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Left ventricular enlargement in coxsackievirus-B3 induced chronic myocarditis — ongoing inflammation and an imbalance of the matrix degrading system
European Journal of Pharmacology 630 (2010) 145–151
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European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / e j p h a r
Immunopharmacology and Inflammation
Left ventricular enlargement in coxsackievirus-B3 induced chronic myocarditis — ongoing inflammation and an imbalance of the matrix degrading system Susanne Rutschow a,⁎,1, Sebastian Leschka c,1, Dirk Westermann a, Kerstin Puhl a, Anneke Weitz a, Leonid Ladyszenskij a, Sebastian Jaeger a, Heinz Zeichhardt b, Michel Noutsias a, Heinz-Peter Schultheiss a, Carsten Tschope a, Matthias Pauschinger d a
Department of Cardiology and Pneumonology, Medizinische Klinik II, Charité – Universitätsmedizin Berlin, Campus Benjamin Franklin, Hindenburgdamm 30, D-12200 Berlin, Germany Institute of Infectious Diseases Medicine, Charité – Universitätsmedizin Berlin, Campus Benjamin Franklin, Berlin, Germany c Institute of Diagnostic Radiology, University Hospital of Zurich, Zurich, Switzerland d Department of Cardiology, Medizinische Klinik 8, Klinikum Nürnberg Süd, Nürnberg, Germany b
a r t i c l e
i n f o
Article history: Received 6 May 2009 Received in revised form 3 December 2009 Accepted 15 December 2009 Available online 24 December 2009 Keywords: Myocarditis DCM Matrix MMP Inflammation
a b s t r a c t Enteroviruses, especially Coxsackie B3 virus (CVB-3), cause acute viral myocarditis, but the detailed mechanisms leading to chronic left ventricular dysfunction and dilatation remain elusive. Myocardial tissues of CVB-3 infected and sham infected male swr/J mice were analyzed after hemodynamic evaluation on days 4, 7, and 28 p.i. by RT-PCR, gelatin zymography, ELISA, immunohistochemistry, sirius red staining, and luxol fast blue staining. In the early phase after infection an abnormal diastolic function was the main hemodynamic finding. CVB-3 infection caused impairment of left ventricular function combined with ventricular dilatation 7 and 28 days post-infection. These hemodynamic findings were associated with relevant upregulation of different cytokines (IL-1β, IL-6, IL-10, INF-γ, and TNF-α) in the acute phase with persistent over-expression of IL-6, IL-10, and INF-γ in the chronic phase. This virus induced myocardial inflammation was linked to a significant induced MMP/TIMP system (MMP-2,-3,-8, TIMP-1, uPA, tPA–mRNA expression, and MMP-2-activity) in the acute and chronic phase leading to imbalance in the MMP/TIMP-ratio at day 28. This imbalance in the MMP/TIMP system was significantly correlated to the development of ventricular dilatation. Viral persistence induces chronic myocardial inflammation and an imbalance of the matrix degrading system, associated with the development of left ventricular dysfunction and dilatation in chronic murine myocarditis. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Acute myocarditis is often caused by cardiotropic viruses in men (Kuhl et al., 2003). In addition, viral persistence and chronic inflammation are present in the majority of patients presenting with dilated cardiomyopathy (Kuhl et al., 2005; Noutsias et al., 1999), and patients with dilated cardiomyopathy with viral persistence have a worse prognosis (Why et al., 1994). The detailed mechanisms responsible for the progressive deterioration of left ventricular function and dilatation are still elusive though. Coxsackie B virus (CVB) infection of susceptible mice induces acute myocarditis at days 7 to 14 post-infection, which later progresses to a chronic phase (Fairweather et al., 2001). After infection with CVB-3, SWR/J mice typically develop a persistent infection with ongoing ⁎ Corresponding author. Tel.: +49 30/84452349; fax: +49 30/84454648. E-mail address: [email protected] (S. Rutschow). 1 The first two authors contributed equally to this work. 0014-2999/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2009.12.019
chronic inflammation as also observed in patients (Klingel et al., 1992). Acute left ventricular dysfunction without dilatation in experimental induced myocarditis is associated with induction of proinflammatory cytokines, intramyocardial inflammation, and an imbalance of the matrix metalloproteinase (MMP) and the tissue inhibitors of MMP (TIMP) system (Li et al., 2002). The pathogenic importance of the regulation and balance of the MMP/TIMP system and the ECM-turnover in correlation to the development of acute to chronic myocarditis with dilated ventricle and left ventricular dysfunction are poorly understood, particularly in SWR/J mice. The extracellular matrix (ECM) proteins, especially the fibrillar collagens, are crucial for the structural integrity of the heart. Modulation of the balance between ECM-synthesis and ECMdegradation is an important process of myocardial remodeling and left ventricular function. Proinflammatory cytokines such as TNF-α and IL-1β contribute not only to depression of the left ventricular function and cardiomyocyte loss by apoptosis, they also play a critical role in maintaining the balance in ECM remodeling (Siwik et al., 2000;
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Mauviel, 1993). In the present study, we investigated the association between left ventricular dilatation, intramyocardial inflammation, components of the ECM and components of the MMP/TIMP system in the chronic phase of myocarditis. 2. Methods 2.1. Virus and animals Pathogen-free, inbred, 6 week old, male swr/J mice obtained from Jackson Laboratory (Bar Harbor, ME, USA), were used for the experiments described in this study (n = 60). Animals (n = 60) were infected and sham infected as described previously (Li et al., 2002). Suitable controls and infected animals were maintained and sacrificed in accordance with the German Animal Protection Law of 1993. 2.2. Hemodynamic characterization of left ventricular function Four, seven, and twenty-eight days after infection, hemodynamic parameters were measured in anesthetized, intubated, artificially ventilated, and closed-chest animals as previously described (Li et al., 2002; Tschope et al., 2004). Systolic function was quantified by left ventricular end-systolic pressure (LVESP), dP/dt max as an index of left ventricular contractility, ejection fraction, end-systolic volume (LVESV), stroke volume, cardiac output, and heart rate. Diastolic function was measured by left ventricular end-diastolic pressure (LVEDP) and dP/dt min. Reliable hemodynamic data could be determined in a subgroup of animals: controls (day 4 (C 4 day) n = 6), controls (day 7 (C 7 day) n = 6), controls (day 28 (C 28 day) n = 6), infected (day 4 (I 4 day) n = 7), infected (day 7 (I 7 day) n = 7), and infected (day 28 (I 28 day) n = 9). 2.3. RNA isolation and reverse transcription Immediately after the hemodynamic parameters were obtained, the hearts were rapidly removed, transversely dissected, and myocardial tissue was collected for various measurements. Total RNA extraction and reverse transcription were performed as previously described (Li et al., 2002). 2.4. Reverse transcriptase-polymerase chain reaction Semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was used to detect the mRNA abundance of MMP-2,-3,-8, extracellular-MMP-inducer, TIMP-1, uPA (urokinase type plasmin activator), tPA (tissue type plasmin activator), IL-6, INF-γ, IL-1β, TNF-α, IL-10, collagen type I, collagen type III, collagen type IV, and CVB-3 genome, according to the methods described previously (Li et al., 2002; Pauschinger et al., 1999a,b).
2.6. Zymography of MMP activity Gelatin zymography was performed to determine the gelatinolytic activities of MMP-2 and MMP-9 as described before (Li et al., 2007). The gelatinolytic activities were detected as clear bands against a blue background and analyzed using Scion Image software as relative optical densities. 2.7. Luxol fast blue staining Luxol fast blue staining was performed in order to determine myocytolysis in myocardial tissue as described before (Li et al., 2007). The area percent of myocytolysis in different sections was measured by digital image analysis (Lucia G, Version 3.51) adapted to previously published techniques (Noutsias et al., 2002, 2003). 2.8. ELISA IL-1β, IL-6, and Interferon-γ were determined by ELISA from (Pharmingen/BD Biosciences, San Diego, CA, USA) from myocardial tissue according to the manufacturer's instructions. All assays were done twice, and the absorbance values were equalized with absorbance values of GAPDH (Active Motif, Carlsbad, CA, USA). 2.9. Sirius red staining Picrosirius red staining was used for the histological assessment of the total collagen content 5 μm thick paraffin embedded tissue as previously described (Pauschinger et al., 1999a,b). Total collagen was measured as the area fraction (AF) as described previously (Li et al., 2002). 2.10. Statistical analysis Statistical analysis was performed using SPSS (SPSS Inc., Chicago, IL, USA). All data are expressed as S.E.M. Significant differences in quantitative parameters across the different groups were determined by Kruskal–Wallis test followed by Mann–Whitney–U test. Bivariate correlations were performed by Pearson's correlation. Values of P b 0.05 were considered as statistically significant. 3. Results 3.1. Left ventricular function of CVB-3-infected mice After diastolic dysfunction at 4 day post-infection (LVEDP: 198%; P b 0.05; versus control), left ventricular systolic dysfunction developed at 7 and 28 days post-infection with significantly increased LVESV (7 days p.i.: 180%, 28 days p.i.: 182%; and P b 0.05) and LVEDV (7 days p.i. 121% and 28 days p.i. 121%; P b 0.05) and significantly reduced left ventricular ejection fraction (7 days p.i.: 63%; P b 0.05 and 28 days p.i.: 68%; P b 0,05) (Fig. 1A–D). Furthermore dP/dt min was significantly reduced at 28 days p.i. without any effects of cardiac heart rate (data not shown).
2.5. Immunohistochemistry 3.2. Course of CVB-3 genomes Immunohistochemical analysis for CD3-T-lymphocytes was performed as described previously (Li et al., 2002). Immunocompetent infiltrates were quantified by digital analysis (Lucia G, Version 3.51) as described elsewhere (Noutsias et al., 2002, 2003). For quantitative analysis, the coded slides were evaluated in a blinded fashion. Total number of CD3 cells in different sections was counted at 200× accounted 0.39 mm2. For each sample, six tissue sections (3 sections per slide, 2 slides per heart) were evaluated. The mean cell number per mm² was calculated and represented as extent of intramyocardial inflammation.
PCR analysis confirmed the presence of the CBV-3 genomes in the myocardium of all infected mice at day 4 and 7 p.i., whereas CBV-3 was proven only in 4 animals (40%) at days 28 p.i. In contrast, all sham infected hearts were CBV-3 negative. 3.3. Myocardial cytokine profiles Semi-quantitative RT-PCR revealed a significant upregulation of the mRNA abundances of IL-1β by 290% (P b 0.0001), of INF-γ by 200%
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Fig. 1. Hemodynamic characterization on days 4, 7, and 28 after CVB-3 infection in swr/J mice; LVEDP (A), dP/dt max, dP/dt min (B), LVEDV, LVESV, stroke volume (C), and ejection fraction (D). Data are expressed as S.E.M. and P b 0.05 was considered as significant.
(P b 0.0001), of IL-6 by 200% (P b 0.0001), and of IL-10 by 208% (P b 0.0001) in the myocardium of infected mice compared to controls at day 4 p.i. (Fig. 2A). On the protein level, we found a significant induction of IL-6 by 334% (P b 0.05) and INF-γ by 145%. Likewise at day 7 p.i., cytokines was significantly upregulated (IL-1β by 154% (P b 0.05), INF-γ by 208% (P b 0.0001), IL-6 by 276% (P b 0.0001), IL-10 by 271% (P b 0.0001), and TNF-α by 193% (P b 0.0001)) (Fig. 2A). At the protein level we found a significant induction of IL-6 by 241% (P b 0.05) and INF-γ by 159% (P b 0.05) in infected mice versus controls. Also in the chronic phase at day 28 p.i., we detected a significant upregulation of the mRNA abundances of INF-γ by 153% (P b 0.001), of IL-6 by 148% (P b 0.001), and of IL-10 by 150% (P b 0.001) compared to controls with significantly increased protein levels of IL-6 by 262% (P b 0.05) and IL-1β by 156% (P b 0.05, Fig. 2).
3.4. MMP/TIMP profiles In the MMP/TIMP system, we found a significant induction of the mRNA abundances of MMP-3 by 376% (P b 0.002), of extracellularMMP-inducer by 163% (P b 0.0001) and of TIMP-1 by 335% (P b 0.0001) compared to controls (Fig. 2B). Furthermore, the mRNA abundance of tPA increased significantly by 147% (P b 0.001, Fig. 2C). At day 7 p.i. we found a significant upregulation of the mRNA abundances of MMP-2 by 124% (P b 0.007), of MMP-3 by 452% (P b 0.0001), of MMP-8 by 305% (P b 0.0001) and of TIMP-1 by 409% (P b 0.0001) compared to controls, as well as a significant upregulation of MMP-3 by 163% (P b 0.03) and of MMP-8 by 196% (P b 0.0001) compared to day 4 p.i. Additionally, we found an upregulation of tPA by 147% (P b 0.007) and uPA by 173% (P b 0.001) compared to controls (Fig. 2C). MMP-2 activity, showed a significant increase by 129% (P b 0.03) compared to controls (Fig. 3D). At day 28 p.i. we could show a significant increase of the mRNA abundance of MMP-2 by 142%, (P b 0.035) and a significant reduction of TIMP-1 to 73% (P b 0.007) compared to controls (Fig. 2B). The MMP-3/TIMP-1 mRNA ratio showed a significant upregulation at 28 days p.i. by 276% (P b 0.019, Fig. 2B). Furthermore, tPA (229%, P b 0.0001) and uPA (211%, (P b 0.0001)) were significantly upregulated compared to controls
(Fig. 2C). The MMP-2 activity analysis revealed an increase at 143% (P b 0.001) compared to controls (Fig. 2D). 3.5. Myocardial collagen content at 4 day post-infection On day 4 p.i., we observed a significant downregulation of collagen III mRNA abundance by 74% (P b 0.03) compared to controls (Fig. 3A). On day 7 p.i., we also found a downregulation of collagen III mRNA abundances by 67% (P b 0.02) compared to controls, with nonsignificant trend towards an increase of collagen IV mRNA abundance, at an unchanged level of collagen I mRNA abundance. These changes on the mRNA-level were associated with a significant upregulation of total collagen content by 208% (P b 0.006) compared to controls and by 152% (P b 0.001) compared to 4. day p.i. (Fig. 3A/B). On day 28 p.i., we observed a significant upregulation of collagen I mRNA abundances by 127% (P b 0.03) and collagen IV by 128% (P b 0.05), and a downregulation of collagen III by 77% (P b 0.01). These changes on the mRNA-level were linked to significantly increased total collagen content by 208% on the protein level (P b 0.001). This led to an increased collagen I/III ratio on the mRNA-level at day 28 postinfection by 166% (P b 0.05). 3.6. T-lymphocytic infiltration and myocytolysis CVB-3 infection led to a significant intramyocardial CD3+ T-lymphocytic infiltration at 7 days p.i. (1345%, P b 0.008) and at 28 days p.i. (247%; P b 0.1) compared to controls (Fig. 4A/B) and also to a significantly increased myocytolysis at 7 day p.i. (2880%, P b 0.002) and 28 day p.i. (761%; P b 0.002) compared to controls (Fig. 5A/B). 3.7. Associations between cytokine expression, MMP expression, myocardial cell infiltration, and myocardial function The mRNA abundance of IL-6, IL-10, and INF-γ as well as the myocardial content of CD-3 cells were significantly correlated to the expression of MMP-3, MMP-8, and TIMP-1, and myocytolysis (P b 0.05) (Table 1). We found a significant correlation between the mRNA abundances of MMP-8 and left ventricular end-systolic volume and stroke volume. There was a significant correlation between mRNA
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Fig. 2. Abundance of cytokine mRNAs (A), MMP/TIMP mRNAs (B), and uPA/tPA mRNAs (C) measured by semi-quantitative RT-PCR, and MMP-2 activity (D) measured by gelatin zymography.
abundance of uPA and left ventricular diameters and ejection fraction. We also found a significant correlation between mRNA abundance of tPA and ejection fraction. Furthermore the increased MMP activity was correlated to the impairment of stroke volume and ejection fraction and the increased left ventricular end-systolic diameters, thus underlining the importance of left ventricular extracellular matrix remodeling in the development of left ventricular dysfunction (Table 2). 4. Discussion This study in a murine CVB-3 myocarditis model in SWR/J mice showed after the acute phase of infection an ongoing activated immune process with subsequent viral persistence and persisting myocardial inflammation. This ongoing inflammatory process is associated with an imbalance of the MMP/TIMP system, progression
of left ventricular dilatation, and reduction of left ventricular systolic function. The relevance of the MMP/TIMP imbalance for the progression of left ventricular dilatation and impairment of left ventricular dysfunction is shown by the documented correlation between uPA–mRNA expression, MMP-2 activity and LVESV, as well as ejection fraction. Therefore, the imbalance of the matrix degrading system may cause a loss of structural integrity of the heart and may be an important factor for an ongoing impairment of left ventricular function with subsequent ventricular dilatation in chronic myocarditis. 4.1. Hemodynamic characterization In CVB-3 murine myocarditis, hemodynamic changes differ during the three phases of viral myocarditis. A diastolic dysfunction with an elevated LVEDP, but no impairment of systolic parameters such as
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Fig. 3. Total collagen content measured by sirius red staining (A/B).
ejection fraction was found on day 4 (acute phase). This diastolic dysfunction may be induced by an interstitial edema of the left ventricel, also indicated by a temporary reduction in LVEDV, as it is also well-known for patients with acute myocarditis. Diastolic dysfunction in the acute phase may also be caused by direct viral effects with focal necrotic myofibers without changes in myocardial inflammation and remodeling (Shioi et al., 1996). Hemodynamically, the subacute phase is characterized by impaired systolic and diastolic function and by an increase in left ventricular chamber diameters, according to the clinical diagnosis of
dilated cardiomyopathy. Alterations of ventricular function are likely to have different causes in ongoing myocarditis. The subacute phase is characterized by the onset of inflammatory infiltrations, cytotoxic virological effects, along with the inflammatory response, results in myocytolysis and early changes of ECM components. In the chronic phase of myocarditis, viral persistence triggers an ongoing inflammatory process. This in turn may cause an imbalance of the MMP/TIMP system, which would lead to a progressive breakdown of the extracellular matrix components, which might cause impairment of ventricular function.
Fig. 4. CD3-T-lymphocytes (A). A representative immunohistochemical staining of control heart sections and CVB-3 infected heart sections (B) on day 7 p.i. Original magnifications, ×20.
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Fig. 5. Luxol fast blue staining (A). Representative control and CVB-3 infected sections (B) on day 28. p.i.
4.2. Inflammatory response For the pathogenesis of myocarditis, Kawai (1999) described a triphasic model of the pathogenesis of myocarditis with acute, subacute, and chronic phases. The acute phase is characterized by the early release of proinflammatory cytokines, such as IL-1β and interferon-γ (Shioi et al., 1996; Matsumori, 1997), with the first appearance of histological changes in the form of myocyte necrosis and apoptosis (Rose and Hill, 1996; Wilson et al., 1969). In this phase, cellular infiltrations are barely detected and usually consist of natural killer cells and macrophages (Seko et al., 1991; Feldman and McNamara, 2000). Consequently, on the 4th day of infection, we found an increased expression of proinflammatory cytokines, including IL-1β, IL-6, IL-10, and Interferon-γ on the mRNA and protein levels, with no significant changes in myocardial T-lymphocytes infiltration. During the subacute phase (day 7), we observed the maximum peaks of IL-2, TNF-α, INF-γ, and IL-10 expression. The inflammatory response with elevated inflammatory cytokines and myocardial T-cell infiltration persisted at a lower intensity to day 28 post-infection. This inflammatory response was associated with a progressive increase of myocytolysis from day 4 to day 28 p.i. (Arnold et al., 1985). The impact of the inflammatory response on myocardial necrosis is once again demonstrated by the significant correlation between myocytolysis and myocardial cellular infiltration (Table 1). 4.3. Extracellular matrix turnover Disruption of the ECM is an important factor in the transition of acute myocarditis to dilated cardiomyopathy (Pauschinger et al., 1999a,b; D'Armiento, 2002; Thomas et al., 1998). As inflammatory
Table 1 Pearson's correlation between inflammation and the MMP/TIMP system from all timepoints. Expressed by r and a: P b 0.05 and b: P b 0.001.
INF-γ IL-6 Il-10 CD-3
MMP-8
MMP-3
TIMP-1
MFD
0.687a 0.732b 0.708b 0.638a
0.823b 0.694a 0.718b 0.469a
0.701a 0.705a 0.717b 0.465a
0.447a 0.587a 0.581a 0.718b
cytokines are involved in the tightly regulated system of collagen expression and degradation, increased inflammatory response is likely to contribute to a pathologic collagen turnover. Proinflammatory cytokines regulate the expression of ECM components such as collagen types I and III and fibronectin (Bouluyt et al., 1994). They also induce the expression of MMP's and plasminogen activators (Siwik et al., 2000; Mauviel, 1993), which causes a degradation of the ECM. Over-expression of TNF-α and IL-1β correlates with an increased expression of MMPs and reduced expression of TIMPs. Furthermore, inhibition of TNF-α leads to a reduced expression and activity of MMPs, with improved left ventricular function (Bradham et al., 2002). These findings agree with our data, which demonstrates a correlation between proinflammatory cytokine expression (IL-1β and TNF-α) and the expression of MMPs (Table 1). Li et al. (2002)have reported an imbalance of the MMP/TIMP system in the acute phase of myocarditis resulting in a functional defect of the ECM without an increase of total collagen. In addition, Woodiwiss et al., 2001. have demonstrated that a reduction in collagen cross-links from increased MMP activity is responsible for an increase of soluble collagen, which results in left ventricular dysfunction, remodeling, and chamber dilatation. This study analyzed different phases of myocarditis (Rutschow et al., 2006). We were able to demonstrate an imbalance in the matrix degradation system, with induced expression of MMPs over the acute, subacute, and chronic phases and a significant increase in the MMP/TIMP-ratio in the chronic phase. This imbalance was also shown by Cheung et al. (2005) in CVB-3 infected A/J mice. The increase in MMPs expression and activity were significantly correlated with development of left ventricular dilatation and reduced stroke volume and ejection
Table 2 Pearson's correlation between matrix degradation system and left ventricular function from all timepoints. Expressed by r and a: P b 0.05 and b: P b 0.001.
MMP-3 MMP8 MMP-2 activity uPA
EF
VES
SV
0.396a 0.396a 0.458a 0.493a
0.312 0.407a 0.551b 0.539b
0.353a 0.388a 0.431a 0.230
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References
Fig. 6. Possible signal pathway in viral myocarditis associated induction of inflammation resulting in matrix degradation.
fraction, underlining the importance of matrix integrity and left ventricular function. Plasminogen activators (urokinase type (uPA) and tissue type (tPA)) take more interest in ECM remodeling, due to activation of proMMP (Li et al., 2007; Heymans et al., 2006). In the model of chronic myocarditis used in this study, uPA and tPA increased significantly over 7 days with no relevant decrease to day 28 p.i. Therefore, the elevated ability of MMP-activation also in the chronic phase of myocarditis is observed by induced MMP-2 activity. Further studies suggest that an active plasminogen–MMP system is essential for the ability of immunocompetent cell to infiltrate tissues. In a murine model of myocardial infarction in plasminogen knockout mice, inflammatory cells were unable to infiltrate the infarct zone, resulting in reduced fibrosis and cellular colonization (Creemers et al., 2000). Also in our model of chronic myocarditis, the increase in uPA–mRNA expression was significantly correlated with myocardial CD-3 infiltration and left ventricular dysfunction, underlining the importance of the activation of MMPs to the development of left ventricular dilatation. 5. Conclusions In CVB-3 induced chronic myocarditis, viral persistence, chronic inflammation, and their influence on the extracellular matrix lead to left ventricular dysfunction with dilated ventricle and reduced left ventricular ejection fraction, resembling human dilated cardiomyopathy. The chronic inflammatory response and ECM remodeling are the pathways involved in this pathogenesis. The imbalance of the matrix degrading system with induced expression of MMPs and plasminogen activators, as well as the reduced expression of TIMPs in the chronic phase, suggest a pathologic collagen turnover, leading to a loss of structural integrity of the heart and an impairment of left ventricular function and dilatation (Fig. 6). In light of these insights, pharmacologic intervention with inhibiting MMPs or plasminogen activators might be promising approaches to abrogate the progressive left ventricular dysfunction in chronic myocarditis with CVB-3 persistence. Acknowledgement This research was supported by the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich Transregio 19, A2). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ejphar.2009.12.019.
Arnold, G., Kaiser, C., Fischer, R., 1985. Myofibrillar degeneration — a common type of myocardial lesion and its selective identification by a modified luxol fast blue stain. Pathol. Res. Pract. 180, 405–415. Bouluyt, M.C., O'Neil, L., Mededith, A.L., 1994. Alteration in cardiac gene expression during the transition from stable hypertrophy to heart failure: Marked upregulation of genes encoding extracellular components. Circ. Res. 75, 25–32. Bradham, W.S., Moe, G., Wendt, K.A., 2002. TNF-α . Am. J. Physiol. Heart Circ. Physiol. 282, H1288–H1295. Cheung, C., Luo, H., Yanagawa, B., 2006. Matrix metalloproteinases and tissue inhibitors of metalloproteinases in coxsackievirus-induced myocarditis. Cardiovasc. Pathol. 15, 63–74. Creemers, E., Cleutjens, J., Smits, J., 2000. Disruption of the plasminogen gene in mice abolishes wound healing after myocardial infarction. Am. J. Pathol. 156, 1865–1873. D'Armiento, J., 2002. Matrix metalloproteinase disruption of the extracellular matrix and cardiac dysfunction. Trends Cardiovasc. Med. 12, 97–101. Fairweather, D., Kaya, Z., Shellam, G.R., 2001. From infection to autoimmunity. J. Autoimmun. 16, 175–186. Feldman, A.M., McNamara, D., 2000. Myocarditis. N. Engl. J. Med. 9 (343), 1388–1398. Heymans, S., Pauschinger, M., Palma, A., 2006. Inhibition of urokinase-type plasminogen activator or matrix metalloproteinases prevents cardiac injury and dysfunction during viral myocarditis. Circulation 114, 565–573. Kawai, C., 1999. From myocarditis to cardiomyopathy: Mechanisms of inflammation and cell death. Learning from the past to the future. Circulation 96, 3549–3554. Klingel, K., Hohenadl, C., Canu, A., 1992. Ongoing enterovirus-induced myocarditis is associated with persistent heart muscle infection: quantitative analysis of virus replication, tissue damage, and inflammation. Proc. Natl. Acad. Sci. U. S. A. 89, 314–318. Kuhl, U., Pauschinger, M., Bock, T., 2003. Parvovirus B19 infection mimicking acute myocardial infarction. Circulation 108, 945–950. Kuhl, U., Pauschinger, M., Noutsias, M., 2005. High prevalence of viral genomes and multiple viral infections in the myocardium of adults with “idiopathic” left ventricular dysfunction. Circulation 111, 887–893. Li, J., Schwimmbeck, P.L., Tschope, C., 2002. Collagen degradation in a murine myocarditis model: relevance of matrix metalloproteinase in association with inflammatory induction. Cardiovasc. Res. 56, 235–247. Li, J., Leschka, S., Rutschow, S., 2007. Immunomodulation by interleukin-4 suppresses matrix metalloproteinases and improves cardiac function in murine myocarditis. Eur. J. Pharmacol. 554, 60–68. Matsumori, A., 1997. Molecular and immune mechanisms in the pathogenesis of cardiomyopathy: role of viruses, cytokines and nitric oxide. Jpn. Circ. J. 61, 275–291. Mauviel, A., 1993. Cytokine regulation of metalloproteinase gene expression. J. Cell. Biochem. 53, 288–295. Noutsias, M., Seeberg, B., Schultheiss, H.P., Kuhl, U., 1999. Expression of cell adhesion molecules in dilated cardiomyopathy: evidence for endothelial activation in inflammatory cardiomyopathy. Circulation 99, 2124–2131. Noutsias, M., Pauschinger, M., Ostermann, K., 2002. Digital image analysis system for the quantification of infiltrates and cell adhesion molecules in inflammatory cardiomyopathy. Med. Sci. Monit. 8, MT59–MT71. Noutsias, M., Hohmann, C., Pauschinger, M., 2003. sICAM-1 correlates with myocardial ICAM-1 expression in dilated cardiomyopathy. Int. J. Cardiol. 91, 153–161. Pauschinger, M., Doerner, A., Kuehl, U., 1999a. Enteroviral RNA replication in the myocardium of patients with left ventricular dysfunction and clinically suspected myocarditis. Circulation 99, 889–895. Pauschinger, M., Knopf, D., Petschauer, S., 1999b. Dilated cardiomyopathy is associated with significant changes in collagen type I/III ratio. Circulation 99, 2750–2756. Rose, N.R., Hill, S.L., 1996. The pathogenesis of postinfectious myocarditis. Clin. Immunol. Immunopathol. 80, S92–S99. Rutschow, S., Li, J., Schultheiss, H.P., Pauschinger, M., 2006. Myocardial proteases and matrix remodeling in inflammatory heart disease. Cardiovasc. Res. 69, 646–656. Seko, Y., Shinkai, Y., Kawasaki, A., 1991. Expression of perforin in infiltrating cells in murine hearts with acute myocarditis caused by coxsackievirus B3. Circulation 84, 788–795. Shioi, T., Matsumori, A., Sasayama, S., 1996. Persistent expression of cytokine in the chronic stage of viral myocarditis in mice. Circulation 94, 2930–2937. Siwik, D.A., Chang, D.L., Colucci, W.S., 2000. Interleukin-1beta and tumor necrosis factor-alpha decrease collagen synthesis and increase matrix metallopro-teinase activity in cardiac fibroblasts in vitro. Circ. Res. 86, 1259–1265. Thomas, C.V., Coker, M.L., Zellner, J.L., 1998. Increased matrix metallo-proteinase activity and selective upregulation in LV myocardium from patients with endstage dilated cardiomyopathy. Circulation 97, 1708–1715. Tschope, C., Westermann, D., Steendijk, P., 2004. Hemodynamic characterization of left ventricular function in experimental coxsackieviral myocarditis: effects of carvedilol and metoprolol. Eur. J. Pharmacol. 491, 173–179. Why, H.J., Meany, B.T., Richardson, P.J., 1994. Clinical and prognostic significance of detection of enteroviral RNA in the myocardium of patients with myocarditis or dilated cardiomyopathy. Circulation 89, 2582–2589. Wilson, F.M., Miranda, Q.R., Chason, J.L., 1969. Residual pathologic changes following murine coxsackie A and B myocarditis. Am. J. Pathol. 55, 253–265. Woodiwiss, A.J., Tsotetsi, O.J., Sprott, S., 2001. Reduction in myocardial collagen crosslinking parallels left ventricular dilatation in rat models of systolic chamber dysfunction. Circulation 103, 155–160.
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European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / e j p h a r
Immunopharmacology and Inflammation
Left ventricular enlargement in coxsackievirus-B3 induced chronic myocarditis — ongoing inflammation and an imbalance of the matrix degrading system Susanne Rutschow a,⁎,1, Sebastian Leschka c,1, Dirk Westermann a, Kerstin Puhl a, Anneke Weitz a, Leonid Ladyszenskij a, Sebastian Jaeger a, Heinz Zeichhardt b, Michel Noutsias a, Heinz-Peter Schultheiss a, Carsten Tschope a, Matthias Pauschinger d a
Department of Cardiology and Pneumonology, Medizinische Klinik II, Charité – Universitätsmedizin Berlin, Campus Benjamin Franklin, Hindenburgdamm 30, D-12200 Berlin, Germany Institute of Infectious Diseases Medicine, Charité – Universitätsmedizin Berlin, Campus Benjamin Franklin, Berlin, Germany c Institute of Diagnostic Radiology, University Hospital of Zurich, Zurich, Switzerland d Department of Cardiology, Medizinische Klinik 8, Klinikum Nürnberg Süd, Nürnberg, Germany b
a r t i c l e
i n f o
Article history: Received 6 May 2009 Received in revised form 3 December 2009 Accepted 15 December 2009 Available online 24 December 2009 Keywords: Myocarditis DCM Matrix MMP Inflammation
a b s t r a c t Enteroviruses, especially Coxsackie B3 virus (CVB-3), cause acute viral myocarditis, but the detailed mechanisms leading to chronic left ventricular dysfunction and dilatation remain elusive. Myocardial tissues of CVB-3 infected and sham infected male swr/J mice were analyzed after hemodynamic evaluation on days 4, 7, and 28 p.i. by RT-PCR, gelatin zymography, ELISA, immunohistochemistry, sirius red staining, and luxol fast blue staining. In the early phase after infection an abnormal diastolic function was the main hemodynamic finding. CVB-3 infection caused impairment of left ventricular function combined with ventricular dilatation 7 and 28 days post-infection. These hemodynamic findings were associated with relevant upregulation of different cytokines (IL-1β, IL-6, IL-10, INF-γ, and TNF-α) in the acute phase with persistent over-expression of IL-6, IL-10, and INF-γ in the chronic phase. This virus induced myocardial inflammation was linked to a significant induced MMP/TIMP system (MMP-2,-3,-8, TIMP-1, uPA, tPA–mRNA expression, and MMP-2-activity) in the acute and chronic phase leading to imbalance in the MMP/TIMP-ratio at day 28. This imbalance in the MMP/TIMP system was significantly correlated to the development of ventricular dilatation. Viral persistence induces chronic myocardial inflammation and an imbalance of the matrix degrading system, associated with the development of left ventricular dysfunction and dilatation in chronic murine myocarditis. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Acute myocarditis is often caused by cardiotropic viruses in men (Kuhl et al., 2003). In addition, viral persistence and chronic inflammation are present in the majority of patients presenting with dilated cardiomyopathy (Kuhl et al., 2005; Noutsias et al., 1999), and patients with dilated cardiomyopathy with viral persistence have a worse prognosis (Why et al., 1994). The detailed mechanisms responsible for the progressive deterioration of left ventricular function and dilatation are still elusive though. Coxsackie B virus (CVB) infection of susceptible mice induces acute myocarditis at days 7 to 14 post-infection, which later progresses to a chronic phase (Fairweather et al., 2001). After infection with CVB-3, SWR/J mice typically develop a persistent infection with ongoing ⁎ Corresponding author. Tel.: +49 30/84452349; fax: +49 30/84454648. E-mail address: [email protected] (S. Rutschow). 1 The first two authors contributed equally to this work. 0014-2999/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2009.12.019
chronic inflammation as also observed in patients (Klingel et al., 1992). Acute left ventricular dysfunction without dilatation in experimental induced myocarditis is associated with induction of proinflammatory cytokines, intramyocardial inflammation, and an imbalance of the matrix metalloproteinase (MMP) and the tissue inhibitors of MMP (TIMP) system (Li et al., 2002). The pathogenic importance of the regulation and balance of the MMP/TIMP system and the ECM-turnover in correlation to the development of acute to chronic myocarditis with dilated ventricle and left ventricular dysfunction are poorly understood, particularly in SWR/J mice. The extracellular matrix (ECM) proteins, especially the fibrillar collagens, are crucial for the structural integrity of the heart. Modulation of the balance between ECM-synthesis and ECMdegradation is an important process of myocardial remodeling and left ventricular function. Proinflammatory cytokines such as TNF-α and IL-1β contribute not only to depression of the left ventricular function and cardiomyocyte loss by apoptosis, they also play a critical role in maintaining the balance in ECM remodeling (Siwik et al., 2000;
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Mauviel, 1993). In the present study, we investigated the association between left ventricular dilatation, intramyocardial inflammation, components of the ECM and components of the MMP/TIMP system in the chronic phase of myocarditis. 2. Methods 2.1. Virus and animals Pathogen-free, inbred, 6 week old, male swr/J mice obtained from Jackson Laboratory (Bar Harbor, ME, USA), were used for the experiments described in this study (n = 60). Animals (n = 60) were infected and sham infected as described previously (Li et al., 2002). Suitable controls and infected animals were maintained and sacrificed in accordance with the German Animal Protection Law of 1993. 2.2. Hemodynamic characterization of left ventricular function Four, seven, and twenty-eight days after infection, hemodynamic parameters were measured in anesthetized, intubated, artificially ventilated, and closed-chest animals as previously described (Li et al., 2002; Tschope et al., 2004). Systolic function was quantified by left ventricular end-systolic pressure (LVESP), dP/dt max as an index of left ventricular contractility, ejection fraction, end-systolic volume (LVESV), stroke volume, cardiac output, and heart rate. Diastolic function was measured by left ventricular end-diastolic pressure (LVEDP) and dP/dt min. Reliable hemodynamic data could be determined in a subgroup of animals: controls (day 4 (C 4 day) n = 6), controls (day 7 (C 7 day) n = 6), controls (day 28 (C 28 day) n = 6), infected (day 4 (I 4 day) n = 7), infected (day 7 (I 7 day) n = 7), and infected (day 28 (I 28 day) n = 9). 2.3. RNA isolation and reverse transcription Immediately after the hemodynamic parameters were obtained, the hearts were rapidly removed, transversely dissected, and myocardial tissue was collected for various measurements. Total RNA extraction and reverse transcription were performed as previously described (Li et al., 2002). 2.4. Reverse transcriptase-polymerase chain reaction Semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was used to detect the mRNA abundance of MMP-2,-3,-8, extracellular-MMP-inducer, TIMP-1, uPA (urokinase type plasmin activator), tPA (tissue type plasmin activator), IL-6, INF-γ, IL-1β, TNF-α, IL-10, collagen type I, collagen type III, collagen type IV, and CVB-3 genome, according to the methods described previously (Li et al., 2002; Pauschinger et al., 1999a,b).
2.6. Zymography of MMP activity Gelatin zymography was performed to determine the gelatinolytic activities of MMP-2 and MMP-9 as described before (Li et al., 2007). The gelatinolytic activities were detected as clear bands against a blue background and analyzed using Scion Image software as relative optical densities. 2.7. Luxol fast blue staining Luxol fast blue staining was performed in order to determine myocytolysis in myocardial tissue as described before (Li et al., 2007). The area percent of myocytolysis in different sections was measured by digital image analysis (Lucia G, Version 3.51) adapted to previously published techniques (Noutsias et al., 2002, 2003). 2.8. ELISA IL-1β, IL-6, and Interferon-γ were determined by ELISA from (Pharmingen/BD Biosciences, San Diego, CA, USA) from myocardial tissue according to the manufacturer's instructions. All assays were done twice, and the absorbance values were equalized with absorbance values of GAPDH (Active Motif, Carlsbad, CA, USA). 2.9. Sirius red staining Picrosirius red staining was used for the histological assessment of the total collagen content 5 μm thick paraffin embedded tissue as previously described (Pauschinger et al., 1999a,b). Total collagen was measured as the area fraction (AF) as described previously (Li et al., 2002). 2.10. Statistical analysis Statistical analysis was performed using SPSS (SPSS Inc., Chicago, IL, USA). All data are expressed as S.E.M. Significant differences in quantitative parameters across the different groups were determined by Kruskal–Wallis test followed by Mann–Whitney–U test. Bivariate correlations were performed by Pearson's correlation. Values of P b 0.05 were considered as statistically significant. 3. Results 3.1. Left ventricular function of CVB-3-infected mice After diastolic dysfunction at 4 day post-infection (LVEDP: 198%; P b 0.05; versus control), left ventricular systolic dysfunction developed at 7 and 28 days post-infection with significantly increased LVESV (7 days p.i.: 180%, 28 days p.i.: 182%; and P b 0.05) and LVEDV (7 days p.i. 121% and 28 days p.i. 121%; P b 0.05) and significantly reduced left ventricular ejection fraction (7 days p.i.: 63%; P b 0.05 and 28 days p.i.: 68%; P b 0,05) (Fig. 1A–D). Furthermore dP/dt min was significantly reduced at 28 days p.i. without any effects of cardiac heart rate (data not shown).
2.5. Immunohistochemistry 3.2. Course of CVB-3 genomes Immunohistochemical analysis for CD3-T-lymphocytes was performed as described previously (Li et al., 2002). Immunocompetent infiltrates were quantified by digital analysis (Lucia G, Version 3.51) as described elsewhere (Noutsias et al., 2002, 2003). For quantitative analysis, the coded slides were evaluated in a blinded fashion. Total number of CD3 cells in different sections was counted at 200× accounted 0.39 mm2. For each sample, six tissue sections (3 sections per slide, 2 slides per heart) were evaluated. The mean cell number per mm² was calculated and represented as extent of intramyocardial inflammation.
PCR analysis confirmed the presence of the CBV-3 genomes in the myocardium of all infected mice at day 4 and 7 p.i., whereas CBV-3 was proven only in 4 animals (40%) at days 28 p.i. In contrast, all sham infected hearts were CBV-3 negative. 3.3. Myocardial cytokine profiles Semi-quantitative RT-PCR revealed a significant upregulation of the mRNA abundances of IL-1β by 290% (P b 0.0001), of INF-γ by 200%
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Fig. 1. Hemodynamic characterization on days 4, 7, and 28 after CVB-3 infection in swr/J mice; LVEDP (A), dP/dt max, dP/dt min (B), LVEDV, LVESV, stroke volume (C), and ejection fraction (D). Data are expressed as S.E.M. and P b 0.05 was considered as significant.
(P b 0.0001), of IL-6 by 200% (P b 0.0001), and of IL-10 by 208% (P b 0.0001) in the myocardium of infected mice compared to controls at day 4 p.i. (Fig. 2A). On the protein level, we found a significant induction of IL-6 by 334% (P b 0.05) and INF-γ by 145%. Likewise at day 7 p.i., cytokines was significantly upregulated (IL-1β by 154% (P b 0.05), INF-γ by 208% (P b 0.0001), IL-6 by 276% (P b 0.0001), IL-10 by 271% (P b 0.0001), and TNF-α by 193% (P b 0.0001)) (Fig. 2A). At the protein level we found a significant induction of IL-6 by 241% (P b 0.05) and INF-γ by 159% (P b 0.05) in infected mice versus controls. Also in the chronic phase at day 28 p.i., we detected a significant upregulation of the mRNA abundances of INF-γ by 153% (P b 0.001), of IL-6 by 148% (P b 0.001), and of IL-10 by 150% (P b 0.001) compared to controls with significantly increased protein levels of IL-6 by 262% (P b 0.05) and IL-1β by 156% (P b 0.05, Fig. 2).
3.4. MMP/TIMP profiles In the MMP/TIMP system, we found a significant induction of the mRNA abundances of MMP-3 by 376% (P b 0.002), of extracellularMMP-inducer by 163% (P b 0.0001) and of TIMP-1 by 335% (P b 0.0001) compared to controls (Fig. 2B). Furthermore, the mRNA abundance of tPA increased significantly by 147% (P b 0.001, Fig. 2C). At day 7 p.i. we found a significant upregulation of the mRNA abundances of MMP-2 by 124% (P b 0.007), of MMP-3 by 452% (P b 0.0001), of MMP-8 by 305% (P b 0.0001) and of TIMP-1 by 409% (P b 0.0001) compared to controls, as well as a significant upregulation of MMP-3 by 163% (P b 0.03) and of MMP-8 by 196% (P b 0.0001) compared to day 4 p.i. Additionally, we found an upregulation of tPA by 147% (P b 0.007) and uPA by 173% (P b 0.001) compared to controls (Fig. 2C). MMP-2 activity, showed a significant increase by 129% (P b 0.03) compared to controls (Fig. 3D). At day 28 p.i. we could show a significant increase of the mRNA abundance of MMP-2 by 142%, (P b 0.035) and a significant reduction of TIMP-1 to 73% (P b 0.007) compared to controls (Fig. 2B). The MMP-3/TIMP-1 mRNA ratio showed a significant upregulation at 28 days p.i. by 276% (P b 0.019, Fig. 2B). Furthermore, tPA (229%, P b 0.0001) and uPA (211%, (P b 0.0001)) were significantly upregulated compared to controls
(Fig. 2C). The MMP-2 activity analysis revealed an increase at 143% (P b 0.001) compared to controls (Fig. 2D). 3.5. Myocardial collagen content at 4 day post-infection On day 4 p.i., we observed a significant downregulation of collagen III mRNA abundance by 74% (P b 0.03) compared to controls (Fig. 3A). On day 7 p.i., we also found a downregulation of collagen III mRNA abundances by 67% (P b 0.02) compared to controls, with nonsignificant trend towards an increase of collagen IV mRNA abundance, at an unchanged level of collagen I mRNA abundance. These changes on the mRNA-level were associated with a significant upregulation of total collagen content by 208% (P b 0.006) compared to controls and by 152% (P b 0.001) compared to 4. day p.i. (Fig. 3A/B). On day 28 p.i., we observed a significant upregulation of collagen I mRNA abundances by 127% (P b 0.03) and collagen IV by 128% (P b 0.05), and a downregulation of collagen III by 77% (P b 0.01). These changes on the mRNA-level were linked to significantly increased total collagen content by 208% on the protein level (P b 0.001). This led to an increased collagen I/III ratio on the mRNA-level at day 28 postinfection by 166% (P b 0.05). 3.6. T-lymphocytic infiltration and myocytolysis CVB-3 infection led to a significant intramyocardial CD3+ T-lymphocytic infiltration at 7 days p.i. (1345%, P b 0.008) and at 28 days p.i. (247%; P b 0.1) compared to controls (Fig. 4A/B) and also to a significantly increased myocytolysis at 7 day p.i. (2880%, P b 0.002) and 28 day p.i. (761%; P b 0.002) compared to controls (Fig. 5A/B). 3.7. Associations between cytokine expression, MMP expression, myocardial cell infiltration, and myocardial function The mRNA abundance of IL-6, IL-10, and INF-γ as well as the myocardial content of CD-3 cells were significantly correlated to the expression of MMP-3, MMP-8, and TIMP-1, and myocytolysis (P b 0.05) (Table 1). We found a significant correlation between the mRNA abundances of MMP-8 and left ventricular end-systolic volume and stroke volume. There was a significant correlation between mRNA
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Fig. 2. Abundance of cytokine mRNAs (A), MMP/TIMP mRNAs (B), and uPA/tPA mRNAs (C) measured by semi-quantitative RT-PCR, and MMP-2 activity (D) measured by gelatin zymography.
abundance of uPA and left ventricular diameters and ejection fraction. We also found a significant correlation between mRNA abundance of tPA and ejection fraction. Furthermore the increased MMP activity was correlated to the impairment of stroke volume and ejection fraction and the increased left ventricular end-systolic diameters, thus underlining the importance of left ventricular extracellular matrix remodeling in the development of left ventricular dysfunction (Table 2). 4. Discussion This study in a murine CVB-3 myocarditis model in SWR/J mice showed after the acute phase of infection an ongoing activated immune process with subsequent viral persistence and persisting myocardial inflammation. This ongoing inflammatory process is associated with an imbalance of the MMP/TIMP system, progression
of left ventricular dilatation, and reduction of left ventricular systolic function. The relevance of the MMP/TIMP imbalance for the progression of left ventricular dilatation and impairment of left ventricular dysfunction is shown by the documented correlation between uPA–mRNA expression, MMP-2 activity and LVESV, as well as ejection fraction. Therefore, the imbalance of the matrix degrading system may cause a loss of structural integrity of the heart and may be an important factor for an ongoing impairment of left ventricular function with subsequent ventricular dilatation in chronic myocarditis. 4.1. Hemodynamic characterization In CVB-3 murine myocarditis, hemodynamic changes differ during the three phases of viral myocarditis. A diastolic dysfunction with an elevated LVEDP, but no impairment of systolic parameters such as
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Fig. 3. Total collagen content measured by sirius red staining (A/B).
ejection fraction was found on day 4 (acute phase). This diastolic dysfunction may be induced by an interstitial edema of the left ventricel, also indicated by a temporary reduction in LVEDV, as it is also well-known for patients with acute myocarditis. Diastolic dysfunction in the acute phase may also be caused by direct viral effects with focal necrotic myofibers without changes in myocardial inflammation and remodeling (Shioi et al., 1996). Hemodynamically, the subacute phase is characterized by impaired systolic and diastolic function and by an increase in left ventricular chamber diameters, according to the clinical diagnosis of
dilated cardiomyopathy. Alterations of ventricular function are likely to have different causes in ongoing myocarditis. The subacute phase is characterized by the onset of inflammatory infiltrations, cytotoxic virological effects, along with the inflammatory response, results in myocytolysis and early changes of ECM components. In the chronic phase of myocarditis, viral persistence triggers an ongoing inflammatory process. This in turn may cause an imbalance of the MMP/TIMP system, which would lead to a progressive breakdown of the extracellular matrix components, which might cause impairment of ventricular function.
Fig. 4. CD3-T-lymphocytes (A). A representative immunohistochemical staining of control heart sections and CVB-3 infected heart sections (B) on day 7 p.i. Original magnifications, ×20.
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Fig. 5. Luxol fast blue staining (A). Representative control and CVB-3 infected sections (B) on day 28. p.i.
4.2. Inflammatory response For the pathogenesis of myocarditis, Kawai (1999) described a triphasic model of the pathogenesis of myocarditis with acute, subacute, and chronic phases. The acute phase is characterized by the early release of proinflammatory cytokines, such as IL-1β and interferon-γ (Shioi et al., 1996; Matsumori, 1997), with the first appearance of histological changes in the form of myocyte necrosis and apoptosis (Rose and Hill, 1996; Wilson et al., 1969). In this phase, cellular infiltrations are barely detected and usually consist of natural killer cells and macrophages (Seko et al., 1991; Feldman and McNamara, 2000). Consequently, on the 4th day of infection, we found an increased expression of proinflammatory cytokines, including IL-1β, IL-6, IL-10, and Interferon-γ on the mRNA and protein levels, with no significant changes in myocardial T-lymphocytes infiltration. During the subacute phase (day 7), we observed the maximum peaks of IL-2, TNF-α, INF-γ, and IL-10 expression. The inflammatory response with elevated inflammatory cytokines and myocardial T-cell infiltration persisted at a lower intensity to day 28 post-infection. This inflammatory response was associated with a progressive increase of myocytolysis from day 4 to day 28 p.i. (Arnold et al., 1985). The impact of the inflammatory response on myocardial necrosis is once again demonstrated by the significant correlation between myocytolysis and myocardial cellular infiltration (Table 1). 4.3. Extracellular matrix turnover Disruption of the ECM is an important factor in the transition of acute myocarditis to dilated cardiomyopathy (Pauschinger et al., 1999a,b; D'Armiento, 2002; Thomas et al., 1998). As inflammatory
Table 1 Pearson's correlation between inflammation and the MMP/TIMP system from all timepoints. Expressed by r and a: P b 0.05 and b: P b 0.001.
INF-γ IL-6 Il-10 CD-3
MMP-8
MMP-3
TIMP-1
MFD
0.687a 0.732b 0.708b 0.638a
0.823b 0.694a 0.718b 0.469a
0.701a 0.705a 0.717b 0.465a
0.447a 0.587a 0.581a 0.718b
cytokines are involved in the tightly regulated system of collagen expression and degradation, increased inflammatory response is likely to contribute to a pathologic collagen turnover. Proinflammatory cytokines regulate the expression of ECM components such as collagen types I and III and fibronectin (Bouluyt et al., 1994). They also induce the expression of MMP's and plasminogen activators (Siwik et al., 2000; Mauviel, 1993), which causes a degradation of the ECM. Over-expression of TNF-α and IL-1β correlates with an increased expression of MMPs and reduced expression of TIMPs. Furthermore, inhibition of TNF-α leads to a reduced expression and activity of MMPs, with improved left ventricular function (Bradham et al., 2002). These findings agree with our data, which demonstrates a correlation between proinflammatory cytokine expression (IL-1β and TNF-α) and the expression of MMPs (Table 1). Li et al. (2002)have reported an imbalance of the MMP/TIMP system in the acute phase of myocarditis resulting in a functional defect of the ECM without an increase of total collagen. In addition, Woodiwiss et al., 2001. have demonstrated that a reduction in collagen cross-links from increased MMP activity is responsible for an increase of soluble collagen, which results in left ventricular dysfunction, remodeling, and chamber dilatation. This study analyzed different phases of myocarditis (Rutschow et al., 2006). We were able to demonstrate an imbalance in the matrix degradation system, with induced expression of MMPs over the acute, subacute, and chronic phases and a significant increase in the MMP/TIMP-ratio in the chronic phase. This imbalance was also shown by Cheung et al. (2005) in CVB-3 infected A/J mice. The increase in MMPs expression and activity were significantly correlated with development of left ventricular dilatation and reduced stroke volume and ejection
Table 2 Pearson's correlation between matrix degradation system and left ventricular function from all timepoints. Expressed by r and a: P b 0.05 and b: P b 0.001.
MMP-3 MMP8 MMP-2 activity uPA
EF
VES
SV
0.396a 0.396a 0.458a 0.493a
0.312 0.407a 0.551b 0.539b
0.353a 0.388a 0.431a 0.230
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References
Fig. 6. Possible signal pathway in viral myocarditis associated induction of inflammation resulting in matrix degradation.
fraction, underlining the importance of matrix integrity and left ventricular function. Plasminogen activators (urokinase type (uPA) and tissue type (tPA)) take more interest in ECM remodeling, due to activation of proMMP (Li et al., 2007; Heymans et al., 2006). In the model of chronic myocarditis used in this study, uPA and tPA increased significantly over 7 days with no relevant decrease to day 28 p.i. Therefore, the elevated ability of MMP-activation also in the chronic phase of myocarditis is observed by induced MMP-2 activity. Further studies suggest that an active plasminogen–MMP system is essential for the ability of immunocompetent cell to infiltrate tissues. In a murine model of myocardial infarction in plasminogen knockout mice, inflammatory cells were unable to infiltrate the infarct zone, resulting in reduced fibrosis and cellular colonization (Creemers et al., 2000). Also in our model of chronic myocarditis, the increase in uPA–mRNA expression was significantly correlated with myocardial CD-3 infiltration and left ventricular dysfunction, underlining the importance of the activation of MMPs to the development of left ventricular dilatation. 5. Conclusions In CVB-3 induced chronic myocarditis, viral persistence, chronic inflammation, and their influence on the extracellular matrix lead to left ventricular dysfunction with dilated ventricle and reduced left ventricular ejection fraction, resembling human dilated cardiomyopathy. The chronic inflammatory response and ECM remodeling are the pathways involved in this pathogenesis. The imbalance of the matrix degrading system with induced expression of MMPs and plasminogen activators, as well as the reduced expression of TIMPs in the chronic phase, suggest a pathologic collagen turnover, leading to a loss of structural integrity of the heart and an impairment of left ventricular function and dilatation (Fig. 6). In light of these insights, pharmacologic intervention with inhibiting MMPs or plasminogen activators might be promising approaches to abrogate the progressive left ventricular dysfunction in chronic myocarditis with CVB-3 persistence. Acknowledgement This research was supported by the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich Transregio 19, A2). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ejphar.2009.12.019.
Arnold, G., Kaiser, C., Fischer, R., 1985. Myofibrillar degeneration — a common type of myocardial lesion and its selective identification by a modified luxol fast blue stain. Pathol. Res. Pract. 180, 405–415. Bouluyt, M.C., O'Neil, L., Mededith, A.L., 1994. Alteration in cardiac gene expression during the transition from stable hypertrophy to heart failure: Marked upregulation of genes encoding extracellular components. Circ. Res. 75, 25–32. Bradham, W.S., Moe, G., Wendt, K.A., 2002. TNF-α . Am. J. Physiol. Heart Circ. Physiol. 282, H1288–H1295. Cheung, C., Luo, H., Yanagawa, B., 2006. Matrix metalloproteinases and tissue inhibitors of metalloproteinases in coxsackievirus-induced myocarditis. Cardiovasc. Pathol. 15, 63–74. Creemers, E., Cleutjens, J., Smits, J., 2000. Disruption of the plasminogen gene in mice abolishes wound healing after myocardial infarction. Am. J. Pathol. 156, 1865–1873. D'Armiento, J., 2002. Matrix metalloproteinase disruption of the extracellular matrix and cardiac dysfunction. Trends Cardiovasc. Med. 12, 97–101. Fairweather, D., Kaya, Z., Shellam, G.R., 2001. From infection to autoimmunity. J. Autoimmun. 16, 175–186. Feldman, A.M., McNamara, D., 2000. Myocarditis. N. Engl. J. Med. 9 (343), 1388–1398. Heymans, S., Pauschinger, M., Palma, A., 2006. Inhibition of urokinase-type plasminogen activator or matrix metalloproteinases prevents cardiac injury and dysfunction during viral myocarditis. Circulation 114, 565–573. Kawai, C., 1999. From myocarditis to cardiomyopathy: Mechanisms of inflammation and cell death. Learning from the past to the future. Circulation 96, 3549–3554. Klingel, K., Hohenadl, C., Canu, A., 1992. Ongoing enterovirus-induced myocarditis is associated with persistent heart muscle infection: quantitative analysis of virus replication, tissue damage, and inflammation. Proc. Natl. Acad. Sci. U. S. A. 89, 314–318. Kuhl, U., Pauschinger, M., Bock, T., 2003. Parvovirus B19 infection mimicking acute myocardial infarction. Circulation 108, 945–950. Kuhl, U., Pauschinger, M., Noutsias, M., 2005. High prevalence of viral genomes and multiple viral infections in the myocardium of adults with “idiopathic” left ventricular dysfunction. Circulation 111, 887–893. Li, J., Schwimmbeck, P.L., Tschope, C., 2002. Collagen degradation in a murine myocarditis model: relevance of matrix metalloproteinase in association with inflammatory induction. Cardiovasc. Res. 56, 235–247. Li, J., Leschka, S., Rutschow, S., 2007. Immunomodulation by interleukin-4 suppresses matrix metalloproteinases and improves cardiac function in murine myocarditis. Eur. J. Pharmacol. 554, 60–68. Matsumori, A., 1997. Molecular and immune mechanisms in the pathogenesis of cardiomyopathy: role of viruses, cytokines and nitric oxide. Jpn. Circ. J. 61, 275–291. Mauviel, A., 1993. Cytokine regulation of metalloproteinase gene expression. J. Cell. Biochem. 53, 288–295. Noutsias, M., Seeberg, B., Schultheiss, H.P., Kuhl, U., 1999. Expression of cell adhesion molecules in dilated cardiomyopathy: evidence for endothelial activation in inflammatory cardiomyopathy. Circulation 99, 2124–2131. Noutsias, M., Pauschinger, M., Ostermann, K., 2002. Digital image analysis system for the quantification of infiltrates and cell adhesion molecules in inflammatory cardiomyopathy. Med. Sci. Monit. 8, MT59–MT71. Noutsias, M., Hohmann, C., Pauschinger, M., 2003. sICAM-1 correlates with myocardial ICAM-1 expression in dilated cardiomyopathy. Int. J. Cardiol. 91, 153–161. Pauschinger, M., Doerner, A., Kuehl, U., 1999a. Enteroviral RNA replication in the myocardium of patients with left ventricular dysfunction and clinically suspected myocarditis. Circulation 99, 889–895. Pauschinger, M., Knopf, D., Petschauer, S., 1999b. Dilated cardiomyopathy is associated with significant changes in collagen type I/III ratio. Circulation 99, 2750–2756. Rose, N.R., Hill, S.L., 1996. The pathogenesis of postinfectious myocarditis. Clin. Immunol. Immunopathol. 80, S92–S99. Rutschow, S., Li, J., Schultheiss, H.P., Pauschinger, M., 2006. Myocardial proteases and matrix remodeling in inflammatory heart disease. Cardiovasc. Res. 69, 646–656. Seko, Y., Shinkai, Y., Kawasaki, A., 1991. Expression of perforin in infiltrating cells in murine hearts with acute myocarditis caused by coxsackievirus B3. Circulation 84, 788–795. Shioi, T., Matsumori, A., Sasayama, S., 1996. Persistent expression of cytokine in the chronic stage of viral myocarditis in mice. Circulation 94, 2930–2937. Siwik, D.A., Chang, D.L., Colucci, W.S., 2000. Interleukin-1beta and tumor necrosis factor-alpha decrease collagen synthesis and increase matrix metallopro-teinase activity in cardiac fibroblasts in vitro. Circ. Res. 86, 1259–1265. Thomas, C.V., Coker, M.L., Zellner, J.L., 1998. Increased matrix metallo-proteinase activity and selective upregulation in LV myocardium from patients with endstage dilated cardiomyopathy. Circulation 97, 1708–1715. Tschope, C., Westermann, D., Steendijk, P., 2004. Hemodynamic characterization of left ventricular function in experimental coxsackieviral myocarditis: effects of carvedilol and metoprolol. Eur. J. Pharmacol. 491, 173–179. Why, H.J., Meany, B.T., Richardson, P.J., 1994. Clinical and prognostic significance of detection of enteroviral RNA in the myocardium of patients with myocarditis or dilated cardiomyopathy. Circulation 89, 2582–2589. Wilson, F.M., Miranda, Q.R., Chason, J.L., 1969. Residual pathologic changes following murine coxsackie A and B myocarditis. Am. J. Pathol. 55, 253–265. Woodiwiss, A.J., Tsotetsi, O.J., Sprott, S., 2001. Reduction in myocardial collagen crosslinking parallels left ventricular dilatation in rat models of systolic chamber dysfunction. Circulation 103, 155–160.