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Cardiovascular Research 2002 56(2):235-247; doi:10.1016/S0008-6363(02)00546-1
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Copyright © 2002, European Society of Cardiology

Collagen degradation in a murine myocarditis model: relevance of matrix metalloproteinase in association with inflammatory induction

Jun Lia, Peter Lothar Schwimmbecka, Carsten Tschopea, Sebastian Leschkaa, Lars Husmanna, Susanne Rutschowa, Florian Reichenbacha, Michel Noutsiasa, Ursula Kobalza, Wolfgang Pollera, Frank Spillmanna, Heinz Zeichhardtb, Heinz-Peter Schultheissa and Matthias Pauschingera,*

aDepartment of Internal Medicine II, University Hospital Benjamin Franklin, Free University Berlin, Hindenburgdamm 30, D-12200 Berlin, Germany
bInstitute for Infectious Diseases Medicine, University Hospital Benjamin Franklin, Free University Berlin, Hindenburgdamm 30, D-12200 Berlin, Germany

* Corresponding author. Department of Cardiology and Pneumonology, University Hospital Benjamin Franklin, Free University Berlin, Hindenburgdamm 30, D-12200 Berlin, Germany. Tel.: +49-30-8445-2349; fax: +49-30-8445-4648. pauschinger{at}ukbf.fu-berlin.de

Received 11 February 2002; accepted 24 June 2002


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusion
 References
 
Objective: Myocardial collagen degradation is regulated by matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinase (TIMPs). The possible relevance of MMPs in association with the inflammatory induction was investigated in a murine coxsackievirus B3 myocarditis model. Methods: Hearts from viral infected and sham-infected BALB/c mice were analyzed by semi-quantitative RT-PCR, picrosirius red staining, Western blot analysis, and immunohistochemistry. Results: In viral infected mice, both mRNA and protein abundance for collagen type I remained unaltered. In addition, picrosirius red staining showed the unchanged total collagen content. However, degraded soluble fraction of collagen type I protein was increased. Moreover, the mRNA abundance for MMP-3 and MMP-9 was upregulated, whereas the mRNAs for TIMP-1 and TIMP-4 were downregulated, respectively. The upregulation of MMP-3/MMP-9 and downregulation of TIMP-4 were confirmed at the protein level, and were associated with significantly increased mRNA levels of interleukin 1β, tumor necrosis factor-{alpha}, transforming growth factor-β1 and interleukin-4. Conclusion: The increment of MMPs in the absence of counterbalance by TIMPs may lead to a functional defect of the myocardial collagen network by posttranslational mechanisms. This may contribute significantly to the development of left ventricular dysfunction in murine viral myocarditis. The inflammatory response with induction of cytokines may mediate the dysregulation of the myocardial MMP/TIMP systems.

KEYWORDS Connective tissue; Cytokines; Extracellular matrix; Gene expression; Myocarditis


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 5. Conclusion
 References
 
Myocardial extracellular matrix collagen (Col), composed mainly of collagen type I (Col I), forms a unique cross-linked collagen network that maintains the structural integrity of the heart and provides a fine scaffold for myocyte alignment. Normally, the synthesis and degradation of collagen are tightly controlled [1], and matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinase (TIMPs) are the major regulators of collagen degradation [3,7,11]. MMPs belong to a family of zinc-dependent enzymes, which share a zinc-binding catalytic domain [17,27]. All members of MMPs are involved in the regulation of collagen degradation. Collagenases, such as MMP-1, MMP-8 and MMP-13, can cleave the triple helical collagen fibrils into gelatin fragments [12]. Gelatinases, such as MMP-2 and MMP-9, digest gelatin further [12,18]. Stromelysins, such as MMP-3, can degrade a broad range of extracellular matrix components including Col I [7], Col type IV, and proteoglycans [17]. In addition, MMP-3 can activate other MMPs [21,22]. The activities of MMPs can be specifically inhibited by TIMPs. All TIMPs share conserved structural features including a N-terminal domain, which interacts with the zinc-binding sites of MMPs [19].

Recent studies have indicated that abnormal remodeling of myocardial collagen can be caused by imbalances between myocardial MMPs and TIMPs [3,11]. However, the mechanism by which MMPs mediate myocardial collagen remodeling in vivo, and the relevance of inflammatory induction, is still unclear. The present study was designed to investigate the myocardial mRNA and protein expression of collagen, MMP-3, MMP-9, TIMP-1 and TIMP-4 in a murine myocarditis model. Moreover, the mRNA expression levels of IL-1β, TNF-{alpha}, TGF-β1 and IL-4 were investigated to evaluate the inflammatory reaction.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 5. Conclusion
 References
 
2.1 Animals and virus
Eight-week-old male BALB/c (H-2d) mice were obtained from The Jackson Laboratory, Bar Harbor, USA. Before infection, the mice were housed under constant temperature and humidity and exposed to a 12 h light/dark cycle. Coxsackievirus B3 (CVB3, Nancy strain; VR-30, Manassas, USA) was propagated on Hela cells and stored at –80 °C until use. At 8 weeks of age, mice (n=22) were inoculated intraperitoneally with 5x105 plaque forming units (PFU) of CVB3 virus in 0.2 ml of saline. Eight-week-old control mice (n=22) were sham-infected with 0.2 ml of saline intraperitoneally. Animals were sacrificed at day 10 after virus inoculation. The investigation was performed in accordance with the German law on animal protection as released in 1993.

2.2 Hemodynamic evaluation and tissue processing
Ten days after infection, hemodynamic parameters were measured in anesthetized, artificially ventilated and open-chest animals. Left ventricular pressure (LVP), maximal rate of pressure increase over time (dP/dt max) as a parameter of LV contractility and minimal rate of pressure decrease over time (dP/dt min) as a parameter of LV relaxation were determined via a 1.2 F Milar-Tip catheter introduced in the LV chamber. Immediately after the hemodynamic parameters were obtained, the hearts were rapidly removed and transversely dissected. The heart sections were either directly frozen in liquid nitrogen or embedded in Tissue TecTM (SLEE, Haan, Germany) and snap-frozen in liquid nitrogen.

2.3 Semi-quantitative RT-PCR analysis
The primers used for PCR amplification are listed in Table 1. Nested PCR was employed for the detection of CVB3 genome as described before [25]. Total RNA was extracted from myocardial samples by Trizol method (GIBCOL BRL) and treated with RNase-free DNase I (Roche, Mannheim, Germany). mRNA was reverse transcribed using the first-strand cDNA synthesis kit (Promega, Mannheim, Germany). Cycle-per-cycle monitoring of PCR products was performed in a Mastercycler gradient (Eppendorf, Hamburg, Germany) to monitor the amplification efficiency and determine the exponential linear phase. Each set of PCR amplification contained β-actin as internal control. PCR reactions were carried out in 50 µl 10 mM Tris–HCl (pH 8.3), 1.5 mM MgCl2, 50 mm KCl, 0.2 mM dNTP, 0.5 µM each of the primers, 1.25 U Taq DNA Polymerase (Rapidozym, Berlin, Germany). PCR cycling condition was 94 °C for 1 min, 61 °C for 1 min and 72 °C for 1 min. PCR products were examined on a 1.2% agarose gel containing 0.4 µg/ml ethidium bromide, and the specific bands of PCR products were measured as optical densities (OD) using a software program (Scion Image, Maryland, USA). Ratio of target amplicon (OD)/β-actin amplicon (OD) was calculated as the relative mRNA level. By sequence analysis of the PCR fragment subcloned into pGEM®-T vector (Promega), the nucleic acid sequences of all PCR products were confirmed to be identical to the published GenBank data.


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  		Table 1 Primer sequences used for semi-quantitative RT-PCR

 
2.4 Histological analysis
Picrosirius red staining was used for the histological assessment of the total collagen content by use of computer-assisted morphometry as described before [26]. Briefly, formalin-fixed tissue was processed for paraffin embedding, serially cutted into sections of 5 µm thickness, and stained with 0.1% picrosirius red F3BA dissolved in saturated picric acid. All fields from coded section magnified in a Leica DMRDTM microscope (Leica, Bensheim, Germany) at 100-fold magnification were transferred to a Sony 3CCDTM color video camera using a Leica C-mount adapter under circularly polarized light condition. The total collagen content of 5 sections per sample was measured and expressed as area fraction (AF), which was calculated as the ratio of thresholded chromogen area to net myocardial area [20]. The investigator responsible for the quantitative morphometrical analysis was blinded as to the experimental samples.

2.5 Immunohistochemistry
Immunohistochemical analysis was performed as described previously [20]. The frozen specimens were cut serially into 6 cryosections of 5 µm thickness per sample. Following fixation and subsequent air drying, endogenous peroxidase activity was quenched by incubating cryosections with 0.3% H2O2 in phosphate-buffered saline (PBS) for 10 min. After rinsing thrice in PBS, slides were blocked with 1.5% heat-treated goat serum in PBS. After removing blocking serum, slides were incubated with rabbit anti-collagen type I (Col I) polyclonal antibody (Calbiochem, 1:50) diluted in PBS containing 1.5% goat serum for 30 min. Slides were then rinsed thrice with PBS and subsequently incubated with goat biotinylated anti-rabbit IgG (Vector, 1:200) diluted in PBS containing 1.5% goat serum for 30 min. After rinsing thrice in PBS, slides were incubated with VECTASTAIN® ABC reagent (Vector) for 30 min. After rinsing thrice with PBS, immunoreactive staining was visualized using 3-amino-9-ethylcarbazole (Merck, Darmstadt, Germany) as chromogenic substrate. The staining and peroxidase reactions in all samples were done identically and in parallel. To exclude any heart tissue cross-reactivity against goat biotinylated anti-rabbit IgG, serial sections from each specimen were incubated with PBS containing 1.5% heat-treated goat serum omitting anti-Col I antibody. To quantitatively analyze Col I expression, the coded slides were evaluated in a blinded fashion. Employing the software LUCIA GTM (Version 3.52ab, Nikon, Düsseldorf, Germany), Col I expression was evaluated by a multistep macro programmed to measure Col I ‘area fraction’ (Col I-AF). Fields magnified in a Leica DMRDTM microscope at 100-fold magnification (Leica) were transferred to a Sony 3CCDTM color (red-green-blue/RGB) video camera under standard light conditions. The resulting measurement frame accounted 1.2 mm2. All available fields were evaluated (more than 30 fields per sample). The macro grabbed, sharpened, and contrasted the image. Subsequently, the threshold was set to detect first the area not captured by cardiac tissue (minimum RGB-values: 230,250,244; maximum RGB-values: 255, 255, 255). Based on this, the net myocardial area was calculated omitting artifacts and areas free of any tissue. The second threshold detected specifically the red chromogen covered area within the sections (minimum RGB-values: 92,0,0; maximum RGB-values: 255,182,162). Col I-AF was calculated as the ratio of thresholded chromogen area to net myocardial area.

Immunohistochemical staining of CD3 was performed using rat anti-CD3 monoclonal antibody (Pharmingen, Heidelberg, Germany) as described previously [24].

2.6 Western blot analysis
The following primary antibodies were used: Rabbit anti-Col I polyclonal antibodies (Calbiochem); rabbit anti-MMP-3, rabbit anti-MMP-9 (Chemicon) and goat anti-TIMP-4 polyclonal antibodies (Santa Cruz, Heidelberg, Germany). Total protein was prepared with Trizol reagent (Gibcol-BRL) and protein pellet was dissolved in sample buffer containing 5% sucrose, 5% SDS, 60 mM Tris–HCl. Following incubation at 95 °C for 5 min, 5 µg of total protein per lane were separated on a 7.5% or 12% SDS–PAGE gel, respectively, and transferred onto a PVDF membrane (Bio-Rad). Equal protein loading was verified by both staining the gel and the membrane with Coomassie brilliant blue and Ponceau S, respectively. The membranes were incubated with primary antibody, followed by the appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (DAKO, Hamburg, Germany). Target proteins were detected by enhanced chemiluminescence (ECL) detection system (Amersham Pharmacia Biotech, Braunschweig, Germany) and recorded on a film in the linear detectable range. Area quantification (OD) of the relevant band was represented as the relative protein level. All protein samples for western blot analysis were prepared without cleavage by enzymes. Therefore, only soluble Col I proteins can be detected by this method.

2.7 Statistical analysis
For quantitative analysis, all data were expressed as means±SD, The significance of differences in the data was evaluated by two-sided Student’s t-test. A value of P<0.05 was considered statistically as significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 5. Conclusion
 References
 
3.1 Characterization of murine myocarditis
CVB3 infected BALB/c mice showed a marked decrease in body weight and a ruffled fur. Persistence of CVB3 genome was confirmed in all CVB3-infected mice by nested RT-PCR analysis, and all sham-infected control mice were proved to be CVB3 virus negative (Fig. 1a). Immunohistochemical staining of CVB3-infected heart sections revealed significant CD3+ T lymphocyte infiltration in loose interstitial space and cardiomyocyte hypertrophy. No significant lymphocytic infiltration or cardiomyocyte hypertrophy was detected in sham-infected control mice (Fig. 1b).


Figure 1
Figure 1
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  		Fig. 1 (a) Coxsackievirus B3 (CVB3) persistence. CVB3 genomic RNA was detected by nested RT-PCR. This representative gel depicts negative PCR signals in samples 1–8 (sham-infected) and positive signals in samples 9–16 (infected). M, molecular weight marker; (+), CVB3-positive control; (–) negative control. (b) CD3+ T lymphocyte infiltration. A representative immunohistochemical staining of CVB3-infected heart sections showed the inflammatory CD3+ T lymphocyte infiltration in loose interstitial space and cardiomyocyte hypertrophy. Original magnifications, x200.

 
CVB3-infected mice developed a significant impairment of left ventricular (LV) function as shown by a significant reduction in LVP (81.6% of control; P<0.01), dP/dt max (76.1% of control; P<0.05) and dP/dt min (73.5% of control; P<0.01) (Table 2).


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  		Table 2 Hemodynamic analysis

 
3.2 Myocardial collagen remodeling
At mRNA level, semi-quantitative RT-PCR analysis did not show significant change of myocardial Col I mRNA abundance (1.0±0.2) in myocarditis when compared to that (1.0±0.1) in control mice (P>0.05; Fig. 2a).


Figure 2
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  		Fig. 2 (a) Unaltered mRNA abundance of myocardial Col I. Semi-quantitative RT-PCR analysis showed no difference (P>0.05) for Col I mRNA between CVB3-infected (n=8) and sham-infected (n=8) mice. M, molecular weight marker; samples 1–8 (sham-infected); samples 9–16 (infected); (–) negative control. (b) Unaltered expression of total myocardial Col I. Quantitative immunohistochemical analysis showed no difference (P>0.05) for the total Col I protein as represented by AF between CVB3-infected (n=16) and sham-infected (n=17) mice. (c) Unaltered myocardial collagen content. Quantitative histological analysis of the total collagen content as represented by AF showed no significant difference (P>0.05) between CVB3-infected (n=13) and sham-infected (n=8) mice. (d) Increased myocardial soluble Col I. Western blot analysis showed the increased soluble Col I (n=11) in CVB3-infected mice in comparison to that in sham-infected mice (n=11). Representative samples 1 and 3 (sham-infected); samples 2 and 4 (infected); (+), Hs 68 WCL (positive control for Col I) (Santa Cruz).

 
At protein level, immunohistochemical quantification of total Col I expression further demonstrated the unaltered Col I proteins, which were stained as large strands or dense waves, predominantly distributed within the myocardial interstitial space surrounding cardiomyocytes of both myocarditis and control mice (Fig. 3). The total Col I expression as expressed as AF was 0.056±0.032 in myocarditis and 0.053±0.026 in sham-infected control mice, respectively (P=0.73; Fig. 2b). Moreover, the histological analysis of the total collagen by picrosirius red staining visualized in circularly polarized light further showed no significant change in total collagen content between myocarditis (0.0060±0.0017) and sham-infected control mice (0.0076±0.0009) (P=0.26; Fig. 2c). The relative collagen content as measured in picrosirius red and immunohistochemical staining is proportional to the absolute collagen content. In contrast, Western blot analysis of soluble proteins, which include only the soluble fraction of total Col I protein, showed the relative levels of myocardial soluble Col I protein significantly increased 2.0-fold in myocarditis mice when compared to that in sham-infected control mice (P<0.05; Fig. 2d).


Figure 3
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  		Fig. 3 Similar expression of total myocardial Col I protein in CVB3-infected and sham-infected control mice. The representative immunohistochemical staining demonstrated the Col I proteins, which were stained as large strands or dense waves, distributed within the myocardial interstitial space surrounding cardiomyocytes.

 
3.3 Upregulation of myocardial MMPs
The mRNA and protein expressions of myocardial MMP-3 and MMP-9 were analyzed by semi-quantitative RT-PCR and Western blot analysis. The relative mRNA expression level for myocardial MMP-3 and MMP-9 in myocarditis mice was significantly upregulated 1.97-fold (P<0.01) and 2.0-fold (P<0.01), respectively (Fig. 4). This enhancement of transcription was accompanied by a significant upregulation of protein expression as demonstrated by Western blot analysis. In myocarditis mice, a 2.3-fold (P<0.01) and 2.6-fold (P<0.01) increase of MMP-3 and MMP-9 protein was detected, respectively (Fig. 5).


Figure 4
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  		Fig. 4 Differential mRNA abundance of myocardial MMPs and TIMPs. (A) A representative RT-PCR data; M, molecular weight marker; (–) negative control; samples 1–8 (sham-infected); samples 9–16 (infected). (B) Semi-quantitative analysis showed the upregulated mRNA of MMP-3 (n=22) and MMP-9 (n=14) but downregulated mRNA of TIMP-1 (n=22) and TIMP-4 (n=14) in CVB3-infected mice. *P<0.01 versus sham-infected.

 

Figure 5
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  		Fig. 5 Differential protein abundance of myocardial MMPs and TIMP-4. (5) A representative Western blot; samples 1 and 3 (sham-infected); samples 2 and 4 (infected); (+), positive controls (MMP-3/MMP-9) (Chemicon) and MCF7 WCL (TIMP-4). (5) Quantitative analysis showed the increased MMP-3 (n=6) and MMP-9 (n=10) but reduced TIMP-4 protein (n=6) in CVB3-infected mice. *P<0.01 versus sham-infected.

 
3.4 Downregulation of myocardial TIMPs
Myocardial TIMP-1 and TIMP-4 were analyzed by semi-quantitative RT-PCR and Western blot analysis. In myocarditis mice, the relative mRNA expression level of myocardial TIMP-1 and TIMP-4 was significantly down-regulated to 18.9% (P<0.01) and 40.03% (P<0.01) of controls, respectively (Fig. 4). In addition, the protein expression of myocardial TIMP-4 was also significantly reduced (39.4% of control) (P<0.01) as shown by Western blot analysis (Fig. 5).

3.5 Cytokine induction
Semi-quantitative RT-PCR analysis showed that the relative mRNA expression level of IL-1β, TNF-{alpha}, TGF-β1 and IL-4 in myocarditis mice was significantly increased 1.4-fold (P<0.01), 1.6-fold (P<0.01), 1.7-fold (P<0.01) and 1.8-fold (P<0.01), respectively, in comparison to that of control mice (Fig. 6).


Figure 6
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  		Fig. 6 Upregulated mRNA abundance of myocardial cytokines. (6) A representative RT-PCR data; M, molecular weight marker; (–) negative control; samples 1–8 (sham-infected); samples 8–16 (infected). (6) Semi-quantitative analysis showed the upregulated mRNA of IL-1β (n=22), TNF-{alpha} (n=8), TGF-β1 (n=22) and IL-4 (n=11) in CVB3-infected mice. *P<0.01 versus sham-infected.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
5. Conclusion
 References
 
CVB3-induced myocarditis in BALB/c mice caused a significant impairment of LV function 10 days after infection. In addition, persistence of CVB3 genome, CD3+ T lymphocyte infiltration, and an increased expression of inflammatory cytokines such as TNF-{alpha} and IL-1β were verified. This myocardial inflammatory reaction was accompanied by a significant upregulation of MMP-3 and MMP-9, but downregulation of TIMP-1 and TIMP-4, at the mRNA and protein level. This imbalance of MMP and TIMP expression may lead to a functional defect of the collagen (Col) network mainly by posttranslational mechanisms, and may contribute significantly to the development of LV dysfunction in myocarditis mice.

4.1 Myocardial collagen remodeling
The myocardial Col network provides a fine scaffold for myocyte alignment, coordinating myocardial contraction and structural integrity of the heart. Recent evidence suggests that myocardial Col remodeling plays an important role in the development of LV diastolic and systolic dysfunction [3,11,24–26,44,45]. The abnormal myocardial Col remodeling mainly concerns the alteration in composition and structure of myocardial Col. Col deposition is associated with the heart less compliant and increased wall stiffness [44,45]. On the other hand, the structure of Col network provides myocardial wall mechanics, and Col disruption contributes to the LV chamber dilatation and eventually impaired LV dysfunction [3,6,31,34]. Col, in particular collagen type I (Col I), is the most abundant protein in the mammals and the major component of the myocardial extracellular matrix. In vivo, native mature collagens are found as strong insoluble fibrils with extensive cross-links. Because of this insolubility, native mature collagens remain extremely difficult to study at both translational and posttranslational level.

In CVB3-induced myocarditis mice, the mRNA and protein abundance of myocardial Col I was unchanged. In addition, picrosirius red staining also confirmed an unaltered total collagen content. Unexpectedly, Western blot analysis demonstrated a significantly increased soluble fraction of total myocardial Col I protein. The present finding of an increased soluble Col I protein in the presence of unaltered total Col I abundance at the mRNA and protein level indicates myocardial collagen degradation mainly at the posttranslational level.

In vivo, cross-linking between Col molecules is one of the key posttranslational modifications, and the insolubility of functional mature Col is primarily a consequence of covalent cross-links between Col molecules [35]. The present findings may indicate that only cross-links between Col molecules disrupted without further cleavage of collagen molecules into fragments, which may contribute to the LV dysfunction. In this regard, previous studies showed the reduced Col cross-links, which represents the decreased native insoluble Col or increased soluble Col, is associated with detrimental cardiac chamber remodeling and ventricular dilatation in rat models of systolic chamber dysfunction [34], human dilated cardiomyopathy [6], and pig models with tachycardia-induced cardiomyopathy [31]. Therefore, the current findings indicate that native Col degradation at the posttranslational level may play an important role for the LV dysfunction in CVB3-induced myocarditis mice.

However, because of the method limitation in studying insoluble collagens so far, a limitation of this study remains that the present in vivo findings did not directly address the exact site of the collagen matrix degradation. Alternatively, the analytical methods may be insufficiently sensitive to detect the decreased Col protein following MMP-mediated degradation process. On the other hand, the collagen fragments as a consequence of effective collagen degradation were also not well addressed in this study.

The CVB3-induced myocarditis models in BALB/c mice are characterized by the myocardial inflammatory reaction that precedes the development of myocardial fibrosis [38–40]. The present study concentrated on the early phase during inflammation in association with the myocardial Col remodeling. To our knowledge, there is no report on altered mRNA expression of myocardial Col during the early phase after CVB3 infection in BALB/c mice. Col synthesis by cardiac fibroblasts has been regulated by multiple factors involving regulatory cytokines such as TNF-{alpha} and TGF-β [30,46]. The combined effects of regulatory cytokines and other factors may lead to an unaltered mRNA expression for myocardial Col at 10 days after CVB3 infection. Since the major Col deposition in the myocardium develops at 55 days and 30 days after CVB3 infection in A/J and BALB/c mice [39,43], respectively, the increased Col mRNA could not be ruled out during the late phase of CVB3 myocarditis in BALB/c mice with the consistent stimulation of fibrogenic factors including TGF-β, which has been shown to be persistently overexpressed in the chronic stage of CVB3-induced myocarditis [41].

4.2 MMPs and myocardial collagen remodeling
The myocardial MMP-3 and MMP-9 expression was significantly upregulated, whereas downregulated TIMP-1 and TIMP-4 expression was detected at both mRNA and protein level in CVB3-induced myocarditis mice.

Although the cleavage of soluble collagens by different types of MMPs has been extensively characterized, few studies on the proteolytic activity of MMPs focused on the cross-linking of insoluble collagens. Both MMP-3 and MMP-9 are involved in collagenolytic activities. MMP-3 cleaves many extracellular matrix components including Col I, and activates other MMPs such as MMP-8 and MMP-9 [7,12,22]. Both MMP-8 and MMP-9 can degrade insoluble collagens [23,36]. MMP-9 is referred to as gelatinase due to its ability to degrade gelatin effectively. However, recent studies have suggested, that MMP-9 also cleaves Col type I, III, V and IV [22,37]. More importantly, there is convincing evidence that the cross-linked polymers of insoluble Col I are depolymerized by MMP-9 [23]. Moreover, targeted deletion of the MMP-9 gene prevented the LV enlargement caused by myocardial infarction [3]. In human dilated cardiomyopathy, there was an increased myocardial abundance of MMP-3 and MMP-9, which may contribute to the abnormal LV remodeling [33].

To exclude the possibility that the increased MMP production was from possible contamination of circulating blood, the hearts were perfused with saline before harvesting. Moreover, in-situ hybridization in combination with immunohistochemistry identified the sources of increased MMP production were mainly infiltrated macrophages located at myocardial interstitial areas (data not shown). However, we can not rule out the effects of other MMP family members and other proteases such as cathepsin K [42], which may also be activated to perform collagenolytic activity during CVB3-induced inflammatory reaction. Another limitation of this study is that the other matrix components were not adequately addressed. We focused very heavily on collagen type I (Col I) which is understandable for several reasons. First, the reagents that work well on mice and reliably work well in the mice, are scarce. Second, Col I can be picked up a little easier with immunostaining. Thirdly, Col I, which is the predominant Col type characterized by tensile strength, plays a very important role in providing myocardial wall mechanics and supporting the structural integrity of the heart. Since the proteolytic targets of increased MMP-3 and MMP-9 include many matrix components such as proteoglycans [17], collagen type III, IV and V [22,37], the other degradative events involving these matrix components may also occur, which may play a role in inducing an impairment of cardiac function. On the other hand, as CVB3 persistence has been confirmed by nested RT-PCR, we can not exclude the possibility that unidentified proteases produced by virus itself may be directly inducing collagen degradation. Such unexplored question remains to be further investigated.

TIMPs bind to the active sites of the MMPs, thereby blocking their access to extracellular matrix substrates. TIMP-1 can form complexes with MMP-3 and MMP-9 [5,32], and TIMP-4 can effectively inhibit MMP-3 and MMP-9 [14]. In addition, the downregulation of TIMPs in human heart failure is associated with myocardial Col degradation [15].

Taken together, the present study suggests, that increased myocardial MMP-3 and MMP-9 in conjunction with reduced TIMP-1 and TIMP-4 may contribute to an abnormal degradation of the native insoluble Col network mainly at the posttranslational level, by cleaving the cross-links between Col molecules. Similar mechanisms by which MMPs mediate a functional defect of the myocardial Col network have been suggested by two previous studies demonstrating in human dilated cardiomyopathy increased MMPs are associated with a significant decrease in mature cross-linked Col or in the ratio of insoluble to soluble Col [6,16]. Thus, the mechanism by which the increased MMPs in the absence of counterbalance by TIMPs may induce a functional defect of the myocardial Col network at the posttranslational level, which may contribute to the development of LV dysfunction in different forms of heart diseases.

4.3 Inflammatory induction and MMPs/TIMPs system
The increased mRNA abundances of myocardial IL-1β, TNF-{alpha}, TGF-β1 and IL-4 were detected in association with upregulated MMPs and downregulated TIMPs. Many previous studies have shown that an inflammatory reaction is the prerequisite for the abnormal remodeling of the myocardial matrix and the development of LV dysfunction in this murine myocarditis model [38–40]. Cytokines are important regulators of the MMPs/TIMPs systems. In cardiac TNF transgenic mice, the overexpression of TNF upregulated the MMP activity, but downregulated the expression of TIMP-1 during the early LV structural remodeling [29]. IL-1β can upregulate MMP-3 and MMP-9 at the mRNA and protein level in cardiac fibroblasts [30], but inhibit TIMP production in endothelial cells [28]. Furthermore, MMP-3 transcription regulatory elements have been identified as the IL-1 responsive element site, which is induced by IL-1 and TNF, but not IL-4 [2]. In addition, in vitro studies indicate that IL-4 alone, and TGF-β1 in combination with other cytokines, can suppress the expression of MMPs in mononuclear infiltrates and fibroblasts [4,10,13]. Although TGF-β1 and IL-4 were upregulated, the increased inflammatory cytokines IL-1β and TNF-{alpha} may dominate in a predominantly Th1 cell phenotypic response in CVB3-infected BALB/c mice [8,9]. The combined effect of myocardial IL-1β, TNF-{alpha}, TGF-β1 and IL-4 induction may indirectly induce myocardial Col remodeling by the upregulation of MMPs and downregulation of TIMPs. A better understanding of the regulation of the MMP/TIMP systems by inflammatory cytokines may allow the development of new therapeutic strategies. The regulation of the myocardial MMP/TIMP systems and Col remodeling under the immunomodulation by anti-inflammatory cytokines warrants further investigation.


   5. Conclusion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusion
References
 
The current findings suggest, that the increment of MMPs in the absence of counterbalance by TIMPs may lead to a functional defect of the myocardial Col network mainly by posttranslational mechanisms, which may contribute significantly to the development of LV dysfunction in CVB3-induced myocarditis mice. The inflammatory response with induction of IL-1β, TNF-{alpha}, TGF-β1 and IL-4 may indirectly mediate myocardial Col remodeling by upregulation of MMPs and downregulation of TIMPs.

Time for primary review: 24 days.


   Acknowledgements
 
This study was supported by the Deutsche Forschungsgemeinschaft (DFG Pa 369/2-1, Pa 369/2-3, Pa 369/3-1).


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusion
 References
 

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