Increased syndecan expression following myocardial infarction indicates a role in cardiac remodeling

Alexandra Vanessa Finsen1, Per Reidar Woldbaek1,2, Jian Li3, Jiaping Wu3, Torstein Lyberg4, Theis Tønnessen1,2 and Geir Christensen1

1 Institute for Experimental Medical Research
2 Department of Cardiothoracic Surgery, Ullevaal University Hospital, Oslo N-0407, Norway
3 Cardiovascular Division, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215
4 Research Forum, Ullevaal University Hospital, Oslo N-0407, Norway


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 References
 
Finsen, Alexandra Vanessa, Per Reidar Woldbaek, Jian Li, Jiaping Wu, Torstein Lyberg, Theis Tonnessen, and Geir Christensen. Increased syndecan expression following myocardial infarction indicates a role in cardiac remodeling. Physiol Genomics 16: 301-308, 2004. First published November 18, 2003; 10.1152/physi-olgenomics. 00144.2002.—The purpose of this study was to identify essential genes involved in myocardial growth and remodeling following myocardial infarction (MI). Left ventricular noninfarcted tissues from six mice subjected to MI under general anesthesia and from six sham-operated mice were obtained 1 wk after primary surgery and analyzed by means of cDNA filter arrays. Out of a total of 1,176 genes, 641 were consistently expressed, twenty-three were upregulated and thirteen downregulated. Five genes were only expressed following MI. Syndecan-3, a transmembranous heparan sulfate proteoglycan, was found to be upregulated together with a transcriptional activator of syndecans, Wilms tumor protein 1 (WT-1). Northern blotting demonstrated a significant upregulation of syndecan-1, -2, -3, and -4, WT-1, fibronectin, and basic fibroblast growth factor (FGF) receptor 1. Furthermore, Western blot analysis showed statistically significant increases in protein levels for syndecan-3 and -4. In conclusion, we have identified a subset of genes with increased expression in noninfarcted left ventricular tissue following MI, including syndecans 1–4, WT-1, fibronectin, collagen 6A, and FGF receptor 1. Since the syndecans link the cytoskeleton to the extracellular matrix and function as required coreceptors for FGF, we suggest a role for the syndecans in cardiac remodeling following MI.

extracellular matrix; gene expression; hypertrophy


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 References
 
CARDIAC REMODELING FOLLOWING myocardial infarction (MI) is known to be associated with myocyte hypertrophy, collagen deposition, and gradually increasing chronic left ventricular dilatation (3). With time a vicious circle is established in which cellular and molecular changes are themselves perpetuated by the remodeling process. Therefore, understanding the mechanisms involved in the initiation of cardiac remodeling is of particular interest with regard to developing efficient therapeutic strategies for the treatment of heart failure. At present these mechanisms are not fully understood.

The signaling pathways that initiate growth and remodeling have become a focus of great interest. Elucidating these is thought to lead to the development of substances specifically aimed at preventing the development of pathological remodeling. Mechanical stimuli, circulating hormones, as well as autocrine and paracrine factors are possibly involved in initiating myocardial growth. Transduction of mechanical stimuli may involve molecules that bind extracellular matrix molecules to the cytoskeleton, but the specific molecules involved in mechanotransduction have not yet been identified. Moreover, autocrine and paracrine factors, as well as intracellular signal regulators involved in initiating cardiac remodeling are only beginning to be unraveled.

The aim of our study was to identify essential genes involved in the early phase of cardiac remodeling. We (11) and others (10) have previously shown development of cardiac hypertrophy already 1 wk after induction of MI in the mouse. In the present study the gene expression in noninfarcted left ventricular tissue from mice 1 wk after MI was compared with data obtained from sham-operated mice. A cDNA filter hybridization array containing 1,176 genes, representing a wide variety of signaling molecules, was used. Differentially expressed genes were confirmed by Northern blotting. Our study demonstrates differential expression of a large number of extracellular matrix-associated genes and resulted in the identification of a subset of genes, including syndecan-1, -2, -3, and -4, WT-1, fibronectin, collagen 6A, and basic fibroblast growth factor (FGF) receptor 1. Western blot analysis showed increased protein levels of syndecan-3 and -4. Since the syndecans link the cytoskeleton to the extracellular matrix and function as required coreceptors for FGF, we suggest that they are involved in initiating cardiac remodeling.


    METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 References
 

Animal model.
We used 5- to 6-wk-old male BALB/c mice. Surgical procedures were performed as previously described by Iversen et al. (11). Briefly, after intravenous induction of anesthesia, the animals were connected to a rodent ventilator (model 874092; B. Braun, Melsungen, Germany). A left-sided thoracotomy was performed in the third intercostal space, and in MI mice the left coronary artery was ligated. Sham-operated animals underwent the same procedure except ligation of the artery.

One week after primary surgery the animals were anesthetized before the hearts were removed, blotted dry, dissected into left ventricular noninfarcted area, infarcted area, and right ventricle, and weighed. The tissue was then immediately snap frozen in liquid nitrogen, or fixed overnight in 4% formalin if intended for histological analysis. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication No. 85-23, revised 1996).

Histological analysis.
Hearts fixed in formalin were processed into paraffin wax. Six 4-µm thick slices were cut along the short axis from the mid-ventricular level of each tissue sample. Slices were either stained with hematoxylin-eosin for morphology or van Gieson or Mason’s trichrome for identification of collagen. Tissue samples from four mice subjected to coronary artery ligation and three sham-operated mice were evaluated.

Immunohistochemistry.
The excised hearts were placed in ice-cold phosphate-buffered saline (PBS, pH 7.4), quickly embedded in Tissue-Tek OCT compound (Sakura Finetek, Torrance, CA), and frozen in dry ice-cooled isopropanol before storing at -70°C. Sections 5-µm thick were cut using the Cryostat 1720 from Leitz, Wetzlar, Germany, and collected on SuperFrost Plus slides (Menzel-Gläser, Braunschweig, Germany). Sections were fixed for 5 min in acetone at -20°C and air dried. After rinsing in Tris-buffered saline (TBS, pH 7.6) containing 0.05% Tween 20, sections were incubated with the Dako peroxidase blocking reagent (Dako, Glostrup, Denmark) for 15 min at 37°C followed by TBS rinse. Sections were then incubated for 30 min at 37°C with affinity-purified antibodies (rabbit) against collagen type I (1:100 dilution) and III (1:1,200 dilution) (Rockland, Gilbertsville, PA), respectively. After three washes in TBS the primary antibody was labeled for 30 min using the Dako EnVision+ system (peroxidase, rabbit, K4010) and 3,3'-diaminobenzidine tetrahydrochloride as the chromogen. Sections were counterstained with hematoxylin for 1–2 min, mounted, and examined under a Leitz Aristoplan microscope.

cDNA filter array analysis.
The procedure followed instructions in Atlas Pure Total RNA Labeling System provided by Clontech (Palo Alto, CA). In brief, noninfarcted left ventricular tissues from MI mice and left ventricular tissues from sham mice were homogenized, and total RNA was isolated and DNase I treated. The total RNA was added to biotinylated oligo(dT) magnetic beads. The cDNA was then synthesized by means of reverse transcription using MMLV reverse transcriptase and colorless [32P]ATP. Filter membranes (Atlas Mouse 1.2 Array, Clontech) were prehybridized in ExpressHyb (Clontech) with salmon testis DNA (Sigma D7665), before labeled cDNA was hybridized to the filter membranes overnight. Each pair of membranes was exposed on the same screen for 7 days and read with a phosphor imager (Fujifilm Bio-Imaging Analyzer BAS-1800; Fuji Photo Film, Tokyo, Japan).

Interpretation of array results.
The integrated signal intensities of the spots on the scanned filters were determined using AtlasImage 1.0a (Clontech). We performed a pair-wise comparison of signal intensities of filters made from MI hearts and sham hearts. A total of six experiments were included in the analysis. Filters were rejected if the background intensity was not homogenous. Spots were visually inspected to exclude artifacts and bleeding. The average background intensity of the membrane was subtracted from the signal intensity of each spot. Spots with intensities within 10% above background were regarded as not expressed. Spot intensities were normalized to the median intensity of all expressed spots on the membrane. The gene expression ratios for each experiment were ranked, and the frequency of a given gene present in defined percentiles for all experiments was used to identify differentially expressed genes. The binomial probability function was used to calculate the probability of obtaining false positives (type II error). For a data set of 1,176 genes, the genes with MI/sham ratios above the 85th percentile or below the 15th percentile in three or more experiments had a probability of type II error <0.048. Genes fulfilling these criteria were regarded as differentially expressed.

Northern blotting.
Northern blotting was performed as described earlier (34). In brief, mRNA extraction was carried out using Dynabeads oligo(dT)25 (Dynal, Oslo, Norway). Three micrograms of poly(A)+ RNA was size-fractioned on a formaldehyde-agarose gel and transferred to a nylon membrane (Nytran N transfer membrane; Schleicher and Schuell, Dassel, Germany). The membrane was subsequently hybridized with 32P-labeled cDNA probes. Autoradiography of the filters was performed in the same way as with the filter arrays. Densitometric analyses were carried out using Image Gauge V3.12 (Fuji Photo Film) software. The intensity of each band was normalized to its respective GAPDH signal. The mean signal for the sham hearts was set equal to 100. Differential expression found in MI hearts was expressed as mean ± SE percent difference compared with sham-operated animals.

The mouse cDNA probe for WT-1 was a kind gift from Andreas Schedl (School of Biochemistry and Genetics, University of Newcastle upon Tyne, Newcastle upon Tyne, UK), the fibronectin probe was from Richard Hynes (Howard Hughes Medical Institute, Centre for Cancer Research, Department of Biology, Massachusetts Institute of Technology, Cambridge, MA), the syndecan-1 and -4 probes were from Arie Horowitz (Dartmouth Medical Center, Hanover, NH), the syndecan-2 probe was from Guido David (Center for Human Genetics, Campus Gasthuisberg, Leuven, Belgium), the syndecan-3 probe was from Merton Bernfield (Division of Newborn Medicine, Harvard Medical School, Boston, MA), and the FGF receptor 1 probe was from Peter A. Cattini (Department of Physiology, University of Manitoba, Winnipeg, Manitoba, Canada).

Western blotting.
Whole tissue lysates were obtained from the noninfarcted region of mouse hearts or from sham-operated hearts by RIPA solution (Boston Bioproducts, Ashland, MA) containing protease inhibitor cocktail (Roche, Mannheim, Germany). The protein concentration was determined according to the Bradford method (Bio-Rad, Hercules, CA). Forty micrograms of total proteins were fractionated by 8% (for syndecan-3) or 12% (for syndecan-4) SDS-polyacrylamide gel electrophoresis and transferred to PVDF membranes (Millipore, Bedford, MA). Ponceau Red staining (Boston Bioproducts) was used to stain the membranes for 1 min to show loading of proteins. The membranes were incubated with primary antibodies overnight at 4°C. Syndecan-3 was detected by polyclonal rabbit anti-syndecan-3 antibody (1:1,000 dilution; a kind gift from Dr. Sarka Tumova, Neuroscience Center, University of Helsinki, Helsinki, Finland). Syndecan-4 was detected by a 1:3,000 dilution of polyclonal chicken anti-syndecan-4 antibody (Aves Laboratories, Tigard, OR; a kind gift from Dr. Arie Horowitz, Dartmouth Medical Center, Hanover, NH). Following the primary antibody incubation, the membranes were incubated for 1 h at room temperature with horseradish peroxidase-conjugated appropriate goat anti-rabbit IgG (1:4,000; Calbiochem, San Diego, CA) for syndecan-3 or goat anti-chicken IgY for syndecan-4 (1:4,000, Aves Laboratories). Immune complexes were visualized using the ECL detection system (Amersham Biosciences, Buckinghamshire, UK). The film images of syndecan-3, syndecan-4, and their respective Ponceau Red-stained blots were scanned and quantitated with ImageQuant software (Alpha Innotech, San Leandro, CA). The measurements for syndecan-3 and syndecan-4 were subsequently normalized against the respective Ponceau Red measurements. For syndecan-3 analysis five MI hearts and five sham hearts were used, and for syndecan-4 analysis six MI hearts and six sham hearts were used. For calculation of changes in percent, the mean value of the sham hearts was set equal to 100. Differential expression found in MI hearts was expressed as mean ± SE percent change compared with sham hearts.

Statistics.
All appropriate data are presented as means ± SE. Simple comparisons between groups used for Northern and Western blot analysis were made using the Mann-Whitney Rank Sum Test based on raw data obtained by densitometry from the blots.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 References
 

Animal characteristics.
Ligation of the left coronary artery resulted in a significant increase in lung weight (P < 0.05), total heart weight (P < 0.05), and heart weight/body weight ratio (P < 0.05) compared with sham-operated animals. Body weight was not significantly different between the groups (Table 1).


View this table:
[in this window]
[in a new window]
 
Table 1. Total heart and lung weights in sham-operated animals and in animals 1 wk after coronary artery ligation

 
Histology of noninfarcted left ventricular tissue from mice with MI was compared with comparable areas of the left ventricle in sham-operated mice (Fig. 1, AD). MI mice (Fig. 1, B and D) exhibited signs of myocyte hypertrophy with large and varying hyperchromatic nuclei, clearly visible interstitial infiltration of granulocytes, and increased deposition of extracellular matrix compared with sham mice (Fig. 1, A and C).



View larger version (121K):
[in this window]
[in a new window]
 
Fig. 1. AD: myocardial histology in sham-operated mice (sham) and in mice 1 wk after myocardial infarction (MI). Hematoxylin and eosin-stained sections show myocyte hypertrophy, large hyperchromatic nuclei, and increased leukocyte infiltration in noninfarcted region of MI (B), compared with sham (A). Mason’s trichrome-stained sections show increased fibrosis after MI (D), compared with sham (C), indicated by collagen staining (blue). EH: immunohistochemical detection of collagen type I (E and F) and collagen type III (G and H) in the myocardium of sham (E and G) and in the noninfarcted myocardium of mice subjected to MI (F and H). There is a clear increase in the amount of both types of collagen following MI.

 
Immunohistochemical examination of myocardial sections from sham-operated animals showed regular, moderate endomysial staining for collagen type I (Fig. 1E) and III (Fig. 1G), albeit generally a weaker staining for type I. Sections from noninfarcted myocardial tissue of MI hearts (Fig. 1, F and H) demonstrated multiple, localized areas with significantly more endomysial collagen than corresponding areas in sham-operated animals. Apart from these areas, the remaining parts of noninfarcted myocardium were immunohistochemically indistinguishable from sham hearts with regard to collagen deposition.

Array analysis of gene expression.
Of a total of 1,176, we found that 641 genes were expressed in our cDNA filter arrays. Twenty-three genes were, according to the criteria described in the METHODS, significantly upregulated in noninfarcted left ventricular myocardium compared with ventricular tissue from sham-operated mice, and five genes were only expressed after MI. Thirteen genes demonstrated decreased expression (Table 2).


View this table:
[in this window]
[in a new window]
 
Table 2. Differentially expressed genes

 
As many as 43% of upregulated genes and 80% of induced genes could be related to the extracellular matrix. We found the expression of genes coding for fibronectin, fibrillin, osteopontin, collagen 6A, and biglycan to be upregulated following MI. BMP-1, which is known to process procollagen into mature collagen as well as probiglycan into biglycan, also demonstrated a significantly increased expression.

Furthermore, we found FGF receptor 1 to be upregulated after MI. Its required coreceptor syndecan-3, one of a family of four transmembranous heparan sulfate proteoglycans, was also upregulated. Syndecan-1 and -2 showed a trend toward upregulation after MI, but the changes did not reach the level of significance according to our strict criteria for significant upregulation. Syndecan-4 was not present on our filter array. Together with syndecan-3 we found a coordinate upregulation of WT-1, which is known to be a transcriptional factor for syndecans.

Northern blot analysis of gene expression.
In accordance with our array results, Fig. 2A clearly depicts an increase in expression of fibronectin in noninfarcted left ventricular tissue from MI mice compared with left ventricular tissue from sham-operated mice. The blot for FGF receptor 1 also shows an increased expression in noninfarcted left ventricular tissue (Fig. 2B). Densitometric assessment of fibronectin and FGF receptor 1 showed a mean increase of 262 ± 41% (P < 0.05) and 69 ± 11% (P < 0.05), respectively.



View larger version (44K):
[in this window]
[in a new window]
 
Fig. 2. Northern blots for fibronectin (A), FGF receptor 1 (B), syndecan-2 (C), syndecan-3 (D), syndecan-4 (E), GAPDH (F), WT-1 (G), syndecan-1 (H), and GAPDH (I) from noninfarcted left ventricular tissue 1 wk after left coronary artery ligation (MI) and from sham-operated animals.

 
The WT-1 mRNA levels were determined using a probe for WT-1 which detects the KTS+/KTS+ splice variant. Densitometric analysis showed a 92 ± 37% (P < 0.05) enhanced expression of WT-1 in MI hearts (Fig. 2G).

Results for syndecan-3 were also in accordance with the array results and showed a mean increase in expression of 45 ± 16% (P < 0.05) in MI hearts (Fig. 2D). Moreover, syndecan-1 and -4 both showed increased expression in MI hearts compared with sham hearts. As expected the syndecan-1 probe hybridized to two distinct bands, corresponding to 3.4 and 2.6 kb, resulting from processing at alternate polyadenylation sites (8). An increased expression of 90 ± 61% (P < 0.05) and 160 ± 28% (P < 0.05) for the two syndecan-1 bands, respectively (Fig. 2H), and 30 ± 9% (P < 0.05) for syndecan-4 (Fig. 2E) was calculated. The syndecan-2 probe identified three distinct bands corresponding to 3.5, 2.4, and 1.2 kb, known to be produced by differential polyadenylation (22). All three bands depicted an increased signal for MI hearts of 27 ± 9% (P < 0.05), 34 ± 11% (P < 0.05), and 77 ± 31% (P < 0.05), respectively (Fig. 2C).

Western blot analysis of protein levels.
The protein levels of syndecan-3 and -4 were significantly increased in the noninfarcted region after MI (Fig. 3). Immunoblot analysis for syndecan-4 showed a statistically significant increase of 64% (P < 0.05) in MI hearts compared with sham controls. For syndecan-3 we found a 72% increase (P < 0.05).



View larger version (26K):
[in this window]
[in a new window]
 
Fig. 3. A: representative Western blots for syndecan-3 and syndecan-4 in noninfarcted left ventricular tissue 1 wk after left coronary artery ligation (MI) and in left ventricular tissue from sham-operated mice. Pon. Red, Ponceau Red stain. B: data from densitometric analysis of all experiments. For syndecan-3 analysis five MI hearts and five sham hearts were used, and for syndecan-4 six MI hearts and six sham hearts were used. Values are means ± SE. *P < 0.05 compared with Sham.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 References
 
In this study, where we compared gene expression in noninfarcted left ventricular tissue from mice subjected to MI to that of sham-operated mice, we observed increased expression of a number of genes associated with the extracellular matrix. One of these, BMP-1, is able to activate latent growth factors (40) and process procollagen into collagen (14). In addition, we have identified increased expression of a group of genes encoding interacting proteins, including all four members of the syndecan family, WT-1, FGF receptor 1, fibronectin, and collagen 6A.

The cDNA filter array strategy provides an efficient means of simultaneously analyzing a large number of RNA transcripts. We have found that repeated array analysis and stringent criteria for defining genes as differentially expressed are essential for any deduction based on array results. We were able to confirm all upregulated genes tested, by means of Northern blotting, indicating a high degree of reliability of our array and data analysis.

One striking feature of our study was the high relative abundance of extracellular matrix-associated genes showing regulated expression. Fibronectin, fibrillin, osteopontin, biglycan, and collagen, all known important proteins of the extracellular matrix, demonstrated either increased expression or induction following MI. This suggests a marked remodeling and de novo deposition of extracellular matrix during the early phase following MI. Our histological findings indicate the same. Collagen staining of sections of the left ventricle, as well as immunohistochemical detection of collagen type I and III, showed increased deposition of extracellular matrix proteins in noninfarcted left ventricular tissue. The observed upregulation of FGF receptor 1, angiotensin converting enzyme, and tumor necrosis factor-{alpha} receptor 2 may influence this deposition (18, 26, 32). There is also an activation of the collagen degrading system parallel to the increased extracellular matrix deposition in noninfarcted myocardium (35). In line with this notion, we found an increase in the mRNA abundance for the metalloproteinase gelatinase A, indicating a change in quality as well as quantity of the extracellular matrix.

Altered extracellular matrix composition in the noninfarcted region of the left ventricle, which in part results from increased expression of genes encoding extracellular matrix proteins, may have profound effects on the diastolic properties of the myocardium by affecting its elasticity. Increased stiffness of the left ventricle may contribute to the characteristic abnormalities of diastolic function, including reduced passive left ventricular filling (12, 21). The extracellular matrix remodeling process has also been associated with degradation of matrix components and depletion of collagen. This process may contribute to left ventricular dilatation (37).

BMP-1 is one of a family of bone morphogenetic proteins first isolated from osteogenic material and shown able to initiate ectopic bone growth (40). Interestingly, our histological examination of left ventricular tissue 8 wk after ligation of the left coronary artery in the rat has even revealed a transformation of papillary musculature into osteogenic material (data not shown). It has become increasingly clear that BMP-1 differs considerably from the other bone morphogenetic proteins and that the task of BMP-1 might be to activate latent forms of the bone morphogenetic proteins (40, 29). Especially BMP-2 and BMP-4 have been thought to function as growth factors, stimulating the proliferation and orchestrating the deposition of mesenchymal cells (9). In addition to this, it is known that BMP-1 processes procollagen into collagen (14) and probiglycan into biglycan (27). This means that BMP-1 potentially possesses a dual role that could make it important in the remodeling of the extracellular matrix following MI.

Osteopontin is an adhesive glycophosphoprotein that was first identified in bone tissue (7). We observed induction of osteopontin expression in the noninfarcted region. It is known that osteopontin interacts with integrins and with extracellular matrix proteins such as collagen and fibronectin, and thus a role for this protein in extracellular matrix remodeling in myocardial failure has been suggested (31). Since osteopontin is also involved in calcification (23), its induction might have contributed to the accumulation of osteogenic material previously mentioned.

The expression of ß1-integrin was also found to be induced in the noninfarcted region following MI in our study. It is known to be localized in costameres (43) and intercalated discs of cardiomyocytes (36). The integrins have been shown to have several important functions in cell-extracellular matrix adhesion, and may act as mechanotransducers. Mice with cardiac-specific inactivation of ß1-integrin might therefore develop dilated cardiomyopathy because of a reduced cell-extracellular matrix linkage ability (28). The increased expression of integrins in the noninfarcted region may indicate that they have a role as mechanotransducers in the remodeling response.

Also, the expression of fibrillin, a constituent of the extracellular matrix, was induced. Fibrillins form the structural framework of an essential class of extracellular microfibrils that endow dynamic connective tissues with long-range elasticity (25). Their importance is emphasized by the linkage of fibrillin mutations to Marfan syndrome and related connective tissue disorders that are associated with severe cardiovascular, ocular, and skeletal defects (24).

Increased expression of the syndecan family following MI indicates a role for these transmembranous proteoglycans in the postischemic heart. The expression of syndecan-3 was significantly increased after MI on our arrays, and Northern blotting showed an upregulation of all four members of the family. Western blotting furthermore, showed a 64% increase in syndecan-3 and a 72% increase in syndecan-4 protein levels, which were found to be statistically significant. We chose to measure the protein levels of syndecan-3 because of its relatively strong mRNA expression in cardiac tissue and of syndecan-4 because we consider it interesting in the context of myocardial remodeling, particularly since it is found in cardiomyocytes. The increases in the transmembranous proteins syndecan-3 and syndecan-4 were most likely higher in the membrane of syndecan producing cells than measured in whole tissue. Such upregulation of the syndecans in the noninfarcted area after MI has not previously been reported. However, one study (19) showed that a rapid increase in syndecan-1 and -4 gene expression occurred in the ischemic region of the heart, and recently plasma levels of syndecan-4 were found to be elevated in patients with acute MI (16).

The mechanisms responsible for regulation of syndecan gene expression are not known in detail. However, it has been shown that factors secreted by inflammatory cells, such as tumor necrosis factor-{alpha} (13) and PR-39 (6), affect the expression of syndecans. Moreover, both monocytes and tissue-resident macrophages are known to express syndecan-1 (42) and syndecan-4 (41) core proteins. Presently, the relative contribution of inflammatory cells and other cells with regard to altered syndecan gene expression in the noninfarcted region is uncertain. However, in a study examining the peri-infarct region (19), it was found that although infiltrating macrophages accounted for a substantial increase in syndecan-1 and -4 expression, increased expression was also noted in the levels of syndecan-1 mRNA in endothelial cells and syndecan-4 mRNA in cardiac myocytes.

All four syndecans are expressed in fibroblasts (15), whereas only syndecan-4 has so far been identified in cardiomyocytes (19). The syndecans are implicated in several signal transduction cascades that regulate cell proliferation, and they are also at the cross section of mechanical signaling, interacting with molecules thought to be involved in mechanotransduction such as integrins (39). The syndecans bind growth factors and extracellular matrix molecules via their extracellular glycosaminoglycan chains (1). There is evidence that the syndecans have a role in focal adhesion formation, and the localization of syndecan-4 to focal adhesions in a variety of cell types (39) further supports syndecan facilitation of organized cell-matrix interaction. Via their small cytoplasmic domain, the syndecans interact with the cytoskeleton and potential downstream signal transducers. Syndecan-4 has been shown to interact with and activate PKC{alpha} (30), which has previously been shown to be involved in myocyte growth (33). In addition to PKC{alpha}, the syndecan-4 V region binds phosphatidylinositol 4,5-bisphosphate (PIP2), which itself is capable of activating PKC{alpha}, and potentiates PKC{alpha} activity induced by PIP2 10-fold (38). The syndecans are known to bind to FGF, for which they are required coreceptors, contributing to cell proliferation (2). Furthermore, the syndecans enable the binding of cytoskeletal actin filaments to two of the extracellular matrix proteins (2) that we found upregulated in heart failure, namely, fibronectin and type VI collagen. The increased expression of all members of the syndecan family together with upregulation of genes encoding interacting proteins (ß1-integrin, collagen 6A, fibronectin, and FGF receptor 1) increases, in our opinion, the possibility that these play a biological role in the heart following MI. Lastly, a transcription factor for syndecans, WT-1 (4), also demonstrated increased expression. Strikingly, WT-1 knockout mice have been shown to develop dilated cardiomyopathy (17), possibly indicating a role for WT-1 in cardiac remodeling through increasing the transcription of syndecans.

A relatively moderate increase in syndecan expression has been shown to have biological effects on fibroblast migration (20). Moreover, it has been reported that mice heterozygous for a disrupted syndecan-4 gene had statistically significant delayed wound healing and impaired angiogenesis in the granulation tissue compared with wild-type littermates (5). These studies indicate that changes in syndecan expression observed in vivo have effects on biological processes related to those seen in the heart during postischemic remodeling. However, few studies have addressed this particular issue, and further work is needed to assess the role of the syndecans in the myocardium following MI.

In conclusion, this study shows that there is an upregulation of a large number of extracellular matrix-associated genes in the noninfarcted region following MI in the mouse, indicating the importance of remodeling of the extracellular matrix in the early phase after MI. The observed upregulation of BMP-1 may have a particularly important role in this context, since it processes procollagen into collagen and activates latent growth factors. Moreover, we have identified a subset of genes with increased expression following MI, including syndecans 1–4, WT-1, fibronectin, collagen 6A, and FGF receptor 1. Western blotting showed significantly increased protein levels of syndecan-3 and -4. Since the syndecans link the cytoskeleton to the extracellular matrix and function as required coreceptors for FGF, we suggest a role for the syndecans in cardiac remodeling following MI.


    ACKNOWLEDGMENTS
 
We are grateful to Aud Svindland for carrying out histological analysis. We thank Lisbeth Winer, Unni Lie Henriksen, Annlaug Ødegaard, Tove Norén, and Roy Trondsen for expert technical assistance. We also thank Morten Eriksen and Line Solberg for animal care.

This work was supported by Anders Jahre’s Fund for Promotion of Science, Professor Carl Semb’s Medical Research Fund, Rakel and Otto Bruun’s Fund, and the Norwegian Research Council.


    FOOTNOTES
 
Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).

Address for reprint requests and other correspondence: A. Vanessa Finsen, Institute for Experimental Medical Research, Ullevaal Univ. Hospital, Kirkeveien 166, Oslo, Norway (E-mail: alexandra.finsen{at}ioks.uio.no).

10.1152/physiolgenomics.00144.2002.


    References
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 References
 

  1. Bernfield M, Gotte M, Park PW, Reizes O, Fitzgerald ML, Lincecum J, and Zako M. Functions of cell surface heparan sulfate proteoglycans. Annu Rev Biochem 68: 729–777, 1999.[CrossRef][ISI][Medline]
  2. Bernfield M, Kokenyesi R, Kato M, Hinkes MT, Spring J, Gallo RL, and Lose EJ. Biology of the syndecans: a family of transmembrane heparan sulfate proteoglycans. Annu Rev Cell Biol 8: 365–393, 1992.[CrossRef][ISI][Medline]
  3. Colucci WS and Braunwald E. Pathophysiology of heart failure. In: Heart Disease, edited by Braunwald E, Zipes DP, and Libby P. Philadelphia, PA: Saunders, 2001, p. 503–528.
  4. Cook DM, Hinkes MT, Bernfield M, and Rauscher FJ III. Transcriptional activation of the syndecan-1 promoter by the Wilms’ tumor protein WT1. Oncogene 13: 1789–1799, 1996.[ISI][Medline]
  5. Echtermeyer F, Streit M, Wilcox-Adelman S, Saoncella S, Denhez F, Detmar M, and Goetinck PF. Delayed wound repair and impaired angiogenesis in mice lacking syndecan-4. J Clin Invest 107: R9–R14, 2001.[ISI][Medline]
  6. Gallo RL, Ono M, Povsic T, Page C, Eriksson E, Klagsbrun M, and Bernfield M. Syndecans, cell-surface heparan-sulfate proteoglycans, are induced by a proline-rich antimicrobial peptide from wounds. Proc Natl Acad Sci USA 91: 11035–11039, 1994.[Abstract/Free Full Text]
  7. Herring GM and Kent PW. Some studies on mucosubstances of bovine cortical bone. Biochem J 89: 405–414, 1963.[ISI][Medline]
  8. Hinkes MT, Goldberger OA, Neumann PE, Kokenyesi R, and Bernfield M. Organization and promoter activity of the mouse syndecan-1 gene. J Biol Chem 268: 11440–11448, 1993.[Abstract/Free Full Text]
  9. Hogan BL. Bone morphogenetic proteins: multifunctional regulators of vertebrate development. Genes Dev 10: 1580–1594, 1996.[CrossRef][ISI][Medline]
  10. Hwang MW, Matsumori A, Furukawa Y, Ono K, Okada M, Iwasaki A, Hara M, Miyamoto T, Touma M, and Sasayama S. Neutralization of interleukin-1 beta in the acute phase of myocardial infarction promotes the progression of left ventricular remodeling. J Am Coll Cardiol 38: 1546–1553, 2001.[CrossRef][ISI][Medline]
  11. Iversen PO, Woldbaek PR, Tønnessen T, and Christensen G. Decreased hematopoiesis in bone marrow of mice with congestive heart failure. Am J Physiol Regul Integr Comp Physiol 282: R166–R172, 2002.[Abstract/Free Full Text]
  12. Jalil JE, Doering CW, Janicki JS, Pick R, Shroff SG, and Weber KT. Fibrillar collagen and myocardial stiffness in the intact hypertrophied rat left-ventricle. Circ Res 64: 1041–1050, 1989.[Abstract]
  13. Kainulainen V, Nelimarkka L, Jarvelainen H, Laato M, Jalkanen M, and Elenius K. Suppression of syndecan-1 expression tumor necrosis factor-alpha. J Biol Chem 271: 18759–18766, 1996.[Abstract/Free Full Text]
  14. Kessler E, Takahara K, Biniaminov L, Brusel M, and Greenspan DS. Bone morphogenetic protein-1: the type I procollagen C-proteinase. Science 271: 360–362, 1996.[Abstract]
  15. Kim CW, Goldberger OA, Gallo RL, and Bernfield M. Members of the syndecan family of heparan sulfate proteoglycans are expressed in distinct cell-, tissue-, and development-specific patterns. Mol Biol Cell 5: 797–805, 1994.[Abstract]
  16. Kojima T, Takagi A, Maeda M, Segawa T, Shimizu A, Yamamoto K, Matsushita T, and Saito H. Plasma levels of syndecan-4 (ryudocan) are elevated in patients with acute myocardial infarction. Thromb Haemost 85: 793–799, 2001.[ISI][Medline]
  17. Kreidberg JA, Sariola H, Loring JM, Maeda M, Pelletier J, Housman D, and Jaenisch R. WT-1 is required for early kidney development. Cell 74: 679–691, 1993.[ISI][Medline]
  18. Kubota T, McTiernan CF, Frye CS, Slawson SE, Lemster BH, Koretsky AP, Demetris AJ, and Feldman AM. Dilated cardiomyopathy in transgenic mice with cardiac-specific overexpression of tumor necrosis factor-alpha. Circ Res 81: 627–635, 1997.[Abstract/Free Full Text]
  19. Li J, Brown LF, Laham RJ, Volk R, and Simons M. Macrophage-dependent regulation of syndecan gene expression. Circ Res 81: 785–796, 1997.[Abstract/Free Full Text]
  20. Li L, Couse TL, Deleon H, Xu CP, Wilcox JN, and Chaikof EL. Regulation of syndecan-4 expression with mechanical stress during the development of angioplasty-induced intimal thickening. J Vasc Surg 36: 361–370, 2002.[ISI][Medline]
  21. Little WC. Assessment of normal and abnormal cardiac function. In: Heart Disease, edited by Braunwald E, Zipes DP, and Libby P. Philadelphia, PA: Saunders, 2001, p. 479–502.
  22. Marynen P, Zhang J, Cassiman JJ, Van den Berghe H, and David G. Partial primary structure of the 48- and 90-kilodalton core proteins of cell surface-associated heparan sulfate proteoglycans of lung fibroblasts. Prediction of an integral membrane domain and evidence for multiple distinct core proteins at the cell surface of human lung fibroblasts. J Biol Chem 264: 7017–7024, 1989.[Abstract/Free Full Text]
  23. Nagata T, Bellows CG, Kasugai S, Butler WT, and Sodek J. Biosynthesis of bone proteins [Spp-1 (secreted phosphoprotein-1, osteopontin), Bsp (bone sialoprotein) and Sparc (osteonectin)] in association with mineralized-tissue formation by fetal-rat calvarial cells in culture. Biochem J 274: 513–520, 1991.[ISI][Medline]
  24. Robinson PN and Godfrey M. The molecular genetics of Marfan syndrome and related microfibrillopathies. J Med Genet 37: 9–25, 2000.[Abstract/Free Full Text]
  25. Sakai LY, Keene DR, Glanville RW, and Bachinger HP. Purification and partial characterization of fibrillin, a cysteine-rich structural component of connective-tissue microfibrils. J Biol Chem 266: 14763–14770, 1991.[Abstract/Free Full Text]
  26. Scheinowitz M, Abramov D, and Eldar M. The role of insulin-like and basic fibroblast growth factors on ischemic and infarcted myocardium. Int J Cardiol 59: 1–5, 1997.[CrossRef][ISI][Medline]
  27. Scott IC, Imamura Y, Pappano WN, Troedel JM, Recklies AD, Roughley PJ, and Greenspan DS. Bone morphogenetic protein-1 processes probiglycan. J Biol Chem 275: 30504–30511, 2000.[Abstract/Free Full Text]
  28. Shai SY, Harpf AE, Babbitt CJ, Jordan MC, Fishbein MC, Chen J, Omura M, Leil TA, Becker KD, Jiang MH, Smith DJ, Cherry SR, Loftus JC, and Ross RS. Cardiac myocyte-specific excision of the beta 1 integrin gene results in myocardial fibrosis and cardiac failure. Circ Res 90: 458–464, 2002.[Abstract/Free Full Text]
  29. Shimell MJ, Ferguson EL, Childs SR, and O’Connor MB. The Drosophila dorsal-ventral patterning gene tolloid is related to human bone morphogenetic protein 1. Cell 67: 469–481, 1991.[ISI][Medline]
  30. Simons M and Horowitz A. Syndecan-4-mediated signalling. Cell Signal 13: 855–862, 2001.[CrossRef][ISI][Medline]
  31. Singh K, Sirokman G, Communal C, Robinson KG, Conrad CH, Brooks WW, Bing OH, and Colucci WS. Myocardial osteopontin expression coincides with the development of heart failure. Hypertension 33: 663–670, 1999.[Abstract/Free Full Text]
  32. Smits JF, Passier RC, Nelissen-Vrancken HJ, Cleutjens JP, Kuizinga MC, and Daemen MJ. Does ACE inhibition limit structural changes in the heart following myocardial infarction? Eur Heart J 16, Suppl N: 46–51, 1995.
  33. Sugden PH. Signalling pathways in cardiac myocyte hypertrophy. Ann Med 33: 611–622, 2001.[ISI][Medline]
  34. Tønnessen T, Giaid A, Saleh D, Naess PA, Yanagisawa M, and Christensen G. Increased in vivo expression and production of endothelin-1 by porcine cardiomyocytes subjected to ischemia. Circ Res 76: 767–772, 1995.[Abstract/Free Full Text]
  35. Tyagi SC. Proteinases and myocardial extracellular matrix turnover. Mol Cell Biochem 168: 1–12, 1997.[ISI][Medline]
  36. Vanderflier A, Kuikman I, Baudoin C, Vanderneut R, and Sonnenberg A. A novel beta-1 integrin isoform produced by alternative splicing: unique expression in cardiac and skeletal-muscle. FEBS Lett 369: 340–344, 1995.[CrossRef][ISI][Medline]
  37. Weber KT, Pick R, Janicki JS, Gadodia G, and Lakier JB. Inadequate collagen tethers in dilated cardiopathy. Am Heart J 116: 1641–1646, 1988.[ISI][Medline]
  38. Woods A and Couchman JR. Syndecans: synergistic activators of cell adhesion. Trends Cell Biol 8: 189–192, 1998.[CrossRef][ISI][Medline]
  39. Woods A and Couchman JR. Syndecan-4 and focal adhesion function. Curr Opin Cell Biol 13: 578–583, 2001.[CrossRef][ISI][Medline]
  40. Wozney JM, Rosen V, Celeste AJ, Mitsock LM, Whitters MJ, Kriz RW, Hewick RM, and Wang EA. Novel regulators of bone formation: molecular clones and activities. Science 242: 1528–1534, 1988.[ISI][Medline]
  41. Yeaman C and Rapraeger AC. Membrane-anchored proteoglycans of mouse macrophages: P388D1 cells express a syndecan-4 like heparan-sulfate proteoglycan and a distinct chondroitin sulfate form. J Cell Physiol 157: 413–425, 1993.[ISI][Medline]
  42. Yeaman C and Rapraeger AC. Posttranscriptional regulation of syndecan-1 expression by camp in peritoneal-macrophages. J Cell Biol 122: 941–950, 1993.[Abstract]
  43. Zhidkova NI, Belkin AM, and Mayne R. Novel isoform of beta-1 integrin expressed in skeletal and cardiac-muscle. Biochem Biophys Res Commun 214: 279–285, 1995.[CrossRef][ISI][Medline]