1 Institute of Medical Technology and Medical School, University of Tampere,
Tampere, Finland
2 Department of Surgery, Tampere University Hospital, Tampere, Finland
3 Department of Morphology, National Institute of Traumatology, Budapest,
Hungary
4 The Accident and Trauma Research Center, the UKK-Institute and the Tampere
Research Center of Sports Medicine, Tampere; Finland
5 Department of Pathology, University Hospital of Turku and Paavo Nurmi Center,
Turku, Finland
* Author for correspondence (e-mail: blteja{at}uta.fi)
Accepted 4 December 2002
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Summary |
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Tenascin-C was expressed abundantly in the normal myotendinous and myofascial junctions, as well as around the cells and the collagen fibers of the Achilles tendon. Tenascin-C expression was not found in the normal skeletal muscle, although it was found in blood vessels within the muscle tissue. Following the removal of the mechanical loading-induced stimulation on the muscle-tendon unit by cast immobilization for 3 weeks, the immonoreactivity of tenascin-C substantially decreased or was completely absent in the regions expressing tenascin-C normally. Restitution of the mechanical loading by removing the cast and allowing free cage activity for 8 weeks resulted in an increase in tenascin-C expression, but it could not restore the expression of tenascin-C to the normal level (in healthy contralateral leg). In response to the application of a more strenuous mechanical loading stimulus after the removal of the cast (after 8 weeks of low- and high-intensity treadmill running), the expression of tenascin-C was markedly increased and reached the level seen in the healthy contralateral limb. Tenascin-C was abundantly expressed in myotendinous and myofascial junctions and in the Achilles tendon, but even the most strenuous mechanical loading (high-intensity treadmill running) could not induce the expression of tenascin-C in the skeletal muscle. This was in spite of the marked immobilization-induced atrophy of the previously immobilized skeletal muscle, which had been subjected to intensive stress during remobilization. mRNA in situ hybridization analysis confirmed the immunohistochemical results for the expression of tenascin-C in the study groups.
In summary, this study shows that mechanical loading regulates the expression of tenascin-C in an apparently dose-dependent fashion at sites of the muscle-tendon unit normally expressing tenascin-C but can not induce de novo synthesis of tenascin-C in the skeletal muscle without accompanying injury to the tissue. Our results suggest that tenascin-C provides elasticity in mesenchymal tissues subjected to heavy tensile loading.
Key words: Tenascin-C, Mechanical strain, Fibronectin, Skeletal muscle, Tendon, Cartilage, Bone, Tensile, Elastic, Extracellular matrix, Adhesion, Integrin
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Introduction |
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TN-C is abundantly expressed in the musculoskeletal tissues during
organogenesis and embryogenesis (Kardon,
1998; Jones and Jones,
2000a
; Jones and Jones,
2000b
) but somewhat less abundantly in the mature forms of these
tissues (Kannus et al.,
1998a
). However, mature musculoskeletal tissues seem to be unique
in their expression pattern of TN-C: TN-C is expressed abundantly in all
musculoskeletal regions transmitting high mechanical forces from one tissue
component to another, for example, in myotendinous and osteotendinous
junctions (Swasdison and Mayne,
1989
; Hurme and Kalimo,
1992
; Salter,
1993
; Chevalier et al.,
1994
; Mackie and Ramsey,
1996
; Riley et al.,
1996
; Webb et al.,
1997
; Kannus et al.,
1998a
; Mackie et al.,
1998
; Järvinen et al.,
1999
; Järvinen et al.,
2000
; Ireland et al.,
2001
; Theilig et al.,
2001
; Altman et al.,
2002
; Martin et al.,
2003
; Hadjiargyrou et al.,
2002
). Not surprisingly, it was shown that TN-C expression is
elevated in fibroblasts cultured in stressed collagen gels
(Chiquet-Ehrismann et al.,
1994
; Chiquet,
1999
; Kessler et al.,
2001
). And at least two different transcription factors and TN-C
promoter elements responding to mechanical loading have been identified; a
conserved GAGACC stretch-responsive enhancer region
(Chiquet, 1999
) and a separate
signal transduction pathway that involves matrix metalloproteinases (MMPs) and
Egr-1 transcription factor (Jones et al.,
2002
).
We recently extended these in vitro findings by showing that mechanical
stress also regulates the expression of TN-C in vivo
(Järvinen et al., 1999).
In addition, other groups showed that mechanical loading placed upon the
tissues controls TN-C expression in the periosteum, heart, blood vessels, skin
wounds, ligament, osteotendinous junction, during synovial joint formation and
in skeletal muscle (Webb et al.,
1997
; Costa et al.,
1999
; Feng et al.,
1999
; Yamamoto et al.,
1999
; Flück et al.,
2000
; Mehr et al.,
2000
; Mikic et al.,
2000
; Theilig et al.,
2001
; Jones et al.,
2002
; Satta et al.,
2002
; Altman et al.,
2002
; Martin et al.,
2003
).
In this study we investigated whether TN-C expression is influenced by different mechanical loading states in a normal muscle-tendon unit. To study this, we first removed the mechanical loading-induced stimulus from the gastronemius-Achilles tendon complex of a rat by immobilizing its hindlimb in a cast for 3 weeks, and then restored the stimulus by subjecting rats to three exercise protocols differing in their intensity (free cage activity, low- and high-intensity treadmill running). The muscle-tendon unit of the rat calf muscles is especially suited for studying the regulation of TN-C expression, as it contains both regions expressing TN-C abundantly (the MTJ between the muscle and the Achilles tendon and the Achilles tendon) and regions devoid of TN-C (e.g. the skeletal muscle, where TN-C is only expressed around the blood vessels). The mechanical strains generated by the contracting gastrocnemius muscle are first concentrated on the MTJ (a specialized structure tailored for transmitting the mechanical forces generated by muscle contractions into more rigid tendon tissue) and then on the Achilles tendon itself (the strongest tendon in the rat body).
A special emphasis was placed on a recent, somewhat surprising, finding by
Flück et al. suggesting that strenuous mechanical loading induces de novo
synthesis of TN-C in the skeletal muscle itself
(Flück et al., 2000). To
specifically explore this hypothesis, the rat
gastrocnemius-muscleAchilles-tendon complex was first subjected to 3
weeks of inactivity (cast immobilization) to induce a severe atrophy of the
tissues and, thus, to ascertain that the subsequent restitution of mechanical
loading would subject the tissues to sufficient stress to confirm (or oppose)
the hypothesized de novo synthesis of TN-C in the skeletal muscle.
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Materials and Methods |
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The rats were divided into six groups (Fig. 1), of which two were control groups (C3 and C11) and the other four were the study groups (IM3, FR11, LR11 and HR11). In the control groups, the animals were allowed to move freely in the cage (18x35x55 cm), five animals per cage, and the gastrocnemius muscles and the Achilles tendons were analyzed 3 weeks (C3) and 11 weeks (C11) after the starting point (Fig. 1). The rats were killed using carbon dioxide inhalation, and the calf muscle complex (including the gastrocnemius and soleus muscles and the Achilles tendon) was removed from both limbs immediately after death.
|
At entry, the left hind limb of each study animal (groups IM3,
FR11, LR11 and HR11) was immobilized with a
padded tape from toes to above the knee. The knee was fixed in 100°
flexion and the ankle in 60° plantarflexion. The fixation was checked
daily. The immobilization method has been described in detail elsewhere
(Józsa et al., 1990).
The right hind limb was kept free, and its gastrocnemiusAchilles-tendon
complex served as an internal control.
After 3 weeks, the rats of the group IM3 (the immobilization group) were sacrificed, and the samples were taken as described above (Fig. 1). In the remaining groups FR11, LR11 and HR11 (the remobilization groups), the tape was removed and the animals were allowed to remobilize the left hind limb for 8 weeks. The group FR11 rats (the free remobilization group) moved freely in the cage and no additional physical training was used.
Rats in the groups LR11 and HR11 were allowed to move freely in their cage for 1 week, after which they started to run on a treadmill twice a day, 5 days a week for 7 weeks. In the group LR11 (the group with a low intensity running program), the speed of the treadmill was 20 cm/second with an inclination of 10°. During the first running week, there was only one 20 minute session per day, after which there were two sessions per day (the morning and afternoon sessions at least 5 hours apart) for 6 weeks. The program was progressive so that the running time increased from 20 minutes per session in the first 2 weeks to 45 minutes per session in the last week.
In the group HR11 (the group with a high intensity running program), the speed of the treadmill was 30 cm/second with an uphill inclination of 30°. This final speed and inclination was achieved by a gradual increase in speed and inclination during the first week of running. As in the group FR11, there was only one daily session during the first running week, and two thereafter. The running was also progressive, from 20 minutes per session in the first two weeks to 45 minutes per session in the 7th and 8th weeks.
After the remobilization period of 8 weeks (Fig. 1), the remobilized animals in the groups FR11, LR11, and HR11 were also sacrificed using carbon dioxide and the calf muscleAchilles-tendon complexes were dissected.
Sample preparation
The dissected gastrocnemius-muscleAchilles tendon complex (including
the calcanear insertion and thus the osteotendinous junction) were cleared of
the adherent fat and connective tissue and transversely divided in half. The
proximal half of the muscles were snap-froxen immediately in freon 22 cooled
with liquid nitrogen and stored at -35°C until processing and analysis,
whereas the distal half (fixed at the resting length by attaching the samples
by pins to pieces of cork) was fixed in neutral buffered 6% formalin (pH 7.4)
and embedded in paraffin. Both ends of the muscles were attached with pins to
the underlying piece of cork to ensure that the normal resting length of the
muscle was maintained throughout the fixation and embedding process.
TN-C expression in the gastrocnemius-muscletendon unit
Immunohistochemistry
10 to 15 serial longitudinal 5 µm thick sections were cut from the
middle area of each formalin-fixed paraffin-embedded block, the cutting
surface being sagittal (not frontal). Half of the serial longitudinal sections
were stained with hematoxylin-eosin or with modified Herovici method, and the
other half were used for the immunohistochemical examination.
For immunohistochemistry, polyclonal rabbit anti-human tenascin-C (dilution
1:1600) (Telios Pharmaceuticals, Inc., San Diego, CA) was used as a primary
antibody (Hurme and Kalimo,
1992; Kannus et al.,
1998a
; Järvinen et al.,
1999
). TN-C antiserum is crossreactive with the corresponding rat
antigen (manufacturer's information), and we have previously verified its
specificity for TN-C (Järvinen et
al., 1999
).
The bound primary antibody was visualized using the appropriate
avidin-biotin-peroxidase method (Vectastain, Vector Laboratories, Burlingame,
CA or Histostain Plus-kit, Zymed Laboratories, San Francisco, CA) with
diaminobenzidine as a chromogen. After the immunohistochemical reaction,
sections were counterstained with hematoxylin. Negative control sections (i.e.
specimens incubated with rabbit serum or without the polyclonal antibody) were
included in every staining patch. Normal rat skeletal muscle with intact
myotendinous junctions, as well as its injured counterpart, expressing TN were
used as positive controls (Hurme and
Kalimo, 1992; Kannus et al.,
1998a
). Finally, all the histological sections were analyzed and
photographed with a light microscope.
mRNA in situ hybridization
In situ hybridization analysis was performed on several samples from each
study group. Paraffin sections (10 µm) were cut onto Superfrost Plus
(Menzel, Germany) slides. Two synthetic oligonucleotide probes directed
against TN-C mRNA [nucleotides 609-642 and 1531-1564, GenBank accession U09361
(LaFleur et al., 1994)] were
labeled with a specific activity of 1x109 cpm/ug at the
3' end with 33P-dATP (DuPont-New England Nuclear Research
Products, Boston, MA) using terminal deoxynucleotidyltransferase (Amersham
Int., Buckinghamshire, UK). After the xylene and graded alcohol series, the
sections were washed in water, air dried and hybridized at 42°C for 18
hours with 5 ng/ml of the probe in the hybridization cocktail, washed four
times (15 minutes each) in 1xSSC at 55°C, and while in the final
rinse, left to cool to room temperature (for an approximately 1 hour)
(Järvinen et al., 1996
;
Kononen and Pelto-Huikko,
1997
). Autoradiograph films (Amersham ß-max; Amersham Int.,
Buckingshire, UK) were overlaid on slides, exposed for three weeks and then
developed using LX24 developer and AL4 fixative (Kodak, Rochester, NY).
Histology was controlled afterwards by staining the hybridized tissue sections
with hematoxylin.
Gross characteristics of the gastrocnemius muscle-tendon unit
To provide a broader context for the possible changes in the expression of
TN-C, a comprehensive series of microscopic analyses characterizing the
changes induced by altered mechanical loading on the
calf-muscleAchilles tendon complex of the rats was performed.
Histochemistry, histology and immunohistochemistry
Capillary density and cross-sectional area of muscle fibers
Unfixed serial cryostat cross-sections (6 µm in thickness) were obtained
from frozen muscles and stained for myofibrillar ATPase activity, after
preincubation at pH 4.2, 4.6 and 10.2
(Józsa et al., 1993;
Kannus et al., 1998b
). This
staining procedure allowed the identification of muscle fibres, as type I,
type IIA (fast-twitch oxidative glycolytic) or type IIB (fast-twitch
glycolytic), the measurement of fiber cross-sectional area and the
identification of the intramuscular capillaries
(Józsa et al., 1993
).
In each muscle, 300-500 consecutive neighboring capillaries and the number of
simultaneously occurring muscle fibers were calculated from the
above-described ATPase-stained sections (pH 4.2 and 4.6). The cross-sectional
area was determined both for type I and type II fibers as previously described
(Kannus et al., 1998b
;
Kannus et al., 1998c
). The
oxidative enzyme activity of the fibres was demonstrated by the NADH reductase
reaction (Kannus et al.,
1998b
; Kannus et al.,
1998a
). The remaining cryostat sections were stained with
periodic-acidSchiff (PAS), with and without diastase pretreatment, to
detect the glycogen content of the muscle fibres.
From the paraffin blocks, 5 µm thick serial sections were cut and
stained with hematoxylin-eosin, picrosirius and
phosphotungstic-acidhematoxylin for the evaluation of the intramuscular
connective tissue and pathological fiber alterations as described elsewhere
(Kannus et al., 1998b).
Picrosirius stained the connective tissue (endo-, peri- and epimysium) dark
red, which contrasts well with the pale yellow muscle fibres. From each
muscle, two to three picrosirius-stained cross-sections were examined using a
Zeiss microscope and were analyzed using a system consisting of a video
camera, automatic image analyzer and image software (Muscle Image Analysis
System, IBM-KFKI, Budabest, Hungary)
(Kannus et al., 1998b
). In
each section, the connective tissue and muscle fiber areas were recorded by
measuring the optical density of 442,400 points in a microscopic field
(
0.86 mm in x 160 magnification). The percentage of connective
tissue or connective tissue to muscle fiber was calculated from the ratio of
total connective tissue area to muscle fiber area and expressed as a
percentage. To calculate the mean connective tissue area for each muscle,
10-30 images/muscle were analyzed (two to three sections/muscle including 2-10
fields/section). Fields containing blood vessels other than capillaries were
excluded from the analysis.
Pathological fiber alterations
The number (and percentage, %) of fibers with a pathological, morphological
and histochemical alteration was determined by analyzing 500 consecutive
neighboring type II fibers from each control and from experimental
gastrocnemius muscle (Kannus et al.,
1998b). The above-described NADH reductase, PAS, ATPase and
phosphotungstic-acidhematoxylin preparates were used for these
analyses.
According to their characteristic histological and histochemical features,
the alterations were classified as follows
(Kannus et al., 1998b): a
moth-eaten fiber (referring to spiral-type deformation and destruction of the
myofibrillar network of the fiber, the term being derived from the microscopic
moth-eaten appearance of the fiber); a central core formation within the fiber
(referring to abnormally increased oxidative enzyme activity and abnormal
aggregation of the myofibrils in the central area of the fiber); a loss of
oxidative enzyme activity in the central part of the fiber (referring to a
reduced number of mitochondria and thus reduced aerobic energy production in
that area of the fiber); an increased oxidative enzyme activity in the
peripheral areas of the fiber (referring to the increased number of
mitochondria and thus increased aerobic production in that area of the fiber);
a shell-like fiber (referring to shell-like degradation and degeneration of
the myofibrillar network of the fiber, the term being derived from the
microscopic shell-like appearance of the fiber); a fiber splitting; any other
(undetermined) alteration; and multiple alterations. The total percentage of
fibers with a pathological alteration was also calculated for each control and
experimental muscle.
Visualization and histometric quantification
To eliminate any bias on the part of the observer during the analyses
described, all data collection and all examinations were performed on a blind
basis with respect to treatment group assignment.
Statistical analysis
For the continuous outcome variables, statistical comparisons were first
done using a two-way ANOVA, with the rat group and hindlimb side being the
grouping variables. When the two-way ANOVA indicated significant
(P<0.05) group and side differences and significant
(P<0.05) group-side interactions, Tukey's post hoc analyses were
used for pairwise comparisons. In the frequency outcome variable (the
percentage of pathological fiber alterations), the groups were compared with
the 2 test. The given significance levels refer to two-tailed
tests. The sample size (8 rats/group with both of the hindlimbs analyzed)
required to detect a 10% difference in muscle morphology between the
experimental and control groups was based on a power analysis by using
alpha=0.05 and beta=0.20 (power 0.80).
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Results |
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|
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The measured variables were selected on the basis of their established role
as markers for physical-inactivity-induced atrophy of skeletal muscle
(Appell, 1990;
Józsa et al., 1990
;
Kannus et al., 1992
;
Kannus et al., 1998b
;
Kannus et al., 1998c
;
Järvinen et al., 2002
);
that is, capillary density as well as the muscle fiber cross-sectional area go
through a substantial decrease in response to physical inactivity, whereas the
simultaneous increase in the amount of intramuscular connective tissue takes
place in atrophic skeletal muscle (Appell,
1990
; Józsa et al.,
1990
; Kannus et al.,
1992
; Kannus et al.,
1998b
; Kannus et al.,
1998c
; Järvinen et al.,
2002
). The measured fiber alterations, in turn, are morphological
changes that have been reported to take place in atrophic skeletal muscle
fibers (Appell, 1990
;
Józsa et al., 1990
;
Kannus et al., 1992
;
Kannus et al., 1998b
;
Kannus et al., 1998c
).
Percentage area of connective tissue
In the gastrocnemius muscle of the contralateral control (non-immobilized)
hindlimbs, the group differences in the amount of connective tissue were small
and insignificant, the percent area being 4.0% in the control group, 3.5% in
the IM3-group, 3.8% in the FR11-group, 4.0% in the
LR11-group, and 4.7% in the HR11-group, respectively
(Fig. 2A). By contrast,
immobilization of the left hindlimb for three weeks created a large and
significant (P<0.01) side-to-side difference in the mean area of
the connective tissue, the area of the connective tissue being 15.3% in the
immobilized left hindlimb (Fig.
2A). Free cage activity (FR11) and especially low- and
high-intensity treadmill running for eight weeks significantly
(P<0.01) restored this value to control levels (14.4%, 13.1% and
9.7%, respectively), the restorative effect being significantly better in the
running groups (P<0.01 for both LR11- and
HR11-groups) than in the free cage activity group (FR11)
(Fig. 2A). Despite the
strenuous nature of the remobilization protocols, the side-to-side difference
was still significant in all three remobilization groups (P<0.001
for all groups) as the amount of the connective tissue was 3.8-, 3.3- and
2.1-fold in the previously immobilized muscles in comparison to the healthy
contralateral muscles (Fig.
2A).
Capillary density
In the gastrocnemius muscle of the healthy, contralateral hindlimbs, the
group differences in the capillary density were small and nonsignificant,
although the animals that had gone through the intensive remobilization
protocols tended to have (not statistically significant) a higher capillary
density than that in the rats with sedentary lifestyle (free cage activity or
immobilization) (Fig. 2B).
Immobilization produced a significant (P<0.001) side-to-side
difference in the gastrocnemius muscle capillary density, the density of the
immobilized left hindlimb being only 64% of the healthy contralateral
gastracnemius muscle (and 62% of the control, C3)
(Fig. 2B). Free cage activity
(FR11) of 8 weeks did not improve the situation at all (left,
immobilized hindlimb density was still only 66% of that of the contralateral
hindlimb (P<0.001 and 64% of the control, C11), but in
the LR11 group, the capillary density of the previously immobilized
left gastrocnemius muscles had improved substantially and had even reached the
control level in the HR11 group (Fig. 9B). In the treadmill-trained
animals, the side-to-side difference of 17% was still significant
(P<0.01) in the LR11-group and remained 6% lower in the
HR11-group (P<0.05)
(Fig. 2B).
Fiber cross-sectional area
In the gastrocnemius muscles of the non-immobilized hindlimbs, the mean
cross-sectional area of type I fibers was, as expected, significantly higher
in the LR11- and HR11-groups (P<0.01 for
both groups) than in the other groups (Fig.
2C). The immobilization period resulted in a significant
side-to-side difference, the mean left (immobilized) hindlimb cross-sectional
area being only 70% of the contralateral (non-immobilized) in the
gastrocnemius muscle (P<0.001)
(Fig. 2C). Free cage activity
did not improve the situation (the left-side area having only 69% of the
cross-sectional area of the right hindlimb) (P<0.001), whereas
after the treadmill training, the hindlimb cross-sectional areas had reached
the age-matched control levels, being 111% (LR11) and 103%
(HR11) of that in the controls (both NS)
(Fig. 2C). However, a
left-to-right difference still existed in the gastrocnemius muscles of the
HR11-group (P<0.01) because of the positive effect of
running on the cross-sectional area (of type I fibers) in the non-immobilized
(right) hindlimbs (Fig.
2C).
Immobilization also produced a significant side-to-side difference in the cross-sectional area of the type II fibers, the cross-sectional area of the immobilized hindlimb being 63% of that in the right hindlimb (P<0.01) (Fig. 2D). Free cage activity could not improve the situation, the left hindlimb value being 58% of that in the right hindlimb (P<0.01). However, the cross-sectional area of type II muscle fibers of the previously immobilized hindlimb muscles reached the control level after low- and high-intensity treadmill running (92% in the LR11- and 99% in the HR11-group as compared to C11), but, similar to type I fibers, a clear side-to-side difference still existed in both of these groups because of the positive effect of running on the non-immobilized (right) hindlimb muscles (P<0.01) (Fig. 2D).
Pathological fiber alterations
The total number of fibers with a pathological alteration was very low in
the gastrocnemius muscle in the control rats (4%)
(Table 1). Immobilization
resulted in a significant increase in the amount of pathological fibers (37%,
P<0.001), and the situation did not improve in the free cage
activity animals (36%). In the LR11- and HR11-groups,
the percentage of the fibers with a pathological alteration was clearly lower
than that in the IM3- and FR11-groups (13% in the
LR11- and 10% in the HR11-group, respectively), but the
above-described control level was not completely attained even in the
HR11-group (Table
1).
TN-C expression in the gastrocnemius muscle-tendon unit
Normal expression of TN-C in the gastrocnemius muscle, MTJ and
Achilles tendon (Groups C3 and C11)
In the gastrocnemius muscle of the control rats, both the myofibers and the
endo- and perimysium stained negative for TN-C
(Fig. 3A,B). The only TN-C
immunoreactivity seen in the gastrocnemius muscle was a few positive stainings
of the myofiber-myofiber (myomuscular) junctions
(Fig. 3C). In addition to this
TN-C immunoreactivity, TN-C was also identified in the intramuscular blood
vessels. However, a strong, irregular band of TN-C immunoreactivity was found
at the interface of the MTJ, and, additionally, the microtendon part of the
MTJ also stained strongly for TN-C (Fig.
3A). A band of TN-C immunoreactivity was also seen in the
myofascial junction (Fig. 3B),
whereas no TN-C was identified in the microtendon roots extending from the MTJ
to the lateral sides of the muscle fibers. TN-C could also be visualized by
mRNA in situ hybridization in the MTJ and myofascial junction (MFJ).
|
In the normal Achilles tendons, TN-C was detected in small amounts in the tenocyte collagen fiber interface (Fig. 3D) and on the surface of the parallel-oriented collagen bundles of the tendon (Fig. 3D). Intense TN-C immunoreactivity was seen in the tendon, fibrocartilage and mineralized fibracartilage zones of the osteotendinous junction (OTJ) of the Achilles tendon, but none was seen in the actual bone tissue. mRNA in situ hybridization analysis confirmed the strong expression of TN-C in the tendon (Fig. 4C).
|
Inactivity-induced changes in the expression of the TN-C (Group
IM3)
After three weeks of immobilization, the muscle cells and their sarcolemmal
membrane stained negative for TN-C in the immobilized and contralateral
(non-immobilized) gastrocnemius muscles
(Fig. 5A). The MTJs of the
immobilized muscles expressed no or only very mild TN-C immunoreactivity, the
only remaining traces of TN-C being in the microtendon part of the MTJ
(Fig. 5A). A strong TN-C
staining remained in the contralateral MTJs. An identical decrease to that
seen in the MTJ was also identified in the expression of TN-C at the MFJ.
|
Only faint expression of TN-C was detected in the tenocyte-collagen fiber interface of the immobilized Achilles tendons, and no expression was seen around the tendineal collagen fiber bundles (Fig. 5B). Also, the OTJs of the immobilized Achilles tendons showed no or only very mild TN-C expression. In the mRNA in situ hybridization analysis, no expression of TN-C was detected in the immobilized muscle (Fig. 4A), whereas a weak mRNA signal for TN-C was detected in the tendon (Fig. 4D). The healthy contralateral limb had mRNA expression comparable to that in control animals (data not shown).
TN-C expression after free cage activity (Group FR11)
The expression pattern of TN-C returned to the same level at the MTJ of the
previously immobilized limbs after eight weeks of free remobilization.
However, the TN-C expression remained at substantially lower levels in the
previously immobilized MTJs and Achilles tendons than in the MTJs and Achilles
tendons of the contralateral limbs (data not shown). The signal for TN-C did
not exceed that of the background in mRNA in situ hybridization analysis of
the previously immobilized limbs.
TN-C expression after low- and high-intensity treadmill running
(Groups LR11 and HR11)
There were no visual differences in the TN-C immunoreactivity between the
groups of animals remobilized with two different intensive remobilization
protocols. A very strong band of TN-C expression was found in both the
previously immobilized and the healthy contralateral MTJs of the gastrocnemius
muscle, whereas no TN-C immunoreactivity could be detected in the muscle
itself (Fig. 6A,B). A strong
irregular band of TN-C expresssion was seen also in the myofascial junction of
the remobilized animals (Fig.
6A,B).
|
Large deposits of TN-C were identified in the tenocyte collagen fiber interface and on the surface of the parallel-oriented collagen bundles of the Achilles tendon. mRNA analysis by in situ hybridization confirmed the expression of TN-C in the MFJ, MTJ and in the tendon, both in the previously immobilized and healthy contralateral limbs (Fig. 4E). No TN-C mRNA expression could be detected in the muscle belly after the strenuous remobilization (Fig. 4B).
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Discussion |
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Blocking of the normal muscle contractive activity (by cast immobilization) resulted in a marked atrophy of the gastrocnemius muscle, which was not completely reversed even by intensive treadmill running for eight weeks. Even at the end of the remobilization period, the muscles of the previously immobilized limbs showed an increased percentage of muscle fibers with clear atrophic features and an increased amount of intramuscular connective tissue. Despite the persisting atrophy of the previously immobilized muscles, and, consequently, the atrophied muscle fibers being subjected to higher mechanical stresses than their contralateral (non-immobilized) muscle fibers, we could not detect any TN-C expression in the skeletal muscle after the remobilization period, although the expression of TN-C was regulated by mechanical loading in an apparently dose-dependent fashion in other parts of the muscle-tendon unit.
Our finding is in agreement with previous studies on skeletal muscle
showing that TN-C is not expressed in the normal skeletal muscle, except
around the blood vessels within the muscle tissue
(Hurme and Kalimo, 1992;
Irintchev et al., 1993
;
Settles et al., 1996
;
Kannus et al., 1998a
;
Ringelmann et al., 1999
;
Järvinen et al., 2000
).
However, in response to such pathological stimuli as trauma to the skeletal
muscle, TN-C has been shown to be expressed at the injury site
(Hurme and Kalimo, 1992
;
Irintchev et al., 1993
;
Settles et al., 1996
;
Ringelmann et al., 1999
). This
is especially evident in muscular dystrophies
(Gullberg et al., 1997
;
Settles et al., 1996
;
Ringelmann et al., 1999
;
Chen et al., 2000
), where the
disease itself results in sarcolemmal injury in response to normal muscle
contractions and strong, focal expression of TN-C at sites of injury and
especially at those sites enriched with inflammatory cells and activated
fibroblasts (Settles et al.,
1996
; Gullberg et al.,
1997
; Ringelmann et al.,
1999
; Chen et al.,
2000
).
However, contrary to all the above-desribed data on the expression of TN-C
in the skeletal muscle, Flück et al. recently reported a rapid induction
(within 4 hours) of TN-C expression in the skeletal muscle in response to
fixing a weight equal to 10% of the total weight of the animal to the
latissimus dorsi muscle of the chicken wing
(Flück et al., 2000).
However, it is worth noting that Flück et al.
(Flück et al., 2000
)
reported an almost 50% increase in the mass of the loaded muscle as soon as 4
hours after the beginning of elongation, concomitant with a marked early
infiltration of inflammatory cells (macrophages and neutrophils) and widening
of the endomysial spaces in the loaded muscle. It is virtually impossible for
a mechanical load to induce an anabolic effect of such magnitude on the
synthesis rate of the skeletal muscle proteins that it could explain the rapid
and massive increase in the mass of the skeletal muscle (+44% at 4 hours, +42%
at 10 hours, +92% at 24 hours after the beginning of loading) as reported by
Flück et al. (Flück et al.,
2000
). The increased muscle mass, the endomysial widening and the
massive inflammatory cell reaction are probably attributable to a
loading-induced over-extension injury to the skeletal muscle, causing
disruption of the intramuscular capillaries and extravasation of inflammatory
cells into endometrial spaces of the skeletal muscle. On the basis of the
above description, we feel that the TN-C expression reported by Flück et
al. is most probably the normal response to tissue injury in the skeletal
muscle.
TN-C is enriched at certain locations in the musculoskeletal tissue that
are exposed to heavy mechanical loading. In this study we show that in three
such sites, the MTJ, the MFJ and the tendon, the prevailing level of
mechanical loading (stress) regulates the expression of TN-C. Considering, on
one hand, the recently proposed function of TN-C as an elastic protein, and,
on the other, the sites where TN-C is expressed, it can be proposed that TN-C
provides some elastic properties for the muscle-tendon unit. The
myofiber-microtendon interface, which is mechanically the most vulnerable site
for injury in the muscle-tendon unit
(Kääriäinen et al.,
2000a;
Kääriäinen et al.,
2000b
), is the site of highest TN-C expression.
In summary, this study shows that the expression of matricellular protein TN-C is regulated in the muscle-tendon unit by mechanical strain. Our study also shows that mechanical loading alone (without accompanying injury to the tissue) cannot induce de novo synthesis of TN-C in the skeletal muscle. Our results further support the current concept that TN-C provides elasticity for mesenchymal tissues subjected to heavy tensile loading. Thus, we propose that mechanical loading regulates the normal expression of TN-C in the musculoskeletal tissues, but disruption of the mechanical integrity of the tissue is required for the induction of the de novo synthesis of this protein.
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