Increased muscle proteolysis after local trauma mainly
reflects macrophage-associated lysosomal proteolysis
Marie-Chantal
Farges1,
Denis
Balcerzak1,
Brian D.
Fisher2,
Didier
Attaix3,
Daniel
Béchet4,
Marc
Ferrara4, and
Vickie E.
Baracos1
Departments of 1 Agricultural, Food and Nutritional Science
and 2 Physical Education and Sports Studies, University of
Alberta, Edmonton, Alberta, T6G 2P5, Canada; and
3 Unité de Nutrition et Métabolisme Proteique and
4 Unité de Nutrition Cellulaire et Moléculaire,
Institut National de la Recherche Agronomique, Theix, 63122 Ceyrat,
France
 |
ABSTRACT |
Rat gastrocnemius showed increased
protein degradation (+75-115%) at 48 h after traumatic
injury. Injured muscle showed increased cathepsin B activity (+327%)
and mRNA encoding cathepsin B (+670%), cathepsin L (+298%), cathepsin
H (+159%), and cathepsin C (+268%). In in situ hybridization,
cathepsin B mRNA localized to the mononuclear cell infiltrate in
injured muscle, and only background levels of hybridization were
observed either over muscle cells in injured tissue or in uninjured
muscle. Immunogold/electron microscopy showed specific staining for
cathepsin B only in lysosome-like structures in cells of the
mononuclear cell infiltrate in injured muscle. Muscle cells were
uniformly negative in the immunocytochemistry. Matrix
metalloproteinase-9 (granulocyte-macrophage gelatinase) mRNA and
activity were not present in uninjured muscle but were expressed after
trauma. There was no activation of the
ATP-ubiquitin-proteasome-dependent proteolytic pathway in injured
muscle, by contrast to diverse forms of muscle wasting where the
activity of this system and the expression of genes encoding ubiquitin
and proteasome elements rise. These results suggest that proteolytic
systems of the muscle cells remain unstimulated after local injury and
that lysosomal enzymes of the inflammatory infiltrated cells are likely
to be the major participant in protein catabolism associated with local trauma.
injury; protein degradation
 |
INTRODUCTION |
IN A MODEL OF BLUNT TRAUMA
TO MUSCLE, we demonstrated a period of degeneration lasting ~3
days, characterized by gross disruption of muscle cells, hemorrhage,
inflammation, invasion of the injured site by mononuclear cells, and a
26% loss of previously existing muscle protein (12). A
large increase in the process of protein catabolism occurs in injured
muscle; however, it is not known which of the several distinct
intracellular proteolytic systems of muscle (1, 3)
might participate in this response. Lysosomal proteinases are thought
to be responsible for degradation of membrane protein and certain
soluble proteins in normal muscle (1). A lack of insulin
and amino acids leads to activation of the lysosomal process
(10). Cathepsin levels are elevated in denervation
atrophy, muscular dystrophy, and inflammatory myopathies (23,
25). Lysosomal enzymes of mononuclear phagocytes that enter the
tissue during inflammation may be present (22). The
elevation of lysosomal enzymes in muscle may not be confined to
conditions of muscle wasting but also to differentiation and
development processes occurring during regeneration (26,
37). Muscle also contains Ca2+-activated
proteinases, which promote overall degradation under conditions that
raise cytosolic Ca2+ levels (4, 8). Cellular
disruption would allow focal entry of extracellular Ca2+,
possibly provoking Ca2+-dependent proteolysis after injury
(4, 8). The bulk of proteolysis in normal muscle and
degradation of the contractile proteins (1) involves an
ATP-dependent system involving the cofactor ubiquitin and the
proteasome complex (17). This may be the primary mechanism
by which breakdown of muscle proteins increases in association with
diverse forms of atrophy (1, 3, 17, 35, 38, 39, 42).
Matrix metalloproteinases (MMP) responsible for degradation of
connective tissue are found in muscle. MMP are regulated at the levels
of transcription and zymogen activation by plasmin or membrane type MMP
and by tissue inhibitors of metalloproteinases (TIMP)
(31). Skeletal muscle shows multiple MMP activities on gelatin zymography and also expresses mRNA encoding MMP-1, -2, -9, -14, and-16 and TIMP-1, -2, and -3, as well as plasminogen activator and its
receptor (2). Physiological regulation of this system in
muscle has not been extensively characterized; however, the activity,
expression, and localization of MMP-9 have recently been reported after
experimental injury induced in normal muscle by cardiotoxin injection
and denervation (20, 21). This proteinase is mainly
produced by inflammatory cells, including polymorphonuclear leukocytes,
macrophages, eosinophils (36), and lymphocytes
(29) and is involved in the migratory process of these
cells in acute inflammation with remodeling and neovascularization (41).
The contribution of proteolytic systems to tissue catabolism after
injury to nonmuscle tissue (i.e., systemic response to injury) has been
studied. For example, muscle wasting after burn injury in rats has been
attributed to the ATP-ubiquitin-proteasome-dependent system
(11). Increased gene expression of elements of this system was also observed in peripheral muscle of head trauma victims (27). The relative roles of proteolytic systems after
direct trauma to muscle are unknown, and we sought to clarify their
nature through study of proteolytic activity, quantitation of mRNA
encoding proteinases and their cofactors, and muscle incubation with
specific proteinase inhibitors. Because initial studies (12,
37) suggested the participation of proteinases potentially
derived from muscle cells and/or mononuclear cells of the inflammatory
infiltrate, we also determined the localization of the most increased
proteolytic activity, cathepsin B, by immunocytochemistry and of its
mRNA by in situ hybridization.
 |
METHODS |
Experimental animals.
Studies were carried out in compliance with the guidelines of the
Canadian Council on Animal Care. Male Sprague-Dawley rats (200-300
g) from a colony maintained at the University of Alberta were used.
Rats were housed in individual wire mesh cages in a temperature
(24°C) and humidity (80%)-controlled room on a 12:12-h light-dark
cycle. Rats were fed ground laboratory chow (Continental Grain,
Chicago, IL) containing 24% crude protein. Rats were killed by
CO2 asphyxiation. Animals were allocated by initial body
weight to the two treatment groups (control and injured) such that the mean body weights and SE of the groups were similar. Injured rats were
administered a single impact trauma to the medial aspect of the right
hindlimb. The procedure produced a moderate contusion of the medial
gastrocnemius and was conducted while the rats were under general
anesthesia (12). Control uninjured rats were also anesthetized. In some experiments, the tissue receiving the direct impact of the device (right medial gastrocnemius) as well as uninjured muscle on the contralateral (left) limb of the same animal were studied. Experiments were carried out with 6-10 rats per
treatment. All of the described experiments were repeated at least
twice. The results of each treatment are presented as mean values ± SE. Statistical comparisons were made by ANOVA followed by Duncan's test.
A time course study (6, 24, 48, and 72 h posttrauma) was done
initially to determine the temporal sequence of induction of proteolysis. Because in vitro protein turnover measurement entails between-day variation, animals were injured at different times and then
killed on the same day so that all incubations could be conducted at
the same time. In all subsequent studies, control uninjured and injured
rats were studied at 48 h after injury, when net protein
mobilization and the process of protein catabolism occurred at the most
rapid rate.
Lysosomal enzyme activities.
The presence of cytosolic inhibitors of cysteine proteinases
(31) precludes direct assay of lysosomal cathepsins in
unfractionated muscle extracts. Preparation and purification of
lysosomal extracts were done as described previously (6,
30). Muscles were homogenized with a polytron in 10 mM potassium
phosphate buffer, pH 7.4, containing 0.25 M sucrose, 50 mM KCl, and 1 mM EDTA. An aliquot of homogenate was brought to 0.25% Triton X-100 in
acetate buffer, pH 5.0, and stored at
20°C until further analysis
of N-acetyl-
-D-glucosaminidase activity and
protein content. The homogenate was centrifuged 10 min at 1,000 g and then for 10 min at 2,500 g. The supernatant was centrifuged at 20,000 g for 20 min, and the pellet was
resuspended in 30 mM sodium phosphate buffer, pH 5.8, and frozen
overnight. After thawing, an aliquot was also made up to 0.2% in
Triton X-100 and stored for determination of
N-acetyl-
-D-glucosaminidase activity. The
supernatant recovered after 20-min centrifugation at 60,000 g was designated the lysosomal extract and was used for
determination of cathepsin B and B + L activity.
N-acetyl-
-D-glucosaminidase activities were
determined in lysosomal fractions to estimate the yield of lysosomes
(7). Protein concentration was determined according to
Bradford (9). Assays for Z-Arg-Arg-aminomethylcoumarin (NMec; cathepsin B) and Z-Phe-Arg-NMec (cathepsins B and L) hydrolysis were carried out according to Barrett (5).
Gelatin zymography.
To detect MMP-2 and MMP-9 activities present in control and injured
muscles, samples were prepared and gelatin zymography conducted as
described by Balcerzak et al. (2). Briefly, after extraction, soluble proteins (15 µg) were separated on a 15%
SDS-PAGE gel containing gelatin (1 mg/ml). After migration, gels were
washed in a Triton X-100 solution (2.5% in distilled water), incubated 20 h at 36°C in enzyme buffer (50 mM Tris · HCl, pH 7.5, 10 mM CaCl2, 0.05% Brij-35), and stained with naphthol
blue-black solution.
Tissue RNA isolation, Northern hybridization analysis, and
RT-PCR.
Total RNA was extracted from frozen samples by the guanidinium
isothiocyanate-CsCl method (34). Purity and quantitation of RNA were determined by measures of absorbance at 260 and 280 nm. A
cDNA insert encoding rat cathepsin B was subcloned into the
EcoRI site of pGEM-blue (Promega, Madison, WI). Antisense riboprobes suited to Northern hybridization and in situ hybridization were generated from this plasmid pGEM rat cathepsin after
HindIII digestion and synthesis with T7 RNA polymerase. The
riboprobe is complementary to 289 bases of rat cathepsin B mRNA.
Northern hybridization with cathepsin B [32P]cRNA was
performed as previously described (6, 42).
Dot blot hybridization was performed as described previously
(38) using probes encoding rat cathepsins C and H, mouse
cathepsins B, L, and D, rat calpain I, chicken ubiquitin, rat 20S
proteasome subunits C2, C3, C5, C8, and C9, and human subunit S5a of
the 19S complex. A probe encoding rat mitochondrial rRNA 12S (F17) was
used as a control. Radioactivity in dot blots was quantitated using a
PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
RT-PCR was performed using the Superscript One-Step RT-PCR system (Life
Technologies, Burlington, ON, Canada). The MMP-9 product (497 bp)
was amplified using 2 µg of total RNA and 10 mM of the following
primers: sense GGCAAGGATGGTCTACTGGC; antisense
GACGCACATCTCTCCTGCCG. Custom primer synthesis was done for the
specified sequences by the DNA Synthesis Laboratory in the Department
of Biochemistry at the University of Alberta. The identity of the
amplified product was determined by sequencing and comparison with the
rat MMP-9 sequence (GenBank accession no. NM031055).
Light microscopy and in situ hybridization analysis.
For light and electron microscopy, control (uninjured) and injured
animals were killed and immediately perfused in a single pass with
phosphate-buffered physiological saline (PBS) and then with 0.5%
glutaraldehyde-4% p-formaldehyde in 0.1 M cacodylate HCl
buffer, pH 7.2. Samples of medial gastrocnemius ~2 mm3
(n = 5/muscle) were dissected from the injured area and
from the same anatomical location on the contralateral (uninjured) limb. Muscle pieces were postfixed for 2 h at 4°C in a solution containing 4% p-formaldehyde and 4% sucrose in 0.1 M
cacodylate HCl buffer, pH 7.2. Finally, tissue pieces were washed in
the same buffer containing 7.5% sucrose. This fixation protocol was developed and employed for immunocytochemistry with the use of affinity-purified anti-cathepsin B (14, 24, 40).
Samples for light microscopy collected after fixation by perfusion were
frozen in isopentane, cooled in liquid nitrogen, and mounted in OTC
compound (Ames, Elkhart, IN) before sectioning (5 µm) in a cryostat
(Reichert-Jung, Nussloch, Germany). To determine the general tissue
architecture, these sections were stained with hematoxylin and eosin
and mounted for light microscopy by standard procedures. In situ
hybridization was carried out as described previously
(15). Briefly, tissue sections were prepared by treatment with proteinase K (20 µg/ml; 8 min), blocking [10 mM dithiothreitol (DTT), 1.85 mg/ml iodoacetamide, 1.25 mg/ml N-ethylmaleimide
for 25 min at 45°C] and acetylation (0.25% acetic anhydride in
triethanolamine HCl, pH 8.0). Sections were incubated in a
prehybridization medium containing 50% formamide, 5× PIPES, pH 6.8, 5× Denhardt's solution, 2% SDS, 0.25 mg/ml salmon sperm DNA, 0.25 mg/ml tRNA, and 0.1 mM DTT at 43°C for 2 h. The hybridization
solution was of the same composition as that used for prehybridization,
except that it contained no DTT and included 0.1 g/ml dextran sulfate
and an antisense [35S]CTP-cathepsin B riboprobe (120,000 dpm/µl). Hybridization was at 43°C for 4-16 h. After
hybridization and rinsing with 4× standard saline citrate (SSC; 0.15 M
NaCl, 0.015 M Na citrate)-0.1% mercaptoethanol, sections were digested
with ribonuclease A (40 µg/ml) and T1 (800 U/ml) for 30 min at
37°C. Finally, sections were counterstained with hematoxylin and
eosin, and liquid emulsion microautoradiography was carried
(15). Photomicrography was performed using light field
optics. To check the specificity of in situ hybridization reactions,
some sections were hybridized with an unrelated antisense riboprobe,
[35S]CTP-glucagon, or with a labeled sense transcript of
the cathepsin B cDNA.
Immunocytochemistry.
For electron microscopy, tissue blocks were washed three times with
PBS, pH 7.4, and then transferred to PBS containing 0.5% OsO4 for 10 min at 25°C. Tissues were dehydrated in a
graded series of ethanol and then transferred to 100% propylene oxide.
Samples were embedded in araldite CY212. Thin sections were cut on a
Reichert Om U2 ultramicrotome and mounted on nickel grids. All of the
following steps were conducted at 25°C. Grids bearing sections were
floated face down on 1% NaIO4 for 1 h and then rinsed
with distilled water three times for 10 min each. To block nonspecific
binding, sections were incubated for 10 min in a solution of PBS that
contained 1% bovine serum albumin (BSA), 1% gelatin, and 0.05%
Tween-20 and transferred to a solution of 0.02 M glycine-PBS for 5 min. Affinity-purified IgG anti-cathepsin B was a generous gift of Dr. E. Kominami (Juntendo University, Tokyo, Japan). Antisera against
cathepsin B were raised in rabbits and purified by affinity chromatography (22). These antibodies have been
characterized by immunoblotting and immunohistochemistry (19,
40). Affinity-purified IgG anti-cathepsin B was diluted to
concentrations of 2.5, 5, or 10 mg/ml in PBS containing 1% BSA, 1%
gelatin, and 0.05% Tween-20. The muscle sections were incubated with
the primary antibody for 1 h at 25°C on a slow shaker and then
washed with PBS containing 1% BSA and 1% gelatin. Protein
A-gold (G-3766; Sigma, Oakville, ON, Canada) was used for
immunocytochemical staining. Protein A was coupled to colloidal
gold particles of the nominal size 10-15 nm. The final solution of
protein A-gold was prepared in 0.5% BSA in 0.01 M PBS, pH 7.4, containing 0.05% Tween-20. The grids were placed on drops of protein
A-gold at a 20-fold dilution of stock solution and incubated at room
temperature for 1 h. The grids were rinsed with PBS and finally
rinsed with distilled water. The sections were subsequently stained
with 2% uranyl acetate for 30 min and with lead citrate for 5 min. The
specimens were examined in a Hitachi H-7000 electron microscope at 75 kV. The specificity of the affinity-purified antibody was determined
by immunoblotting on whole skeletal muscle obtained from
injured and uninjured control animals. For a negative control for
immunocytochemistry, the primary antibody was omitted.
Muscle incubations.
Incubations are routinely done with muscles that are small and thin
enough to permit the diffusion of oxygen and substrates into the
tissue. The use of surgically prepared longitudinal strips of larger
muscles has been validated for studies of muscle protein turnover
(43) as well as glucose transport (13).
Because it would not have been possible to traumatize some of the more
commonly incubated muscles, we dissected four thin longitudinal strips from control and injured medial gastrocnemius. Muscle strips weighed ~20 mg and were ~12 mm in length (i.e., dissected from origin to
insertion of the gastrocnemius muscle) and not thicker than 1.2 mm. The
strips were not mounted on a physical support because of the lack of
long tendons on this muscle. Muscle strips were incubated in vitro as
described extensively in other work from our laboratories (3, 4,
42). Muscles were preincubated in individual flasks containing 3 ml of a modified Krebs-Ringer bicarbonate medium (KRB) composed as
previously described (3). In all studies, tissues were
incubated at 35°C in medium with 95% O2-5%
CO2. KRB buffer contained 1.0 mM CaCl2, 8 nM
bovine insulin, 0.5 mM cycloheximide, and 5 mM glucose. A
control strip from each injured and uninjured muscle was incubated in
this medium, and in a second strip dissected from the same muscles,
lysosomal proteinases were inhibited by adding methylamine HCl
(4). A separate experiment was conducted to study the
possible involvement of proteasome-dependent proteolysis. Media were
formulated as indicated above; however,
Ca2+-dependent proteases were inhibited by deleting
Ca2+ from the medium and adding 10 µg/ml Na dantrolene to
prevent release of intracellular Ca2+ (3);
methylamine HCL was added as indicated to inhibit lysosomal protease
activity. In this approach, the contribution of the
non-proteasome-dependent systems is first eliminated. We used a
proteasome inhibitor MG 132 (carbobenzoxy-L-leucyl-L-leucyl-L-leucinal;
Calbiochem, La Jolla, CA) (10 µM, final concentration) dissolved in
dimethyl sulfoxide (35, 39). Control muscles were
incubated in the same medium containing an equivalent amount of
dimethyl sulfoxide but no inhibitor.
All muscles were preincubated for 1 h and then incubated for
2 h in fresh medium of identical composition. At the end of
incubation, muscles were blotted and frozen in liquid nitrogen and
stored at
20°C until analysis. Muscle protein mass was determined
using the bicinchonic acid procedure (BCA Protein Assay; Pierce
Chemical, Rockford, IL) after tissue solubilization in 1.0 N NaOH at
25°C.
Protein degradation was determined as the amount of tyrosine released
by the tissue into the medium during incubation in the presence of
cycloheximide to prevent amino acid reincorporation into proteins
(3). Preliminary studies established that changes in
intracellular pools of tyrosine during incubation were small and could
be ignored for such measurements. Tyrosine release was linear for up to
120 min of incubation.
 |
RESULTS |
Gastrocnemius muscle preparation and effects of trauma on
proteolysis.
Rates of protein catabolism in control uninjured gastrocnemius strips
fell within a range from 2.09 to 2.55 nmol tyrosine · mg
protein
1 · 2 h
1, and these values
were similar to those reported for incubated epitrochlearis
(3). In preliminary experiments, we determined that the
four strips from each muscle had highly similar rates of catabolism
(coefficient of variation, 3%). Tissue levels of free tyrosine were
0.625 ± 0.031 nmol/mg protein in control muscles and 0.685 ± 0.043 nmol/mg protein in injured muscles, and these values were not
significantly different from each other before or after tissue
incubation. Injury resulted in increased release of tyrosine into the
incubation medium (Fig. 1). Because
tissue levels of tyrosine did not change over the course of incubation and protein synthesis was inhibited, tyrosine appearing in the medium
originated from proteolysis. At 6 h after injury, protein degradation tended to rise (+25%, P = 0.149 vs.
control) and was significantly elevated at 24, 48, and 72 h.
Day 2 posttrauma was the most catabolic day (+ 115% vs.
control, P < 0.0001). In different experiments, the
magnitude of the proteolytic response on day 2 showed some
variation, from +75 to +115%. This time point, which reflected the
peak rate of net protein mobilization (12) and the peak
rate of protein catabolism, was selected for all further studies.

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 1.
Time course of protein breakdown after injury.
Proteolysis was estimated by the release of tyrosine into the
incubation medium in the presence of cycloheximide. Medial
gastrocnemius from control and injured rats (4 thin strips/muscle) were
preincubated for 1 h in Krebs-Ringer bicarbonate buffer containing
cycloheximide (0.5 mM), glucose (5 mM), insulin (8 nM), and
CaCl2 (1.0 mM). Muscles were transferred to fresh media of
the same composition and incubated for a further 3 h. The average
value from each of the 4 strips was calculated for each animal. The 4 strips from each muscle had highly similar rates of catabolism
(coefficient of variation, 3%). Values not sharing the same letters
are significantly different (P < 0.01, n = 4).
|
|
Proteinase activity.
We tested for modifications of lysosomal proteinase activity. Similar
lysosomal yields were obtained from control and injured muscles as
demonstrated by glucosaminidase activity (Table
1). Both cathepsin B and B + L
activities in the medial gastrocnemius increased after trauma. The
large increase in proteolysis observed on day 2 posttrauma
was associated with a maximal activity of both cathepsin B and B + L in muscle homogenates (+327% and +123% vs. control, respectively).
On day 7 posttrauma, cathepsin activities in lysosomal
extracts returned to normal levels compared with control rats; however,
these remained elevated in injured muscle homogenates (+96 and +53%
for cathepsin B and B + L, respectively, vs. control).
Proteinase gene expression.
Injury was associated with increased expression of cathepsin B mRNA
compared with a control gene, glyceraldehyde phosphate dehydrogenase
(Fig. 2). Day 2, the most
catabolic day, corresponded to the maximal expression of cathepsin B. Results of quantitative dot blot for proteinase gene expression are
shown in Table 2. Lysosomal proteinase
mRNAs increased (cathepsins B, H, L) or tended to increase (cathepsins
C and D) in injured muscle. Of these, cathepsin B showed the largest
increase, 6.72-fold, compared with control muscles. No significant
changes were seen in expression of calpain, ubiquitin, or proteasome
subunit mRNAs.

View larger version (40K):
[in this window]
[in a new window]
|
Fig. 2.
Cathepsin B and glyceraldehyde phosphate dehydrogenase
(GAPDH) mRNA in injured (Inj) and uninjured (C, control) gastrocnemius.
RNA was extracted from medial gastrocnemius of control rats and both
injured and uninjured contralateral limbs of injured rats at 1, 2, or 3 days after injury. Each lane represents pooled RNA from 2 rats/treatment. Northern hybridization analysis was used to detect mRNA
encoding cathepsin B and a control gene, GAPDH.
|
|
Light microscopy and in situ hybridization.
In uninjured muscle, only scattered silver grains in a random pattern
were observed (Fig. 3B), and
this was not different from muscle sections that were treated with an
unrelated antisense riboprobe, [35S]CTP-glucagon, or with
a sense transcript of the cathepsin B cDNA. In injured gastrocnemius,
cathepsin B mRNA localized to the area of tissue damage (Fig.
3A). Dense clusters of silver grains were seen over and
around mononuclear cell infiltrates localized in the widened
interstitial spaces and around the damaged myofibers.

View larger version (111K):
[in this window]
[in a new window]
|
Fig. 3.
In situ hybridization analysis of cathepsin B mRNA in
injured and control gastrocnemius. A: injured muscle at a
site of focal damage, showing widened interstitial spaces and
mononuclear cell infiltrate. Dense clusters of silver grains can
be seen in cells comprising the mononuclear cell infiltrate.
B: uninjured muscle. Bar, 50 µm.
|
|
Immunoelectron microscopy.
Because of the energy level of the isotope used in in situ
hybridization, the silver grains are scattered about the source of
radioactivity, and we chose the immunogold technique to more precisely
localize cathepsin B at a higher level of resolution. At 2 days
posttrauma, muscle damage was characterized by the presence of widened
interstitial spaces (endomysial and/or perimysial areas) between the
muscles fibers, mononuclear cell infiltration into the interstitial
spaces, and disorganization of the muscle architecture. Specific
myofiber damage was noted in cross section (Fig.
4). In both control and injured
muscles, muscle cells were scarcely stained with anti-cathepsin
B, and this was not different from sections in which the primary
antibody had been deleted. In the interstitial space, cells of the
mononuclear cell infiltrate were stained, and gold particles were
specifically localized over lysosome-like structures (Fig.
5). Combined with the in situ
hybridization, these results suggest that the observed
increase of cathepsin B activity and mRNA in injured muscle reflects
the invasion into the damaged muscle of phagocytes rich in this
proteinase.

View larger version (172K):
[in this window]
[in a new window]
|
Fig. 4.
Immunocytochemical localization of cathepsin B in injured
gastrocnemius muscle cells. Muscles were treated with affinity-purified
anti-cathepsin B. A and B: injured muscle at site
of focal damage, showing disorganized myofibrillar structures. No
specific staining was observed. Bar, 5 µm.
|
|

View larger version (189K):
[in this window]
[in a new window]
|
Fig. 5.
Immunocytochemical localization of cathepsin B in mononuclear cell
infiltrate of injured gastrocnemius. Muscles were treated with
affinity-purified anti-cathepsin B. A: low-magnification
view of mononuclear cell. B and C: gold particles
are localized in lysosome-like structures of uniform electron density.
Solid bar, 5 µm.
|
|
Gelatinase activities and MMP-9 gene expression.
We obtained the pattern of gelatinase activities in control and
traumatized muscles using gelatin zymography 48 h after injury (Fig. 6). The conditions of SDS
electrophoresis in zymography allow activity of both the uncleaved
proenzymes and their cleaved active forms on the gelatin substrate.
Uninjured muscles showed only MMP-2, with the two latent forms (66 and
60 kDa) and the active form (55 kDa). The pattern and intensity of
MMP-2 activities in injured muscles were identical to those observed in
control muscles. Traumatized muscles differed by the presence a strong band corresponding to pro-MMP-9 (100 kDa; Fig. 6A). The
RT-PCR technique confirmed the absence of MMP-9 expression in the
control muscle and showed a localization of MMP-9 expression locally in the traumatized gastrocnemius (Fig. 6B).

View larger version (55K):
[in this window]
[in a new window]
|
Fig. 6.
Gelatinase activities and ,matrix metalloproteinase
(MMP)-9 gene expression in injured and uninjured gastrocnemius.
A: MMP-2 and MMP-9 activities detected using the gelatin
zymography technique in uninjured and injured muscles. Proteins (15 µg) were separated on a 15% SDS-PAGE gel containing gelatin (1 mg/ml). After migration, the gel was washed in a Triton X-100 solution
(2.5% in distilled water), incubated 20 h at 36°C in enzyme
buffer, and stained with naphthol blue-black solution. B:
determination of MMP-9 expression by RT-PCR. Total RNA (2 µg)
isolated from a positive control tissue rat lung (L), uninjured muscle
(U), and injured muscle (I) was used. The 100-bp DNA ladder is
presented in the lanes denoted M.
|
|
Inhibition of the proteolytic systems.
We compared untreated strips of each gastrocnemius with other strips
from the same muscle incubated with inhibitor. Methylamine (20 mM), an
inhibitor of lysosomal acidification, had no significant effect on
protein degradation in uninjured muscles but suppressed the increase
due to trauma by 67% (Table 3). An
inhibitor of proteasome activity substantially inhibited total
proteolysis in uninjured muscles but had no effect on the activation of
proteolysis induced by injury (Table 4).
 |
DISCUSSION |
Contribution of proteolytic systems to trauma-induced proteolysis.
After the experimental injury used here, a degenerative phase of ~3
days duration is characterized by acute inflammation and muscle protein
loss (12). The present study clearly demonstrates that a
sustained increase in protein breakdown is one determinant of protein
loss previously reported in acute muscle trauma (12). The
specific aim of this study was to determine which proteolytic system(s)
contribute to muscle degeneration after trauma. Using different
approaches, we have shown that it is mainly the lysosomal proteolytic
system that is activated during the degenerative phase. Lysosomal
proteolysis accounted substantially (~67%) for the overall increase
in protein breakdown based on an in vitro approach with the use of an
inhibitor. Concordant results were found with several different
approaches. Increased activity of cathepsin B and B + L was found
in the atrophying muscles. On day 2 after trauma, these
enzyme activities increased both in muscle homogenates and in lysosome
fractions in parallel with the rise in the lysosomal process in
incubated muscles. Moreover, elevated mRNA levels coding for lysosomal
cathepsins were markedly expressed in injured muscle, particularly on
day 2 posttrauma.
Cellular source of lysosomal enzymes.
Various cellular components of the muscle tissue were altered after
injury (12). At 1-2 days after injury, inflammation appeared to be fully established in the muscle, and this period was
characterized by large numbers of mononuclear cells that had not
previously been present. Mononuclear cells were seen both in the
endomysial connective tissue and within some damaged muscle fibers
(12). Mononuclear cells were seen beneath the basement membrane of the muscle cells in focal aggregates. These mononuclear cells had the distinctive morphology of phagocytes and may include tissue macrophages that were previously present, monocytes that were
attracted to the site of injury and crossed the vascular wall to become
macrophages, B lymphocytes, and cytotoxic T lymphocytes. Mononuclear
phagocytes, when stimulated, synthesize and secrete >80 defined
molecules, which serve to mediate the inflammatory, antibacterial, and
antitumor activities of these cells. Hydrolytic enzymes, and
particularly proteinases, figure prominently in the enzymes synthesized
by phagocytes. Thus two potential sources for increased lysosomal
enzyme levels in injured muscle may be considered: 1)
infiltration of mononuclear cells into the muscle tissue and
2) activation of the lysosomal system endogenous to the
muscle cells. The cellular components released from mechanically disrupted muscle cells and vascular walls provide strong stimuli for
the influx of inflammatory cells into the injured site, and these cells
have a large capacity for phagocytosis and lysosomal degradation of
proteins. We localized cathepsin B and cathepsin B mRNA to determine
their potential cellular source(s). Cathepsin B mRNA, studied at the
resolution of light microscopy, localized to cells of the mononuclear
cell infiltrate and not in muscle cells. At the higher resolution of
transmission electron microscopy, immunoreactive cathepsin B was
detected only in lysosome-like structures within mononuclear cells.
The presence of inflammatory cell infiltrate and its possible
contribution to proteolytic events after injury is additionally suggested by the appearance of MMP-9 activity and mRNA in the injured
muscles. MMP-9 activity and mRNA were present only in the traumatized
area dissected from the injured medial gastrocnemius. The infiltration
of inflammatory cells is linked to the presence of the MMP-9 activity
observed, as macrophages and lymphocytes are an important source of
this particular gelatinase (29, 36). MMP-9 may be involved
in the migration of the inflammatory cells into the traumatized area.
By contrast, MMP-2, an MMP constitutively expressed in skeletal muscle
and connective tissue cells (2), showed the same level of
activity before and after injury.
Without the use of specific stains or markers, the origin of all of the
new cells appearing within the injured site cannot be positively
identified. Stauber et al. (37) studied cell infiltrates after forced lengthening of muscle. The authors offered evidence that
some of the mononuclear cells observed in the injured muscle were
phagocytes and some were of myofiber origin (satellite cells), and the
latter were possibly responsible for the reestablishment of the
myofiber after injury. The role of proteinases in the activities of
satellite cells and muscle regeneration following injury is not clearly defined.
Lysosomal proteinases are known to eliminate specific proteins in
normal muscle cells such as membrane proteins and soluble enzymes
(10). The endogenous lysosomal enzymes of the myofiber are
not thought to contribute to the degradation of the myofibrillar proteins actin and myosin under normal physiological and catabolic conditions (1, 3). However, this is likely due to low
overall activity and/or accessibility to the substrate, since lysosomal enzymes such as cathepsin B are clearly capable of attacking myosin heavy chain, actin, and troponin T (28). We observed an
inhibition of the trauma-associated rise in protein degradation of
~70% in the presence of methylamine. This inhibitor neutralizes the
pH of lysosomal vesicles, indicating that ~70% of proteolysis occurs inside lysosome/endosomes and is not due to secreted (pro) cathepsins. That observation suggests a mononuclear cell penetration inside the
muscle fiber and the participation of a phagocytic process in
catabolism rather than liberation of free lysosomal enzymes and is
concordant with our ultrastructural observations.
Methylamine did not inhibit ~30% of the trauma-associated rise in
protein degradation, and this could be due to several alternative types
of proteolysis, which might be difficult to differentiate experimentally. The fraction not inhibited by methylamine could be from
the activation of lysosomal proteinases released outside the
mononuclear cells. It may also be due to muscle cellular disruption, increased Ca2+ entry into cells, and consequent activation
of calpains (4, 8). The
ATP-ubiquitin-proteasome-dependent system degrades myofibrillar proteins and accounts for the bulk of myofibrillar protein
catabolism in normal muscle; this system is activated in a large
variety of muscle-wasting conditions (reviewed in Ref. 1).
By contrast, in our model of local trauma, a proteasome inhibitor,
MG132, had no effect on the rise in protein degradation associated with
injury, and there was no significant increase of mRNA encoding elements
of the ATP-ubiquitin-proteasome system. This is surprising for several
reasons. In skeletal muscles, this pathway was recently described to be
the critical system responsible for the degradation of myofibrillar
proteins in denervation atrophy and fasting, glucocorticoid treatment,
disuse atrophy, metabolic acidosis, cancer, and sepsis. All of these
conditions are associated with a marked rise in mRNA encoding
ubiquitin, enzymes of ubiquitin conjugation, and/or proteasome subunits
(1, 3, 17, 35, 38, 39, 42). This has stimulated interest
in the ATP-ubiquitin-proteasome system as a possible common pathway for
muscle catabolism in diverse forms of atrophy and as a possible site
for therapeutic intervention. Highly localized muscle injury would
appear to be one of the few instances recorded so far where induction
of this system is not a major contributor to catabolic events. However,
the lack of detectable activation of the ubiquitin-proteasome pathway
reported here further supports a limited role for intramuscular
proteolytic systems in the remodeling of muscle after direct injury.
Only a few results are available regarding the participation of
proteolytic systems after trauma. In one of the few studies done to
date in humans (27), mRNAs encoding multiple elements of
the ATP-ubiquitin-proteasome system were increased in peripheral skeletal muscles of head trauma victims. Superficially, these results
would seem to contradict those presented here, which suggest that
proteolysis in traumatized muscle is largely lysosomal and is
associated with mononuclear phagocytes. However, it seems that there is
more likely to be both a systemic and a local proteolytic response to
injury. A severe injury to the head would induce a systemic response by
alterations in hormones such as glucocorticoids, which are known to
activate ATP-ubiquitin-proteasome-dependent catabolism in peripheral
muscle (1). Because the extent of the injury in our model
was limited to a small part of the medial gastrocnemius, it is possible
that a systemic response was small or not present and that, within the
local environs of injury to muscle, proteolytic activity associated
mainly with inflammatory cells prevailed. Although we did not obtain
data on glucocorticoid levels in the injured animals, the absence of
any reduction in food intake after injury (12) suggests
that the overall level of stress was minimal.
A full appreciation of the role of locally and systemically produced
inflammatory mediators in muscle injury and regeneration will permit
therapeutic strategies to limit excess muscle catabolism and enhance
regeneration. The results presented here clarify the role of
proteinases in injury-induced local muscle catabolism. Lysosomal
enzymes accounted in large part for the increased protein catabolism
associated with muscle trauma, and these enzymes and their mRNA
localized to cells of the inflammatory infiltrate, not to muscle cells,
in the injured tissue. The lack of increase in mRNA of elements of the
ATP-ubiquitin-proteasome-dependent proteolytic pathway, considered to
be a key participant in diverse forms of muscle wasting, further
supports a limited role of intramuscular proteolytic pathways in the
remodeling of muscle after local injury.
 |
ACKNOWLEDGEMENTS |
This research was supported by the Natural Sciences and Engineering
Research Council of Canada.
 |
FOOTNOTES |
Address for reprint requests and other correspondence: V. E. Baracos, Dept. of Agricultural, Food and Nutritional Science, Univ. of
Alberta, Edmonton, AB, T6G 2P5, Canada (E-mail:
vickie.baracos{at}ualberta.ca).
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.
10.1152/ajpendo.00345.2001
Received 2 August 2001; accepted in final form 18 September 2001.
 |
REFERENCES |
1.
Attaix, D,
and
Taillandier D.
The critical role of the ubiquitin-proteasome pathway in muscle wasting in comparison to lysosomal and Ca2+-dependent systems.
In: Intracellular Protein Degradation, , edited by Bittar EE,
and Rivett AJ. Greenwich, CT: JAI, 1998, p. 235-266.
2.
Balcerzak, D,
Querengesser L,
Dixon WT,
and
Baracos VE.
Coordinate expression of matrix-degrading proteinases and their activators and inhibitors in bovine skeletal muscle.
J Anim Sci
79:
94-107,
2001[Abstract/Free Full Text].
3.
Baracos, VE,
DeVivo C,
Hoyle DH,
and
Goldberg AL.
Activation of the ATP-ubiquitin-proteasome pathway in skeletal muscle of cachectic rats bearing a hepatoma.
Am J Physiol Endocrinol Metab
268:
E996-E1006,
1995[Abstract/Free Full Text].
4.
Baracos, VE,
Greenberg AE,
and
Goldberg AL.
Influence of calcium and other divalent cations on protein turnover in rat skeletal muscle.
Am J Physiol Endocrinol Metab
250:
E702-E710,
1986[Abstract/Free Full Text].
5.
Barrett, AJ,
Kembhavi AA,
Brown MA,
Kirschke H,
Knight CG,
Tamai M,
and
Hanada K.
L-trans-epoxysuccinyl-leucylamido(4-guanidino)butane (E-64) and its analogues as inhibitors of cysteine proteinases including cathepsins B, H and L.
Biochem J
201:
189-198,
1982[ISI][Medline].
6.
Béchet, DM,
Ferrara MJ,
Mordier SB,
Roux MP,
Deval CD,
and
Obled A.
Expression of lysosomal cathepsin B during calf myoblast-myotube differentiation.
J Biol Chem
266:
14104-14112,
1991[Abstract/Free Full Text].
7.
Béchet, D,
Obled A,
and
Deval C.
Species variations amongst proteinases in liver lysosomes.
Biosci Rep
6:
991-997,
1986[ISI][Medline].
8.
Belcastro, AN,
Shewchuk LD,
and
Raj DA.
Exercise-induced muscle injury: a calpain hypothesis.
Mol Cell Biochem
179:
135-145,
1998[ISI][Medline].
9.
Bradford, M.
Rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.
Anal Biochem
71:
2114-2121,
1976.
10.
Dice, JF.
Molecular determinants of protein half-lives in eukariotic cells.
FASEB J
1:
349-356,
1987[Abstract/Free Full Text].
11.
Fang, CH,
Tiao G,
James H,
Ogle C,
Fischer JE,
and
Hasselgren PO.
Burn injury stimulates multiple proteolytic pathways in skeletal muscle.
J Am Coll Surg
180:
161-170,
1995[ISI][Medline].
12.
Fisher, BD,
Baracos VE,
Shnitka T,
Mendryk S,
and
Reid DC.
Ultrastructural events following acute muscle trauma.
Med Sci Sports Exerc
22:
185-193,
1990[ISI][Medline].
13.
Furnsinn, C,
Brunmair B,
Furtmuller R,
Roden M,
Englisch R,
and
Waldhausl W.
Failure of leptin to affect basal and insulin-stimulated glucose metabolism of rat skeletal muscle in vitro.
Diabetologia
41:
524-529,
1998[ISI][Medline].
14.
Furuhashi, M,
Nakahara A,
Fukutomi H,
Kominami E,
Grube D,
and
Uchiyama Y.
Immunocytochemical localization of cathepsins B, H and L in the rat gastroduodenal mucosa.
Histochemistry
95:
231-239,
1991[ISI][Medline].
15.
Glimm, DR,
Baracos VE,
and
Kennelly JJ.
Nothern and in situ hybridization analyses of the effects of somatotropin on bovine mammary gene expression.
J Dairy Sci
75:
2687-2705,
1992[Abstract/Free Full Text].
16.
Hasselgren, PO.
Pathways of muscle protein breakdown in injury and sepsis.
Curr Opin Clin Nutr Metab Care
2:
155-160,
1999[Medline].
17.
Hershko, A,
and
Ciechanover AA.
Ubiquitin system for protein degradation.
Annu Rev Biochem
67:
425-479,
1998[ISI][Medline].
18.
Hill, AG,
and
Hill GL.
Metabolic response to severe injury.
Br J Surg
85:
884-890,
1998[ISI][Medline].
19.
Im, B,
Kominami E,
Grube D,
and
Uchiyama Y.
Immunocytochemical localization of cathepsins B and H in human pancreatic endocrine cells and insulinoma cells.
Histochemistry
93:
111-118,
1989[ISI][Medline].
20.
Kherif, S,
Dehaupas M,
Lafuma C,
Fardeau M,
and
Alameddine HS.
Matrix metalloproteinases MMP-2 and MMP-9 in denervated muscle and injured nerve.
Neuropathol Appl Neurobiol
24:
309-319,
1998[ISI][Medline].
21.
Kherif, S,
Lafuma C,
Dehaupas M,
Lachkar S,
Fournier JG,
Verdière-Sahuqué M,
Fardeau M,
and
Alameddine HS.
Expression of matrix metalloproteinases 2 and 9 in regenerating skeletal muscle: a study in experimental injured and mdx muscles.
Dev Biol
205:
158-170,
1999[ISI][Medline].
22.
Kominami, E,
Bando Y,
Il K,
Hizawa K,
and
Katunuma N.
Increases in cathepsins B and L and thiol protease inhibitor in muscle of dystrophic hamsters. Their localization in invading phagocytes.
J Biochem (Tokyo)
96:
1841-1848,
1984[Abstract].
23.
Kominami, E,
Il K,
and
Katunuma N.
Activation of the intramyofibral autophagic-lysosomal system in muscular dystrophy.
Am J Pathol
127:
461-466,
1987[Abstract].
24.
Kominami, E,
and
Katunuma N.
Immunological studies on cathepsins B and H from rat liver.
J Biol Chem
257:
14148-14152,
1982.
25.
Kumamoto, T,
Ueyama H,
Sugihara R,
Kominami E,
Goll DE,
and
Tsuda T.
Calpain and cathepsins in the skeletal muscle of inflammatory myopathies.
Eur Neurol
37:
176-181,
1997[ISI][Medline].
26.
Maltz, K,
and
Oron U.
Proteolytic enzyme activities during regeneration of the rat gastrocnemius muscle.
J Neurol Sci
98:
149-154,
1990[ISI][Medline].
27.
Mansoor, O,
Beaufrère B,
Boirie Y,
Rallière D,
Taillandier D,
Aurosseau E,
Schoeffer P,
Arnal M,
and
Attaix D.
Increased mRNA levels for components of the lysosomal, Ca2+-activated, and ATP-ubiquitin-dependent proteolytic pathways in skeletal muscle from head trauma patients.
Proc Natl Acad Sci USA
93:
2714-2718,
1996[Abstract/Free Full Text].
28.
Matsuishi, M,
Matsumoto T,
Okitani A,
and
Kato H.
Mode of action of rabbit skeletal muscle cathepsin B towards myofibrillar proteins and myofibrillar structure.
Int J Biochem
24:
1967-1978,
1992[ISI][Medline].
29.
Montgomery, AMP,
Sabzevary H,
and
Reisfeld RA.
Production and regulation of gelatinase B by human T-cells.
Biochim Biophys Acta
1176:
265-268,
1993[ISI][Medline].
30.
Obled, A,
Ouali A,
and
Valin C.
Cysteine proteinase content of rat muscle lysosome. Evidence for an unusual proteinase activity.
Biochimie
66:
609-616,
1994.
31.
Ouali, A,
Bige A,
Obled A,
Lacourt A,
and
Valin C.
Small and high molecular weight proteinase inhibitors from bovine muscle.
In: Cysteine Proteinases and their Inhibitors, edited by Turk V. Berlin, Germany: Walter de Gruyter, 1986, p. 545-554.
32.
Parsons, SL,
Watson SA,
Brown PD,
Collins HM,
and
Steele RJC
Matrix metalloproteinases.
Br J Surg
84:
160-166,
1997[ISI][Medline].
33.
Rock, KL,
Gramm C,
Rothstein L,
Clark K,
Stein R,
Dick L,
Hwang D,
and
Goldberg AL.
Inhibitors of the proteasome block the degradation of most cell proteins and the generation of peptides presented on MHC class I molecules.
Cell
78:
761-771,
1994[ISI][Medline].
34.
Sambrook, J,
Fritsch EF,
and
Maniatis T.
Molecular Cloning: A Laboratory Manual. New York: Cold Spring Harbor Laboratory, 1989.
35.
Solomon, V,
and
Goldberg AL.
Importance of the ATP-ubiquitin-proteasome pathway in the degradation of soluble and myofibrillar proteins in rabbit muscle extracts.
J Biol Chem
271:
26690-26697,
1996[Abstract/Free Full Text].
36.
Stahle-Backdal, M,
Inooue M,
Giudice GJ,
and
Parks WC.
92-kD Gelatinase is produced by eosinophils at the site of blister formation in bullous pemphigoid and cleaves the extracellular domain of recombinant 180-kD bullous pemphigoid autoantigen.
J Clin Invest
93:
2022-2030,
1994[ISI][Medline].
37.
Stauber, WT,
Fritz VK,
Vogelbach DW,
and
Dahlmann B.
Characterization of muscles injured by forced lengthening. I. Cellular infiltrates.
Med Sci Sports Exerc
20:
345-353,
1988[ISI][Medline].
38.
Taillandier, D,
Aurousseau E,
Meynial-Denis D,
Béchet D,
Ferrara M,
Cottin P,
Ducastaing A,
Bigard X,
Guezennec CY,
Schmid HP,
and
Attaix D.
Coordinate activation of lysosomal, Ca2+-activated and ATP-ubiquitin-dependent proteinases in the unweighted rat soleus muscle.
Biochem J
316:
65-72,
1996[ISI][Medline].
39.
Tawa, NE,
Odessey R,
and
Goldberg AL.
Inhibitors of the proteasome reduce the accelerated proteolysis in atrophying rat skeletal muscles.
J Clin Invest
100:
197-203,
1997[Abstract/Free Full Text].
40.
Uchiyama, Y,
Nakajima M,
Watanabe T,
Waguri S,
Sato N,
Yamamoto M,
Hashizume Y,
and
Kominami E.
Immunocytochemical localization of cathepsin B in rat anterior pituitary endocrine cells, with special reference to its co-localization with renin and prorenin in gonadotrophs.
J Histochem Cytochem
39:
1199-1205,
1991[Abstract].
41.
Uhm, JH,
Dooley NP,
Stuve O,
Francis GS,
Duquette P,
Antel JP,
and
Tong VW.
Migratory behavior of lymphocytes isolated from multiple sclerosis patients: effects of interferon beta-1b therapy.
Ann Neurol
46:
319-324,
1999[ISI][Medline].
42.
Voisin, L,
Breuillé D,
Combaret L,
Pouyet D,
Taillandier D,
Aurousseau E,
Obled C,
and
Attaix D.
Muscle wasting in a rat model of long-lasting sepsis results from the activation of lysosomal, Ca2+-activated, and ubiquitin-proteasome proteolytic pathways.
J Clin Invest
97:
1610-1617,
1996[Abstract/Free Full Text].
43.
Zamir, O,
Hasselgren PO,
Frederick JA,
and
Fischer JE.
Is the metabolic response to sepsis in skeletal muscle different in infants and adults? An experimental study.
J Pediatr Surg
27:
1399-1403,
1992[ISI][Medline].
Am J Physiol Endocrinol Metab 282(2):E326-E335
0193-1849/02 $5.00
Copyright © 2002 the American Physiological Society