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1 Division of Kinesiology and Health and 2 Department of Animal Science, University of Wyoming, Laramie, Wyoming 82071; and 3 Department of Animal Sciences, Ohio State University, Wooster, Ohio 44691
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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We examined the temporal relationship between
messages (type I and type III mRNAs) for the principal fibrillar
procollagens and subsequent collagen accretion, cross-linking, and
decorin expression in the left ventricle (LV) postmyocardial infarction (post-MI). We sought to determine 1) what role the
proteoglycan decorin plays in extracellular matrix (ECM) remodeling
known to take place as a consequence of MI and 2) the extent
skeletal muscle ECM is altered early post-MI. Therefore, after
surgically induced production of small- to moderate-sized infarcts
(~20% of LV mass), extent and time course of ECM remodeling was
evaluated in remaining viable LV free wall and in slow- [soleus
(SOL)] and fast-twitch [gastrocnemius (GAST)] skeletal muscles.
Decorin, collagen, and hydroxylysylpyridinium cross-link concentrations
and 
1(I) (type I) and 1(III) (type III) procollagen mRNAs were
measured in LVs from noninfarcted controls and at 72 h, 1, 2, 5, and 13 wk post-MI. These same data were collected in SOL and GAST
muscles at all time points except 13 wk. Type I procollagen mRNA
increased at both 72-h and 1-wk time points in LVs. Type III
procollagen mRNA was elevated at 1 wk, returning to baseline by 2 wk
post-MI. Collagen concentration was significantly increased by 1 wk,
more than doubled by 5 wk, and was elevated 129% by 13 wk in the
remaining viable LV. LV decorin expression was unaltered at early time
points, but increased 38% at 5 wk post-MI and doubled by 13 wk
post-MI. In skeletal muscle, procollagen mRNAs were transiently altered in SOL and GAST muscles without any demonstrable effect on the measured
ECM parameters. This study reports, for the first time, the
upregulation time course of decorin and its relationship to increased
HP cross-linking and accumulation of collagen in viable myocardium
post-MI.
cardiac remodeling; collagen cross-linking; procollagen mRNA
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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RECOVERY FROM MYOCARDIAL INFARCTION (MI) typically involves significant fibrosis of the remaining viable left ventricle (LV) (5, 19, 26) and a subsequent increase in mature collagen cross-linking [as measured by hydroxylysylpyridinoline (HP)] (19). The fibrillar collagens, specifically types I and III, are the predominant proteins within the extracellular matrix (ECM) of cardiac and skeletal muscle and are thought to be responsible for this fibrotic response. Several groups have investigated the time course of collagen concentration changes post-MI (5, 26). However, there are currently few data regarding the extent to which other components of the ECM (possibly important in collagen fiber organization or stability) are altered in the noninfarcted myocardium post-MI.
The small dermatan sulfate, proteoglycan decorin, has been implicated
in collagen metabolism and structure. Decorin affects the formation of
collagen fibrils in vitro (35) through binding of its core
protein to types I and III collagen (4, 28). Decorin may
also be important in the regulation of fibrosis through binding and
inhibition of transforming growth factor-
(TGF-), a potent
upregulator of collagen production (1, 6). Decorin is
elevated after 8 wk of recovery from MI (11), but the
relationship between collagen and decorin accumulation in the
noninfarcted myocardium during the early healing phase post-MI is unclear.
MI also results in profound alterations in skeletal muscle function altering blood vessels (25, 29) and metabolism (3). Structural changes in skeletal muscle post-MI could result in an altered amount of collagen and a change in the overall properties and functionality of the ECM. In fact, collagen volume fraction of skeletal muscle was found to be increased 1 yr post-MI (25). An increase in the collagen concentration of skeletal muscle could partially explain some of the decreased exercise capacity in MI patients. However, there are no studies that have evaluated skeletal muscle ECM in the early healing phase post-MI.
The purpose of the present study was to examine the extent and time course of collagen, HP cross-linking, and decorin changes in the ECM of noninfarcted myocardium and locomotor skeletal muscle after coronary artery ligation. It was of interest to determine whether the temporal relationship between collagen deposition and decorin expression post-MI would support the tenet that decorin plays an integral role in altered collagen metabolism and/or cross-linking.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Animal selection and surgery. Thirty Charles River rats (6-8 mo old) were obtained from the University of Wyoming breeding population and housed three to five per cage. Rats were maintained on a 12:12-h light-dark cycle and fed ad libitum. Before and after surgery all animals were maintained in an Animal Welfare Assurance-approved facility according to the American Physiological Society guidelines.
Surgeries were successfully performed on 25 rats after the administration of a combination of ketamine hydrochloride (50 mg/kg ip) and xylazine (3 mg/kg ip). Rats were intubated and placed on a rodent respirator (Harvard 683). The heart was exposed through a left thoracotomy between the fifth and sixth ribs. The pericardium was dissected away, and the left anterior descending coronary artery was ligated with a 7-0 Ethilon suture at its highest point caudal to the left atrial appendage. Successful ligation was verified by a blanching of the area distal to the point of occlusion. The lungs were expanded immediately before thoracic closure to reestablish negative pressure. All rats received penicillin and lidocaine after surgery. Rats were allowed to recover and then killed at 72 h, 1, 2, 5, or 13 wk post-MI (5 at each time point). Noninfarcted rats of the same age were also killed at time 0 to serve as a control group. Heart, soleus (SOL), and gastrocnemius (GAST) muscles were excised and weighed. The right ventricle was dissected away from the rest of the heart. The LV was separated into septal and free wall portions, and the infarct scar was dissected away from the remaining viable LV free wall. All tissues were weighed and rapidly frozen in liquid nitrogen and stored at
70°C until analyses could be performed.
Determination of cardiac hypertrophy. Because there were significant differences in age and body mass among the groups, heart weight corrected for body weight was used to estimate extent of cardiac hypertrophy post-MI. It is also recognized that the calculation of cardiac hypertrophy is compounded by the loss of tissue mass occurring with infarct production. Scar weight has been shown to be 87% of the original infarcted mass by 72 h post-MI in the rat and 50% by 15 days post-MI (9). Requirements for tissue mRNA and protein analysis precluded measurement of infarct size expressed as a percentage of the total endocardial and epicardial LV circumference as we have calculated previously (29). Therefore, in the current study, recently infarcted area (72 h) or consolidated scar tissue mass (1, 2, 5 and 13 wk) was adjusted using the Fishbein et al. (9) correction factors indicated above to provide an accurate estimate of original infarct size.
Hydroxyproline and HP cross-link assays. Hydroxyproline concentration was measured colorimetrically using the method of Woessner (36). Collagen concentration was calculated assuming collagen weighs 7.25 times hydroxyproline and has a molecular weight of 300,000. The degree of collagen cross-linking, as measured by HP concentration, was assessed using the reverse-phase, high-performance liquid chromatography methods of Eyre et al. (7) with modifications previously described by this laboratory (19).
Western blot analysis of decorin antibody. Specificity of the decorin antibody was confirmed by Western blot analysis using the procedure previously reported by Velleman et al. (32). Approximately 1 g of rat LV was homogenized in 2 ml buffer of 10 mM Tris and 1 mM EDTA (pH 8.0). The extract was centrifuged, supernatant was collected, and aliquots containing 0.4 mg protein were lyophilized and resuspended in 250 mM Tris, 350 mM NaCl, and 0.05% BSA (pH 8.0) buffer. Samples were digested with chondroitinase ABC (Sigma) to remove the glycosylaminoglycan side chain to allow entry of the decorin core protein into the electrophoretic gel. Undigested samples were also run as internal controls. Samples were electrophoresed and separated proteins were transferred to a nitrocellulose membrane. The dried membrane was blocked, washed, and incubated with a rabbit antidecorin polyclonal antibody specific to the carboxy-terminus of the decorin core protein (Chemicon International, Temecula, CA) at a 1:200 dilution in 1% gelatin. The blot was then incubated in a 1:5,000 dilution in ×1 Tris-buffered saline of alkaline phosphatase-conjugated goat anti-rabbit IgG (Chemicon International). Color was developed by adding the substrate 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT), and the blot was then scanned using a Quantity One scanner (PDI, Huntington Station, NY).
Decorin immunoblot method. Decorin blots were determined by the method of Velleman (31). Muscle portions were homogenized in TE buffer (10 mM Tris and 1 mM EDTA; pH 8.0). Total protein concentrations were assessed using a protein assay kit (Protein Assay Kit II, BioRad, Richmond, CA). Protein (10 mg) from each sample was spotted onto nitrocellulose (NitroBind, Micron Separations; Westboro, MA) using a dot-blot apparatus (Schleicher and Schuell; Keene, NH). Blots were exposed to the rabbit antidecorin polyclonal antibody and to the goat anti-rabbit IgG with an alkaline phosphatase conjugate. Blots were then exposed to the BCIP/NBT color system, and decorin concentrations within each blot were quantified to a relative scale by color intensity after scanning with a digital color scanner (HP ScanJet IIcx/T scanner, Hewlett-Packard; Corvallis, OR) (31).
Procollagen mRNA determination.
Viable LV free wall and SOL and GAST muscles were minced on ice using
sterile procedures. Approximately 100 mg of each tissue were used for
procollagen mRNA analysis, and the rest was set aside for
hydroxyproline, HP cross-link, and decorin analyses. Total RNA was
extracted using Tri-Reagent (Molecular Research Center, Cincinnati, OH)
and the methodology described by Sambrook et al. (24).
Frozen muscle tissue was homogenized on ice in Tri-Reagent (10 volumes). Phases were separated by the addition of chloroform (0.2 ml/ml Tri-Reagent). The upper phase was transferred to a sterile tube
and mixed with isopropanol (0.5 ml/ml Tri-Reagent) to precipitate RNA.
The solution was centrifuged (12,000 g) and the supernatant
discarded. The pellet was washed in 75% ethanol to remove salt,
repelleted, lightly dried, and solubilized in diethyl
pyrocarbonate-treated water. RNA concentration was measured spectrophotometrically (
= 260 nm).
Northern blot and dot-blot preparation. Messenger RNA levels were determined by Northern blot and dot-blot hybridization analyses using methods adapted from Lehrach et al. (16) and Goldberg (10). Total RNA (10 µg) was denatured in 50% formamide, 17.5% formaldehyde, and ×1 MOPS buffer (20 mM MOPS; 5 mM sodium acetate, pH 6.5; 1 mM EDTA, pH 8.0; total solution pH 7.0), electrophoresed through a 1.2% agarose gel, and transferred to a nylon membrane (0.2 µm, Biotrans, Pall BioSupport; East Hills, NY).
For dot-blot preparation, 5 µl of total RNA were incubated (15 min at 65°C) in 50% formamide, 7% formaldehyde, and ×1 standard saline citrate (SSC). The mixture was spotted onto the nylon membrane (0.2 µm, Biotrans, Pall BioSupport) using a Minifold I dot-blot apparatus (Schleicher and Schuell) after the procedure described by Sambrook et al. (24). The membranes, both Northern blot and dot-blot, were baked for 2 h at 80°C. Prehybridization of membranes took place for 4 h at 42°C in prehybridizing buffer of ×5 SSC, 0.1% sodium dodecyl sulfate, ×5 Denhardt's solution, 50% deionized formamide, 0.1 M phosphate buffer (pH 7.0), and 100 mg/ml denatured salmon testes DNA (Sigma D7656). Hybridizations were performed at 42°C using the same buffer without salmon testes DNA and containing the appropriate radioactive probes to obtain 3 × 106 counts · min
1 · ml1
hybridization medium.
cDNA was radioactively labeled by random primer extension
(8). [
-32P]dCTP (specific activity 3,000 Ci/mM; New England Nuclear, Wilmington, DE) was included in the
reaction mixture to obtain a specific activity between 2 and 6 × 108
counts · min1 · mg1 DNA.
DNA fragments were radiolabeled by Klenow DNA polymerase-catalyzed extension of random primers. Rat 1(I) (type I) procollagen (1074 bp)
and 1(III) (type III) procollagen (449 bp) inserts, isolated from
recombinant plasmid PGEM7Zf(), were used as probes.
Procollagen probes were a generous gift from J. A. Last, Dept. of
Internal Medicine, University of California, Davis, CA. The specificity of these probes has been previously documented (2). A
full-length cDNA for human glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) was used as a control. Hybridized membranes were washed and
exposed to film at 70°C. Dot-blots were quantified for relative
mRNA content by scanning densitometry.
Statistical analysis.
Data from LV, SOL, and GAST muscles were each analyzed over time using
a one-way ANOVA design. Overall significant main effects with respect
to time were further examined using Fisher's test (probability least
squares difference). The -level was set at P 
0.05.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Heart weight/body weight, scar weight, original infarct size, SOL
weight, and GAST weight.
When corrected for changes in body weight at any of the 5 time points
over the 13-wk period, a significant increase in heart weight/body
weight was observed in all infarcted groups compared with controls (see
Table 1). Estimated original infarct mass before scar consolidation, expressed as a percentage of heart weight,
was no different among any of the infarcted groups and averaged 14 ± 1% of heart mass. However, mass of actual infarcted area was
significantly heavier at 72 h post-MI (before scar consolidation) compared with any of the later time points. There were no differences in SOL weight or GAST weight among any of the groups in the study (Table 1).
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Procollagen mRNA.
Representative Northern blots of heart and skeletal muscle procollagen
mRNAs demonstrate the specificity of collagen type I and III cDNA
probes employed (see Fig. 1.). Both
1(I) and 1(III) procollagen mRNAs occurred at ~4.5 Kb, with no
significant cross-reactivity of the cDNA probes. GAPDH was also
established as a positive control. 1(I) procollagen mRNA levels were
elevated in rat LV after 72 h post-MI (Fig.
2), remained elevated at 1 wk, and
returned to control levels at 2 wk. 1(III) procollagen mRNA was
increased in LV by 1 wk and returned to control level by 2 wk. Coronary artery ligation also caused a significant decrease in SOL muscle 1(I) procollagen mRNA, which was maintained out to 5 wk post-MI. 1(III) procollagen mRNA levels were similarly depressed in the same
muscle. There was no effect on collagen gene expression in GAST muscle.
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Collagen concentration and cross-linking.
Noninfarcted rat LV showed significant fibrosis within 1 wk of coronary
artery ligation (Fig. 3). LV collagen
concentrations increased 57% by 1 wk, 81% by 2 wk, 106% by 5 wk, and
107% by 13 wk post-MI. There was no such fibrosis in either skeletal
muscle as the result of MI (Fig. 4).
Collagen concentration remained unchanged in both SOL and GAST muscles
through 5 wk post-MI. Slow-twitch SOL muscle contained significantly
more collagen (3.08 ± 0.20% dry weight) than fast-twitch GAST
(1.90 ± 0.08% dry weight).
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Decorin levels.
Western blot analysis confirmed the lack of decorin antibody
cross-reactivity with other muscle proteins (Fig.
5). As shown in the blot, trace
amounts of core protein in the undigested sample indicated
endogenous cleavage of the glycosylaminoglycan chain. Remodeling of
viable LV post-MI resulted in a significant 38% increase in decorin
expression at 5 wk, with a further increment by 13 wk post-MI so that
decorin levels at this time point were elevated 98% over control
values (Fig. 3). Decorin levels were unchanged in either SOL or
GAST skeletal muscle (Fig. 4).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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In the current study, ligation of the left coronary artery and the resulting MI caused significant alterations in collagen gene expression and the measured ECM components of the remaining viable LV. LV procollagen mRNAs for type I and III collagens significantly increased within 72 h and 1 wk of coronary artery ligation, respectively. Subsequently, an increase in collagen concentration was seen 1 wk after infarction. Not until 5 wk post-MI were LV decorin and collagen cross-linking both significantly increased. These data suggest that collagen cross-linking and decorin expression may be linked. In SOL muscle, decreased type I and III procollagen mRNA within 72 h, and 1 wk of coronary artery ligation did not affect collagen concentration.
The most profound changes in the ECM of the remaining myocardium after surgically induced MI are seen to occur in the LV free wall compared with LV septum (19). Therefore, in the present work, the remaining viable LV was separated from the LV septum in an attempt to observe ECM changes solely in that region of the LV most proximal to the infarct.
Decorin significantly alters the morphology of collagen fibrils formed in vitro. Collagen fibrils form more slowly (34) and are of smaller diameter (35) in the presence of decorin than those allowed to form spontaneously. Decorin may be a factor in maintaining spatial order of collagen fibrils. Decorin binds near the d and e bands of type I collagen (21) and also to type III collagen (28) through interaction of the decorin core protein with the collagen molecule. Maintenance of spatial order may be an important factor determining the formation of collagen cross-links (20). In the present work, the failure of decorin levels to increase before 5 wk post-MI was not unexpected. During tissue development, a time when collagen turnover is high, decorin is not expressed at significant levels (28). During recovery from MI, fibrosis of the remaining LV occurs rapidly; collagen concentration more than doubles within 5 wk. Rapid collagen deposition in the remaining viable LV post-MI may be similar to the rapid remodeling of the ECM of developing tissues. Decorin expression seems to occur subsequent to periods of rapid collagen deposition and/or accumulation. Recent research has demonstrated that decorin is increased 8 wk post-MI (11).
The lag period between fibrosis of the LV and a change in decorin
expression may also be due to extracellular effects. Various growth
factors known to affect collagen metabolism have been implicated in the
regulation of decorin. Specifically, TGF- has been shown to
downregulate decorin (23) and is increased in myocardium bordering the infarct within 48 h of coronary artery ligation (30). Increased TGF- levels in the viable myocardium
early post-MI may partially explain the absence of a decorin response to coronary artery ligation before 5 wk of recovery. Treatment of
several cell lines with TGF- increased the production and secretion
of collagen from those cells (13, 14, 22). The later
appearance of decorin may also begin a process slowing fibrosis. The
decorin core protein binds TGF-, sequestering it to the ECM and
inactivating it (12). Thus the possibility exists that
growth factors acting on different components of the cardiac ECM may account for the differential changes in ECM components in the LV
post-MI.
While the findings from most studies would support the contention that collagen concentration increases within the remaining viable LV as a consequence of MI (5, 19, 26), Marijianowski et al. (17) suggested that collagen concentration does not increase in viable human myocardium postinfarction. However, the latter study chose to express myocardial collagen content as a function of the wet tissue weight. The fluid compartment of most tissues is highly variable, and the extraction of water is crucial to the accuracy of whole tissue extrapolation of collagen concentration. Expressing collagen concentrations as dry weight allows comparisons within and among studies. Although collagen concentration within the remaining viable myocardium is seen to increase post-MI, the time course of myocardial fibrosis is not well understood. Cleutjens et al. (5) reported progressive fibrosis of the infarcted LV free wall in rats so that collagen concentration was elevated both 1 wk (threefold) and 2 wk (eightfold) post-MI. These large increases in collagen concentration in infarct scar are similar to those reported previously by us 13 wk post-MI (19). Cleutjens et al. (5) did not distinguish between infarcted and noninfarcted LV free wall in their study, making a comparison of their findings with those of the current study inappropriate. However, the progressive increase in fibrosis in noninfarcted LV septum reported in their study is similar to the increases in percent collagen reported for viable LV free wall in the current investigation. The findings of the current study extend those of the previous work (5) and show that, whereas the majority of the fibrosis occurs in the first week post-MI, it continues to increase out to at least 5 wk post-MI.
In the current study, ventricular fibrosis was preceded by an upregulation of collagen transcription. The time course for procollagen mRNA upregulation in LV was slightly different for the two fibrillar collagen types measured. Type I collagen mRNA was elevated within 72 h of coronary artery ligation and returned to control levels by 2 wk. Collagen type III mRNA was also significantly increased by 1 wk post-MI before returning to preligation levels by 2 wk. These findings with respect to upregulation of collagen type I and III mRNA levels are similar to those seen for rats in which cardiac hypertrophy is produced by aortic banding (33). Although the most plausible explanation for the differences in findings with respect to collagen mRNAs between the current work and the study of Cleutjens et al. (5) is the nature of the tissue being evaluated (infarcted vs. noninfarcted), the difference in size of infarcts produced between the two groups of animals could also have an effect. Cleutjens et al. (5) reported a mean infarct size of ~41% of the entire LV (free wall plus septum). In the current study, we employed the method originally described by Fishbein et al. (9) to calculate the size of the original infarct expressed as a percentage of the combined LV septum and free wall. On the basis of the measured weight of the consolidated scar, the infarct size for the 72-h and 15-day post-MI groups was 25 and 22%, respectively. Thus our infarcts were smaller than those of the Cleutjens et al. (5) study. This difference in infarct size may represent distinct sides of a threshold value for prolonged signaling of the collagen mRNA pathway and may account for the differences in mRNA expression between their study and ours. Likewise, the production of larger infarcts might result in an even more pronounced increase in decorin expression than seen in the current study. Clearly, further work is necessary to elucidate the mechanisms underlying procollagen and decorin transcription post-MI.
Collagen concentration varies among skeletal muscles of different fiber
types. Muscles containing predominantly slow-twitch fibers (e.g., SOL)
contain more collagen than fast-twitch (e.g., GAST) muscles
(37) and this finding is confirmed in the current work.
Skeletal muscle undergoes significant alteration post-MI. In rats,
infarction may ultimately cause a decrease in capillary density, a
thickening of the resistance vessels (25), and impairment of skeletal muscle blood flow (18), as well as changes in
oxidative capacity within the skeletal muscle bed (3).
However, there is a paucity of data regarding the immediate effects of
MI on skeletal muscle ECM. Although MI did not cause alterations in any
of the ECM parameters measured, there does appear to be a change in the
signals controlling transcription that the skeletal muscle is
receiving. Transient alterations in procollagen types I and III mRNA in
both locomotor muscles evaluated would suggest that the signaling
pathways for these fibrillar collagens are affected for unknown
reasons, although these perturbations did not translate into any
alterations in protein content within the time frame of the study,
collagen concentration was seen to be increased 1 yr post-MI in rat
hindlimb skeletal muscle (25). The 5-wk time period
examined in the present study may not be a sufficient amount of time to
observe changes in collagen metabolism in either fast- or slow-twitch
rat skeletal muscle. In the absence of a significant alteration in
collagen concentration and HP cross-linking in skeletal muscle post-MI,
the finding of unaltered decorin levels was also not surprising. There
was no apparent change in collagen metabolism within these tissues and
thus no reason for expression of decorin to be upregulated. Skeletal
muscle fibrosis does not appear to be an early event post-MI, with
collagen apparently accumulating slowly over a prolonged period of
time. It is possible that late fibrosis of skeletal muscle post-MI is a
response to chronic "spill-over" of fibrosis-inducing factors
(i.e., TGF-, angiotensin II) as the heart deteriorates from a
dysfunctional to a failure state. At this time, the mechanisms
responsible for the chronic ECM remodeling seen in skeletal muscle are
unclear and warrant further research.
Decorin concentration increased after a period of fibrosis in noninfarcted LV post-MI. Decorin upregulation appeared at an appropriate time to play a role in the organization of the collagen matrix after increased collagen deposition in cardiac muscle and may impact the nature of the fibrosis within the noninfarcted myocardium post-MI. Infarction appears to be a potent stimulus for fibrosis, subsequent decorin accumulation and increased cross-linking in the noninfarcted myocardium. Clearly, LV fibrosis in viable myocardium post-MI is not paralleled by fibrotic events in skeletal muscle at least within the experimental time frame used in this study. The temporal upregulation of decorin and its putative relationship to increased cross-linking and accumulation of collagen in viable myocardium post-MI require further examination.
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ACKNOWLEDGEMENTS |
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The authors gratefully acknowledge the excellent technical assistance of Kathy Austin and Jim Kaltenbach. The authors also acknowledge the gift of the collagen probes that were kindly donated by Dr. Jerold A. Last, Dept. of Internal Medicine, University of California, Davis, California.
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FOOTNOTES |
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This research was supported by grants from the American Heart Association (Wyoming affiliate) (to D. P. Thomas and R. J. McCormick).
Address for reprint requests and other correspondence: D. P. Thomas, Human Energy Research Laboratory, Division of Kinesiology and Health, College of Health Sciences, Univ. of Wyoming, Laramie, WY 82071-3196 (E-mail: cymru{at}uwyo.edu).
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 7 January 2000; accepted in final form 5 July 2001.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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significant difference from week 5 at P 
