were
centrifuged at 3,000 g for 10 min at 4°C.
p-Nitrophenol (end product, Merck M1.06798) was used as a
standard. The absorbances were measured at the wavelength 420 nm. The
results were calculated per soluble protein and incubation time.
Protein concentration was measured using a commercial kit (Bio-Rad).
Gelatin zymography. Zymography was used to quantify gelatinase activities of MMP-2 and MMP-9 and carried out by minor modification of the methods of Kleiner and Stetler-Stevenson (23). SDS polyacrylamide gels (11%) containing 1 mg/ml gelatin were overlaid with 4% stacking gels. Supernatants from centrifuged muscle homogenates were mixed with 1:1 volume of sample buffer consisting of 50 mM Tris, pH 6.8, 2% SDS, 20% glycerol, and 0.03% bromphenol blue without reducing agent or heat. The samples were loaded into the wells of a gel, and the electrophoresis was carried out at 80 V until the dye front had reached the bottom of the gel. Gels were removed from glass plates and incubated for 30 min in the solution containing 2% Tween 80 and 50 mM Tris, pH 7.5, to remove SDS from gels. The incubation was continued for another 30 min with solution containing 2% Tween 80, 50 mM Tris, pH 7.5, 5 mM CaCl2, and 1 µM ZnCl2. The gels were then incubated at 37°C for 18 h in solution containing 50 mM Tris, pH 7.5, 5 mM CaCl2, and 1 µM ZnCl2. Gelatinase activity was revealed by negative staining with Coomassie brilliant blue. Purified proMMP-2 and proMMP-9 (Diabor, Oulu, Finland) was used for identification of enzyme activity. The degree of digestion was quantified by densitometry (Personal Densitometry SI). The values of integrated optical density were used as results.
Reverse gelatin zymography. Reverse zymography is an electrophoresis technique, in which substrate (gelatin) and protease (MMP-2) are incorporated directly into acrylamide gels. After the gels are stained, inhibitory activity of TIMPs result in the presence of dark blue areas, where TIMPs have inhibited the gelatin-degrading activity of MMP-2. Inhibitory activities of TIMP-1 and TIMP-2 were analyzed with reverse zymography as described by Oliver et al. (34). SDS polyacrylamide gels (12%) were prepared with 2 mg/ml gelatin and 180 ng/ml proMMP-2 (Diabor). A standard stacking gel of 4% was used. Supernatants from centrifuged muscle homogenates were mixed with 1:5 volume of sample buffer consisting of 40 mM Tris, pH 6.8, 5% SDS, 20% glycerol, and 0.03% bromphenol blue without reducing agent or heat. The samples were loaded into the wells of a gel, and the electrophoresis was carried out at 80 V until the dye front had reached the bottom of the gel. After electrophoresis, gels were removed from glass plates and incubated on a rotary shaker for 3 h in 100 ml of 2.5% Triton X-100. The Triton X-100 solution was decanted and replaced with 100 ml of solution containing 50 mM Tris, pH 7.5, 5 mM CaCl2, and 1 µM ZnCl2, in which the gels were incubated at 37°C for 20 h. Gels were stained with Coomassie brilliant blue. TIMPs inhibitory activity resulted in the presence of dark blue bands on a clear background. Purified TIMP-1 and TIMP-2 (Diabor) were run as standards. The intensity of the bands were quantified by densitometry (Personal Densitometry SI). The values of integrated optical density were used for further analysis.
Type IV collagen concentration. Type IV collagen content was measured with RIA as a concentration of the 7S domain of type IV collagen (40). Briefly, in the standard inhibition assay, 200 µl dilution of antibody against 7S domain, capable of binding 40% of labeled 7S antigen, was incubated for 2 h at 37°C together with 100 µl nonlabeled 7S antigen (standard) or samples (supernatants from collagenase and trypsin-digested muscle pellet) and 200 µl 125I-labeled 7S antigen (50,000 cpm). Free and bound antigens were separated by precipitation (16 h at 4°C) with goat antiserum to rabbit immunoglobulin G (Fitzgerald Industries International, Concord, MA). All standard and sample dilutions were performed in PBS, pH 7.2, containing 0.04% Tween 20. 7S antibody and labeled 7S antigen were diluted in PBS containing 0.5% normal rabbit serum and 0.1% BSA, respectively. The antibody against the 7S domain of mouse type IV collagen (39, 44) and labeled 7S antigen was generously provided by Dr. J. Risteli (Department of Clinical Chemistry, University of Oulu, Oulu, Finland).
Statistics. Statistical evaluation of the results was performed by use of nonparametric Mann-Whitney U-test for independent samples. Statistical comparisons were made between the sedentary controls and each exercised group. Variability of the data is expressed as means ± SE. Differences were considered statistically significant at P < 0.05.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Estimation of muscle damage.
-Glucuronidase activity (Table 1) rose
significantly in all muscles after running; the increase was more
pronounced in the red part of MQF than in the white part. In soleus,
only slightly elevated activities were measured 2, 4, and 7 days after
the exercise. Light microscopical examination of
hematoxylin-eosin-stained sections (data not shown) showed signs of
muscle damage, including fiber necrosis and inflammation, in the red
part of MQF 1, 2, and 4 days after the exercise and of the repair
process, such as small-sized fibers with central nuclei, after 4 and 7 days. There were no signs of muscle damage in soleus muscle.
Muscle histology after downhill running with the same exercise protocol
was published earlier by Han et al. (17) and Komulainen et
al. (25).
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Northern and slot-blot analysis.
Total RNA was extracted from muscles to determine specific mRNA levels.
Northern blot analysis was used to test the specificity of different
cDNA probes (Fig. 1), whereas slot-blot
analysis (Fig. 2) was used to quantify
the amount of mRNA per 10 µg of total RNA. mRNAs for basement
membrane type IV collagen and both fibrillar types I and III collagen,
as well as metalloproteinase involved in type IV collagen degradation,
increased in response to exercise. The most pronounced increase in mRNA
levels was observed in the red part of MQF. The relative amount of type
IV collagen mRNA levels (Fig. 2A) increased as early as
6 h after the exercise and tended to remain higher than control
level 7 days after running. mRNA levels of types I and III collagen
(Fig. 2, B and C) showed slower but relatively
greater increases than that of type IV collagen. The steady-state mRNA
level of MMP-9 was under the detection limit in all three muscles and
at every time point (data not shown). A large increase in the relative
amount of MMP-2 mRNA (Fig. 2D) was observed both 4 and 7 days after the exercise in the red part of MQF, whereas only slight
increases were seen in soleus and the white part of MQF. Elevated mRNA
levels of TIMP-1 were observed as early as 6 h after exercise. The
levels peaked 1 or 2 days after running and attained control values 7 days after running. The steady-state mRNA level of TIMP-2 (Fig.
2F) increased at the same time as the mRNA level of MMP-2,
and the greatest increase was observed in the red part of MQF.
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Type IV collagen concentration.
Soleus muscle, which is predominantly composed of type I fibers,
contained the highest type IV collagen concentration (353 ± 26 µg/mg wet wt, measured from eight unexercised control muscles), whereas the white part of MQF, which is predominantly composed of type
IIb fibers, contained the lowest type IV collagen concentration (130 ± 8 µg/mg). Type IV concentration of the red part of MQF, which is
composed of types IIa and IIx fiber, was 245 ± 10 µg/mg. Type
IV collagen concentrations behaved differently in all three studied
muscles after downhill running (Fig. 3).
In the red part of MQF, statistically significant decreases were
observed 1 and 2 days after running, whereas in soleus muscle, type IV
collagen concentration was unchanged, and in the white part of MQF it
increased statistically significantly 7 days after the exercise.
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Gelatinolytic activity of MMP-2 and MMP-9.
Pure proMMP-2 and proMMP-9 were used to identify the bands in gelatin
zymography gels (Fig. 4). The active form
of MMP-2 can be seen (in Fig. 4B) in the red part of MQF 4 and 7 days after the exercise (the lowest band). There were no signs of
proMMP-9 gelatinolytic activity in any of the samples. The most marked increase in gelatinolytic activity of proMMP-2 (Fig.
5A) and intensive bands of
active MMP-2 (Fig. 5B) were observed in the red part of MQF
2, 4, and 7 days after the exercise. A similar increase was also seen
in the gelatinolytic activity of proMMP-2 in soleus muscle 2, 4, and 7 days after running, but gelatinolytic activity of active MMP-2 was
under the detection limit (Fig. 5A). Only slight changes
were observed in proMMP-2 in the white part of MQF (Fig.
5A).
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Reverse gelatin zymography for detection of TIMP-1 and TIMP-2.
TIMP-1 and TIMP-2 were detectable in the red part of MQF (Fig.
6B), whereas in soleus muscle
only TIMP-2 was detectable (Fig. 6A). In the white part of
MQF, both TIMP-1 and TIMP-2 were under the detection limit. In the red
part of MQF, the amount of TIMP-1 increased (Fig.
7A) and TIMP-2 decreased (Fig.
7B) 6 h after the exercise but increased statistically
significantly 4 and 7 days after running. In soleus muscle,
the amount of TIMP-2 was unchanged.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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The results of the present study showed that acute, damaging exercise in animals had an impact on intramuscular type IV collagen concentration. Upregulation of MMP-2, the enzyme that degrades type IV collagen, seemed to be correlated with severity of muscle damage and probably reflected the regeneration of basement membranes in myofibers. TIMP-2, the enzyme that inhibits MMP-2 activity, was observed to increase and decrease at the same time as MMP-2. TIMP-1, the other enzyme that inhibits MMP activity, seemed to be activated during the early phase of muscle damage, whereas TIMP-2 was activated in the later phase of muscle damage.
Types I, III, and IV collagen after acute, damaging exercise. At the pretranslational level, type IV collagen expression showed a rapid upregulation in all studied muscles as early as 6 h after the exercise, whereas mRNA levels of fibrillar types I and III collagen did not increase until 1 day after the exercise (Fig. 2). This is in accordance with our earlier findings, in which the mRNA level of type IV collagen was elevated when measured 12 h after exercise, and fibrillar collagens increased 1 day after exercise (17). In the white part of MQF, type IV collagen concentration was elevated 7 days after the exercise, whereas no changes in type IV collagen concentration were detected in undamaged soleus muscle (Fig. 3). These results suggest that the changes in type IV collagen concentrations depend on severity of muscle damage. Somewhat in contrast, in the red part of MQF, which appeared to be the most damaged, type IV collagen concentration in fact decreased 1 and 2 days after the downhill running and attained the control level after 7 days. Interestingly, the decrease was associated with a drop in TIMP-2 level 6 h after the exercise (Fig. 7), indicating a reduction in TIMP-2 inhibition capacity. This may lead to an increase in MMP-2 degradation capacity, despite no detectable change in gelatinolytic activity of proMMP-2. Our present findings together with our earlier studies (17) demonstrate that both mRNA and protein levels of type IV collagen are regulated by exercise and that the pattern of the changes differs between muscle groups with different fiber types and most likely is related to the severity of muscle damage. Furthermore, the results showed a difference in the expression of basement membrane type IV collagen and fibrillar types I and III collagen. The increase in type IV collagen mRNA level occurred faster than the increase in mRNA levels for types I and III collagen, and exercise had an effect on type IV collagen concentration, whereas no changes in total intramuscular collagen concentration or I/III collagen ratio were observed in our previous study (17).
Type IV collagen concentration in studied muscles. The highest type IV collagen concentration in the sedentary control muscles was measured in soleus and the lowest in the white part of MQF. These findings are in accordance with an earlier study (28). There are potentially two explanations of the type IV collagen concentration differences. First, in rat skeletal muscles, type IIB fibers are at least twice as big as type I fibers (7), which probably explains the higher volume of basement membranes in soleus than in the white part of MQF. As no studies exist with regards to the basement membrane thickness of skeletal muscle fibers and its relationship to different fiber types, this explanation remains speculative. Second, capillary-to-fiber ratio is higher in slow-twitch than fast-twitch muscles (43), which further could contribute to the higher type IV collagen concentration in soleus muscle vs. MQF. Correspondingly, total collagen concentration is higher in slow-twitch than in fast-twitch muscles (27). This indicates that collagen composition in the extracellular matrix diverged from different muscle types.
Steady-state mRNA levels for specific collagens in studied muscles.
mRNA levels of different collagen types in sedentary control muscles
were low compared with maximal levels in response to exercise (Fig. 1).
The lowest intensity of mRNA signals after running was observed in the
white part of MQF. The previous findings showed that slow type I fibers
contain a five- to sixfold higher level of total RNA compared with the
fastest type II fibers (16). This is probably related to a
greater number of myonuclei per millimeter of myofiber in type I fibers
compared with type II fibers (45). On the other hand,
Habets et al. (16) showed that equal amounts of -myosin
heavy chain mRNA are present per microgram of total RNA in rat soleus
and extensor digitorum longus (EDL) muscles despite the fact that
soleus muscle contains severalfold higher amount total RNA than does
EDL muscle. In the present study, we cannot state whether lower mRNA
signals in the white part of MQF compared with soleus muscle are due to
a low number of mRNA copies per microgram of total RNA or to a lesser
number of collagen-synthesizing cells in fast-twitch muscle. However,
our study suggests that amounts of specific collagen mRNAs (per total
RNA) relative to sedentary control levels depend on severity of muscle damage.
Expression of MMP-2 and MMP-9. Our gelatin zymography data showed increased proMMP-2 activities in all three muscles (Fig. 5). The presence of the active form of MMP-2 was observed only in the damaged red part of MQF 2, 4, and 7 days after running (Fig. 4). The data thus suggest that the activation of the MMP-2-dependent proteolytic pathway is associated with the severity of exercise-induced muscle fiber injury. The relative increase in gelatinolytic activity of proMMP-2 was highest in the red part of MQF, followed by soleus muscle, and the white part of MQF. The increased proMMP-2 activity in soleus is interesting as no muscle fiber injury could be observed in light microscopical examination of hematoxylin-eosin-stained sections. Correspondingly, higher gelatinolytic activities of proMMP-2 were observed in paralyzed human muscles and in the same muscles subjected to functional electrical stimulation compared with able-bodied control muscles (26).
At the pretranslational level, there was a clear increase in mRNA level of MMP-2 in the red part of MQF, whereas only slightly elevated mRNA levels were observed in soleus and the white part of MQF (Fig. 2). We were unable to detect MMP-9 either at the mRNA or protein level. Interestingly, in skeletal muscle damage induced by cardiotoxin injection, both MMP-2 and MMP-9 were expressed as latent and/or active forms but with differential patterns related to the time course of muscle necrosis and regeneration (22). We cannot exclude the possibility that the absence of MMP-9 in our study is due to the different natures of exercise-induced muscle damage and toxin-induced muscle damage, respectively. It is known that MMPs are present at low levels in normal tissues and that their expression is tightly regulated by growth factors and cytokines in tissue remodeling and that MMP-2 and MMP-9 are secreted into the extracellular matrix as inactive proenzymes (reviewed in Ref. 2). An important step in the regulation of their activity is the conversion from latent to active form by proteolytic removal of the propeptide. MMP-2 and MMP-9 have a nearly identical digestion profile against extracellular matrix compounds (48), but it has been shown that MMP-9 is involved tissue remodeling during the early inflammatory phase and MMP-2 during the prolonged remodeling phase (1, 22). A wide range of cell types, e.g., connective tissue cells and human myogenic cells (15), have been shown to produce and secrete MMP-2, but its expression in vivo in skeletal muscle has not been documented. Kherif et al. (22) showed by in situ hybridization that MMP-9 is localized in inflammatory cells identified as polymorphonuclear leukocytes and macrophages and in mononucleated cells at the periphery of injured myofibers. Our findings suggest upregulation of MMP-2 mRNA and activity levels 2, 4, and 7 days after damaging exercise and that this is correlated with the regeneration of basement membranes of skeletal muscle.Expression of TIMP-1 and TIMP-2. In the present study, TIMP-1 and TIMP-2 expression was studied, to our knowledge, for the first time in rat skeletal muscle. In the damaged red part of MQF, MMP-2 inhibitor activity of TIMP-1 increased 6 h after the exercise, whereas in the white part of MQF and soleus muscle, it was always under the detection limit (Fig. 6). Somewhat in contrast, MMP-2 inhibitor activity of TIMP-2 decreased at the same observation point and rose 4 and 7 days after running, while TIMP-1 level tended to decrease (Fig. 7). MMP-2 inhibitor activity of TIMP-2 was unchanged in soleus muscle and was under the detection limit in the white part of MQF. TIMPs are secreted by the same cell types as MMP-2 and MMP-9 (15), but their expression in vivo in skeletal muscle is unknown at the moment. TIMP-1 and TIMP-2 are reported to perform different functions in vivo (47). This is in accordance with our findings, which suggest that TIMP-1 is activated during the early phase of muscle damage, whereas TIMP-2 is upregulated during the later phase of muscle damage.
mRNA levels partly reflected the proteins levels. mRNA for TIMP-1 peaked earlier than mRNA for TIMP-2. TIMP-1 mRNA levels increased clearly in all studied muscles, but the increase was most pronounced in the red part of MQF. Moreover, the most marked increase in TIMP-2 mRNA levels was observed in the red part of MQF, but only slight changes were observed in the white part of MQF and soleus muscle. Although the role of TIMPs is clearly important in inhibiting matrix degradation by MMPs, according to cell culture studies it seems that TIMP-1 and TIMP-2 have other functions such as growth factor activity (18, 19), effect on cell morphology (35), and inhibition of angiogenesis (20), but the exact role of their upregulation in response to intensive, eccentric exercise cannot be stated from the present study. Recent results suggest that TIMP-2 is required for efficient activation of proMMP-2 in vivo, although TIMP-2 inhibits MMP-2 activity (3, 46). This is interesting because MMP-2 and TIMP-2 seemed to increase and decrease at same time both at mRNA and protein levels in the damaged red part of MQF. In conclusion, the results of the present study suggest that synthesis of basement membrane type IV collagen changed as a result of exercise-induced skeletal muscle damage at both mRNA and protein levels, whereas our earlier study (17) showed that fibrillar types I and III collagen were affected only at the pretranslational level. MMP-2, the protein that degrades type IV collagen, was upregulated most pronouncedly in the red part of MQF, in which skeletal muscle fiber damage was the greatest. In addition, our study showed that expression of TIMP-1 and TIMP-2, the proteins that inhibit extracellular matrix degradation, is upregulated at different stages of the degeneration and regeneration process of muscle damage.Perspectives
The results of the present study together with those of our earlier study (17) suggest that acute, damaging exercise has an effect on type IV collagen concentration, the major basement membrane compound, whereas total collagen concentration, comprising mainly fibrillar types I and III collagen, remains unchanged. The simultaneous activation of protein synthesis and degradation after acute exercise are similar to the findings on other muscle proteins in response to exercise (36). It indicates that net synthesis of muscle collagen is dependent on an intimate coupling between the activity of synthesis and degradation. Furthermore, TIMP-1 and TIMP-2, the proteins that inhibit extracellular matrix degradation, showed differential responses in relation to stages of the degeneration and regeneration processes of muscle damage, respectively. This could indicate an important relationship of tissue responses to repeated loading such as, e.g., physical training, that may be especially crucial with regards to the necessary recovery period needed after damaging exercise before new exercise can be performed. The findings of the present study illustrated a much more dynamic picture of muscle-connective tissue turnover than hitherto believed, a finding similar to what has recently been found for type I collagen in relation to tendon (30). It could be of importance in integrating the myofibrillar proteins' responses with the connective tissue adaptations, the goal of which is to improve muscular resistance towards loading and tissue damage.| |
ACKNOWLEDGEMENTS |
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The authors thank E. Helkala and L. Kaihlavirta for technical assistance in the preparation of samples. We also thank M. Koistinen, A. Ollikainen, and T. Nykänen for excellent technical assistance in biochemical and molecular biological analysis.
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FOOTNOTES |
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The present study was supported by the Finnish Ministry of Education and the Finnish Graduate School in Musculo-Skeletal Problems.
Address for reprint requests and other correspondence: S. Koskinen, Neuromuscular Research Ctr., Dept. of Biology of Physical Activity, Univ. of Jyväskylä, PO Box 35, 40351 Jyväskylä, Finland (E-mail: koskinen{at}maila.jyu.fi).
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.
Received 29 August 2000; accepted in final form 15 December 2000.
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REFERENCES |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
1.
Ågren, MS.
Gelatinase activity during wound healing.
Br J Dermatol
131:
634-640,
1994[Web of Science][Medline].
1a.
Barrett, AJ.
Lysosomal enzymes: - and -glucosidase, - and -galactosidase, - and -mannosidase, - and -N-acetylglucosaminadase, -N-acetylgalactosaminadase, -L-fucosidase and -glucoronidase.
In: Lysosomes. A Laboratory Handbook, edited by Dingle JT.. Amsterdam: North Holland, 1972, p. 46-126.
2.
Birkedal-Hansen, H,
Moore WG,
Bodden MK,
Windsor LJ,
Birkedal-Hansen B,
DeCarlo A,
and
Engler JA.
Matrix metalloproteinases: a review.
Crit Rev Oral Biol Med
4:
197-250,
1993
3.
Caterina, JJ,
Yamada S,
Caterina NCM,
Longenecker G,
Holmback K,
Shi J,
Yermovsky AE,
Engler JA,
and
Birkedal-Hansen H.
Inactivating mutation of the mouse tissue inhibitor of metalloproteinases-2 (TIMP-2) gene alters proMMP-2 activation.
J Biol Chem
275:
26416-26422,
2000
4.
Chan, YL,
Gutell R,
Noller HF,
and
Wool IG.
The nucleotide sequence of a rat 18 S ribosomal ribonucleic acid gene and a proposal for the secondary structure of 18 S ribosomal ribonucleic acid.
J Biol Chem
259:
224-230,
1984
5.
Chomczynski, P,
and
Sacchi N.
Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.
Anal Biochem
162:
156-159,
1987[Web of Science][Medline].
6.
Corcoran, ML,
Hewitt RE,
Kleiner DE, Jr,
and
Stetler-Stevenson WG.
MMP-2: expression, activation and inhibition.
Enzyme Protein
49:
7-19,
1996[Web of Science][Medline].
7.
Delp, MD,
and
Duan C.
Composition and size of type I, IIA, IID/X, and IIB fibers and citrate synthase activity of rat muscle.
J Appl Physiol
80:
261-270,
1996
9.
Edwards, DR,
Waterhouse P,
Holman ML,
and
Denhardt DT.
A growth-responsive gene (16C8) in normal mouse fibroblasts homologous to a human collagenase inhibitor with erythroid-potentiating activity: evidence for inducible and constitutive transcripts.
Nucleic Acids Res
14:
8863-8878,
1986
10.
Foidart, M,
Foidart JM,
and
Engel WK.
Collagen localization in normal and fibrotic human skeletal muscle.
Arch Neurol
38:
152-157,
1981
11.
Glumoff, V,
Mäkelä JK,
and
Vuorio E.
Cloning of cDNA for rat pro alpha 1(III) collagen mRNA. Different expression patterns of type I and type III collagen and fibronectin genes in experimental granulation tissue.
Biochim Biophys Acta
1217:
41-48,
1994[Medline].
12.
Goldberg, GI,
Marmer BL,
Grant GA,
Eisen AZ,
Wilhelm S,
and
He C.
Human 72-kilodalton type IV collagenase forms a complex with a tissue inhibitor of metalloproteases designated TIMP-2.
Proc Natl Acad Sci USA
86:
8207-8211,
1989
13.
Goldberg, GI,
Strongin A,
Collier IE,
Genrich LT,
and
Marmer BL.
Interaction of 92-kDa type IV collagenase with the tissue inhibitor of metalloproteinases prevents dimerization, complex formation with interstitial collagenase, and activation of the proenzyme with stromelysin.
J Biol Chem
267:
4583-4591,
1992
14.
Gomez, DE,
Alonso DF,
Yoshiji H,
and
Thorgeirsson UP.
Tissue inhibitors of metalloproteinases: structure, regulation and biological functions.
Eur J Cell Biol
74:
111-122,
1997[Web of Science][Medline].
15.
Guerin, CW,
and
Holland PC.
Synthesis and secretion of matrix-degrading metalloproteases by human skeletal muscle satellite cells.
Dev Dyn
202:
91-99,
1995[Web of Science][Medline].
16.
Habets, PE,
Franco D,
Ruijter JM,
Sargeant AJ,
Pereira JA,
and
Moorman AF.
RNA content differs in slow and fast muscle fibers: implications for interpretation of changes in muscle gene expression.
J Histochem Cytochem
47:
995-1004,
1999
17.
Han, XY,
Wang W,
Komulainen J,
Koskinen SOA,
Kovanen V,
Vihko V,
Trackman PC,
and
Takala TES
Increased mRNAs for procollagens and key regulating enzymes in rat skeletal muscle following downhill running.
Pflügers Arch
437:
857-864,
1999[Web of Science][Medline].
18.
Hayakawa, T,
Yamashita K,
Ohuchi E,
and
Shinagawa A.
Cell-growth-promoting activity of tissue inhibitor of metalloproteinase-2 (TIMP-2).
J Cell Sci
107:
2373-2379,
1994[Abstract].
19.
Hayakawa, T,
Yamashita K,
Tanzawa K,
Uchijima E,
and
Iwata K.
Cell-growth-promoting activity of tissue inhibitor of metalloproteinase-1 (TIMP-1) for wide range of cells.
FEBS Lett
298:
29-32,
1992[Web of Science][Medline].
20.
Johnson, MD,
Kim HRC,
Chesler L,
Tsao-Wu G,
Bouck N,
and
Polverini PJ.
Inhibition of angiogenesis by tissue inhibitor of metalloproteinase.
J Cell Physiol
160:
194-202,
1994[Web of Science][Medline].
21.
Karttunen, T,
Risteli J,
Autio-Harmainen H,
and
Risteli L.
Effect of age and diabetes on type IV collagen and laminin in human kidney cortex.
Kidney Int
30:
586-591,
1986[Web of Science][Medline].
22.
Kherif, S,
Lafuma C,
Dehaupas M,
Lachkar S,
Fournier JG,
Verdiere-Sahuque M,
Fardeau M,
and
Alameddine HS.
Expression of matrix metalloproteinases 2 and 9 in regenerating skeletal muscle: a study in experimentally injured and mdx muscles.
Dev Biol
205:
158-170,
1999[Web of Science][Medline].
23.
Kleiner, DE,
and
Stetler-Stevenson WG.
Quantitative zymography: detection of picogram quantities of gelatinases.
Anal Biochem
218:
325-329,
1994[Web of Science][Medline].
24.
Kolkenbrock, H,
Orgel D,
Hecker-Kia A,
Noack W,
and
Ulbrich N.
The complex between a tissue inhibitor of metalloproteinase (TIMP-2) and 72 kDa progelatinase is a metalloproteinase inhibitor.
Eur J Biochem
198:
775-781,
1991[Web of Science][Medline].
25.
Komulainen, J,
Kytölä J,
and
Vihko V.
Running-induced muscle injury and myocellular enzyme release in rats.
J Appl Physiol
77:
2299-2304,
1994
26.
Koskinen, SOA,
Kjaer M,
Mohr T,
Sørensen FB,
Suuronen T,
and
Takala TES
Type IV collagen and its degradation in paralyzed human muscle: effect of functional electrical stimulation.
Muscle Nerve
23:
580-589,
2000[Web of Science][Medline].
27.
Kovanen, V,
Suominen H,
and
Heikkinen E.
Connective tissue of "fast" and "slow" skeletal muscle in rats
effects of endurance training.
Acta Physiol Scand
108:
173-180,
1980[Web of Science][Medline].
28.
Kovanen, V,
Suominen H,
Risteli J,
and
Risteli L.
Type IV collagen and laminin in slow and fast skeletal muscle in ratseffects of age and life-time endurance training.
Coll Relat Res
8:
145-153,
1988[Web of Science][Medline].
29.
Kurkinen, M,
Condon MR,
Blumberg B,
Barlow DP,
Quinones S,
Saus J,
and
Pihlajaniemi T.
Extensive homology between the carboxyl-terminal peptides of mouse alpha 1(IV) and alpha 2(IV) collagen.
J Biol Chem
262:
8496-8499,
1987
30.
Langberg, H,
Skovgaard D,
Petersen LJ,
Bulow J,
and
Kjaer M.
Type I collagen synthesis and degradation in peritendinous tissue after exercise determined by microdialysis in humans.
J Physiol (Lond)
521:
299-306,
1999
31.
Metsäranta, M,
Toman D,
De Crombrugghe B,
and
Vuorio E.
Specific hybridization probes for mouse type I, II, III and IX collagen mRNAs.
Biochim Biophys Acta
1089:
241-243,
1991[Medline].
32.
Murphy, G,
Ward R,
Hembry RM,
Reynolds JJ,
Kühn K,
and
Tryggvason K.
Characterization of gelatinase from pig polymorphonuclear leucocytes. A metalloproteinase resembling tumour type IV collagenase.
Biochem J
258:
463-472,
1989[Web of Science][Medline].
33.
Nagase, H,
and
Woessner JF, Jr.
Matrix metalloproteinases.
J Biol Chem
274:
21491-21494,
1999
34.
Oliver, GW,
Leferson JD,
Stetler-Stevenson WG,
and
Kleiner DE.
Quantitative reverse zymography: analysis of picogram amounts of metalloproteinase inhibitors using gelatinase A and B reverse zymograms.
Anal Biochem
244:
161-166,
1997[Web of Science][Medline].
35.
Ray, JM,
and
Stetler-Stevenson WG.
Gelatinase A activity directly modulates melanoma cell adhesion and spreading.
EMBO J
14:
908-917,
1995[Web of Science][Medline].
36.
Rennie, MJ,
and
Tipton KD.
Protein and amino acid metabolism during and after exercise and the effects of nutrition.
Annu Rev Nutr
20:
457-483,
2000[Web of Science][Medline].
37.
Reponen, P,
Sahlberg C,
Huhtala P,
Hurskainen T,
Thesleff I,
and
Tryggvason K.
Molecular cloning of murine 72-kDa type IV collagenase and its expression during mouse development.
J Biol Chem
267:
7856-7862,
1992
38.
Reponen, P,
Sahlberg C,
Munaut C,
Thesleff I,
and
Tryggvason K.
High expression of 92-kD type IV collagenase (gelatinase B) in the osteoclast lineage during mouse development.
J Cell Biol
124:
1091-1102,
1994
39.
Risteli, J,
Bächinger HP,
Engel J,
Furthmayr H,
and
Timpl R.
7S collagen: characterization of an unusual basement membrane structure.
Eur J Biochem
108:
239-250,
1980[Web of Science][Medline].
40.
Risteli, J,
Rohde H,
and
Timpl R.
Sensitive radioimmunoassays for 7S collagen and laminin: application to serum and tissue studies of basement membranes.
Anal Biochem
113:
272-278,
1981.
41.
Salminen, A,
and
Kihlström M.
Lysosomal changes in mouse skeletal muscle during the repair of exercise injuries.
Muscle Nerve
8:
269-279,
1985.
42.
Shimizu, S,
Malik K,
Sejima H,
Kishi J,
Hayakawa T,
and
Koiwai O.
Cloning and sequencing of the cDNA encoding a mouse inhibitor of metalloproteinase-2.
Gene
114:
291-292,
1992[Web of Science][Medline].
43.
Smith, D,
Green H,
Thomson J,
and
Sharratt M.
Capillary and size interrelationships in developing rat diaphragm, EDL, and soleus muscle fiber types.
Am J Physiol Cell Physiol
256:
C50-C58,
1989
44.
Timpl, R,
Risteli J,
and
Bächinger HP.
Identification of a new basement membrane collagen by the aid of a large fragment resistant to bacterial collagenase.
FEBS Lett
101:
265-268,
1979[Web of Science][Medline].
45.
Tseng, BS,
Kasper CE,
and
Edgerton VR.
Cytoplasm-to-myonucleus ratios and succinate dehydrogenase activities in adult rat slow and fast muscle fibers.
Cell Tissue Res
275:
39-49,
1994[Web of Science][Medline].
46.
Wang, Z,
Juttermann R,
and
Soloway PD.
TIMP-2 is required for efficient activation of proMMP-2 in vivo.
J Biol Chem
275:
26411-26415,
2000
47.
Wang, Z,
and
Soloway PD.
TIMP-1 and TIMP-2 perform different functions in vivo.
Ann NY Acad Sci
878:
519-521,
1999[Web of Science][Medline].
48.
Woessner, JF, Jr.
Matrix metalloproteinases and their inhibitors in connective tissue remodeling.
FASEB J
5:
2145-2154,
1991[Abstract].
49.
Yurchenco, PD,
and
Schittny JC.
Molecular architecture of basement membranes.
FASEB J
4:
1577-1590,
1990[Abstract].
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