J Mol Med (2008) 86:49–60 DOI 10.1007/s00109-007-0249-3 ORIGINAL ARTICLE Connective tissue growth factor: a crucial cytokine-mediating cardiac fibrosis in ongoing enterovirus myocarditis Christine Lang & Martina Sauter & Gudrun Szalay & Giorgia Racchi & Gabriele Grassi & Giuseppe Rainaldi & Alberto Mercatanti & Florian Lang & Reinhard Kandolf & Karin Klingel Received: 9 March 2007 / Revised: 26 June 2007 / Accepted: 5 July 2007 / Published online: 11 September 2007 # Springer-Verlag 2007 Abstract Dilated cardiomyopathy (DCM) as a consequence of viral myocarditis is a worldwide cause of morbidity and death. The deposition of matrix proteins, such as collagen, in the course of ongoing viral myocarditis results in cardiac remodeling and finally in cardiac fibrosis, the hallmark of DCM. To identify mediators of virusinduced cardiac fibrosis, microarray analysis was conducted in a murine model of chronic coxsackievirus B3 (CVB3) myocarditis. By this attempt, we identified connective tissue growth factor (CTGF) as a novel factor highly expressed in infected hearts. Further investigations by quantitative reverse transcription polymerase chain reaction and Western blot analysis confirmed a strong induction of cardiac CTGF expression in the course of CVB3 myocarditis. By in situ hybridization and immunohistochemistry, C. Lang : M. Sauter : G. Szalay : G. Racchi : G. Grassi : R. Kandolf : K. Klingel (*) Department of Molecular Pathology, Institute for Pathology, University Hospital Tübingen, Liebermeisterstr.8, 72076 Tübingen, Germany e-mail: karin.klingel@med.uni-tuebingen.de C. Lang : F. Lang Institute of Physiology, University of Tübingen, Tübingen, Germany G. Grassi Department of Internal Medicine, University Hospital of Trieste, Cattinara, Strada di Fiume 447, 34149 Trieste, Italy G. Rainaldi : A. Mercatanti Istituto di Fisiologia Clinica, Area della Ricerca di Pisa, Pisa, Italy CHRISTINE LANG is currently completing her M.D. at the Medical School, University of Tübingen, Germany. Her research interests comprise the elucidation of cell signaling mechanisms in acute and chronic viral myocarditis and aspects on the pathological development of cardiac fibrosis. KARIN KLINGEL received her M.D. at the University of Heidelberg, Germany. She is currently Professor and associate head of the Department of Molecular Pathology at the University of Tübingen, Germany. Her research interests include regulation of virus–host interactions in viral heart diseases by immune-mediated mechanisms, identification of pathogenicity determinants of inflammatory cardiomyopathy, and determination of cell signaling molecules involved in cardiac fibrosis and dysfunction. basal CTGF messenger ribonucleic acid (mRNA) and protein expression were confined in noninfected control hearts mainly to endothelial cells, whereas in CVB3infected hearts, also numerous fibroblasts were found to express CTGF. Regulation of CTGF is known to be basically mediated by transforming growth factor (TGF)- 50 β. In the course of CVB3 myocarditis, CTGF upregulation coincided with increased cardiac TGF-β and procollagen type I mRNA expression, preceding the formation of fibrotic lesions. In in vitro experiments, we found that downregulation of CVB3 replication by means of small interfering RNAs (siRNAs) reverses the upregulation of CTGF mRNA expression. In contrast, downregulation of CTGF by siRNA molecules did not significantly reduce viral load, indicating that CTGF is not essential for CVB3 life cycle. The significantly enhanced transcript levels of TGF-β, CTGF, and procollagen type I in cultivated CVB3infected primary cardiac fibroblasts substantiate the role of fibroblasts as a relevant cell population in cardiac remodeling processes. We conclude that CTGF is a crucial molecule in the development of fibrosis in ongoing enteroviral myocarditis. Thus, downregulation of cardiac CTGF expression may open novel therapeutic approaches counteracting the development of cardiac fibrosis and subsequent heart muscle dysfunction. Keywords Coxsackievirus B3 . Fibroblasts . TGF-β . Collagen . siRNA Introduction Enteroviruses of the Picornaviridae usually cause mild and self-limiting infections but may also induce a variety of severe acute and chronic forms of diseases, including myocarditis, meningitis, and pancreatitis. In adults, especially coxsackieviruses of group B (CVB) are known to induce chronic myocarditis, which may result in dilated cardiomyopathy (DCM) and severe congestive heart failure [1]. The typical hallmark of DCM is fibrosis, which is characterized by the abundant production of extracellular matrix proteins resulting in a change of the structure and architecture of the myocardium, thus impairing the ventricular contractility and functionality [2]. Despite the clinical relevance of cardiac fibrosis, a leading cause of morbidity and mortality [3], the key components of the virus-induced stimulation of matrix protein formation and deposition are not defined yet. To dissect virus–host interactions in the development of cardiac fibrosis, CVB3-induced murine heart disease represents the most suitable model, as susceptible mice proceed to chronic myocarditis, which is defined by persistence of viral ribonucleic acid (RNA), ongoing inflammation, and the development of severe fibrosis at later stages of the illness [1, 4]. Cardiac fibrosis, which is characterized by excess deposition of mainly collagen type I, is typically seen in susceptible CVB3infected mice later than 12 days postinfection (pi) [4]. Corresponding to the observations in hearts of patients with DCM, the abundant synthesis of collagen in CVB3-infected J Mol Med (2008) 86:49–60 susceptible mouse strains, such as ABY/SnJ, ACA/SnJ, and SWR/J mice [4], is associated with severe alterations of the architecture and functional deficiencies of the heart [5]. Putative mediators underlying cardiac fibrosis include connective tissue growth factor (CTGF), a member of the CCN (ctgf/cyr61/nov) gene family [6]. CTGF has been demonstrated to play a role in fibroblast proliferation and in stimulation of extracellular matrix formation [7, 8]. CTGF is upregulated in several fibrotic disorders including cardiac fibrosis [9–11], skin sclerosis [12], and systemic sclerosis [13]. As a result of mitogenic action on fibroblasts and its capacity to induce generation of extracellular matrix proteins, such as collagen type I and fibronectin [8], CTGF is suggested to play a role in the extracellular matrix deposition as an important downstream mediator of transforming growth factor (TGF)-β [6, 14]. The specific induction of CTGF expression by TGF-β was also shown in cardiac fibroblasts in a rat model of myocardial infarction [15]. In murine viral myocarditis, TGF-β messenger RNA (mRNA) was found to be upregulated in CVB3-infected hearts as soon as 1 day pi and was still detectable 3 months pi, indicating generation of TGF-β at any stage of myocarditis [16]. In a murine skin fibrosis model, it was shown that subcutaneuos injection of TGF-β alone induces a transient fibrotic response, whereas the simultaneous application of TGF-β and CTGF resulted in sustained fibrosis, thus pointing to synergistic effects of these two molecules in the promotion of fibrosis [17]. The aim of the present study was to explore the role of CTGF in the outcome of ongoing CVB3 myocarditis. For this purpose, we have investigated in an in vivo model system of chronic enterovirus myocarditis the spatial and temporal relationships of cardiac infection with CTGF, TGF-β, and procollagen expression and the development of fibrosis. In addition, in vitro experiments were performed to define the relevant cell types involved in these processes and to evaluate potential antiviral/antifibrotic treatment options by small interfering RNA (siRNA) molecules. Materials and methods Virus and mice Complementary deoxyribonucleic acid (cDNA)-generated CVB3 (Nancy strain) was grown and propagated in Vero cells (African green monkey kidney cells) as described previously [4]. CVB3 titers in culture medium were determined by an agar overlay plaque assay according to standard procedures. Immunocompetent inbred ABY/SnJ mice (H-2b) were kept under specific pathogen-free conditions at the animal facilities of the Department of Molecular Pathology, University Hospital Tuebingen, and J Mol Med (2008) 86:49–60 experiments were conducted according to the German animal protection law. Four- to 5-week-old mice were infected intraperitoneally with 5×104 plaque-forming units of purified CVB3 as described [4]. Samples of aseptically removed hearts were either fixed for 12 h in phosphatebuffered (pH 7.2) 4% paraformaldehyde and embedded in paraffin for histology and in situ hybridization or quick frozen in liquid nitrogen for DNA microarray analysis, reverse transcription polymerase chain reaction (RT-PCR), and Western blot analysis. 51 Sabine Werner, ETH Zürich). As control probes, the corresponding sense 35S-labeled RNA probes were used. Pretreatment, hybridization, and washing procedures of dewaxed 5-μm paraffin tissue sections were performed as described previously [4]. Slide preparations were subjected to autoradiography, exposed for 3 weeks at 4°C, and counterstained with HE. Immunohistochemistry Total RNA of murine heart tissue was isolated by using Trizol reagent (Invitrogen, Karlsruhe, Germany) followed by the RNeasy Mini Kit (Qiagen, Duesseldorf, Germany). RNA of five animals was pooled in equal amounts and converted to cDNA and used for hybridization of Affymetrix (Santa Clara, USA) MG-U74Av2 chips as previously described [18]. To localize CTGF protein expression, 5-μm-thick heart tissue sections were deparaffinated and incubated for 1 h at room temperature with CTGF primary antibody (Abcam) followed by three washing steps in Tris-buffered saline and incubation for 1 h with a secondary antibody (biotinylated goat anti-rabbit IgG (Vector, Burlingham, UK). Visualization of positive cells was completed by using the iVIEW™DAB Detection Kit (Ventana, Illkirch, France). Slides were counterstained with hematoxylin. Histopathology CVB3-infection of cultivated cells Histological analysis was performed on deparaffinized 5-μm-thick tissue sections, which were stained with hematoxylin/eosin (HE) to assess inflammation and myocyte injury or with picrosirius red to visualize the degree of fibrosis. For quantitative evaluation of fibrosis, the picrosirius polarization method was applied on transverse tissue sections covering the right and left ventricle. The images were processed by an interactive image analyzing system, applying the Optimas® 6.0 software (Stemmer) and using a color Sony video camera mounted on a microscope at a primary magnification of 10×. Video signals were digitized resulting in images of 784×784 pixels. Results are expressed as percentage of area fractions of fibosis per total areas of transverse heart tissue sections (n=7 mice per time point). For isolation of cardiac mouse fibroblasts, hearts from 8–14week-old mice were dissected and transferred to culture dishes containing Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen, Heidelberg, Germany) supplemented with 10% fetal calf serum, 1% penicillin/streptomycin, and cultured under standard conditions (37°C, 5% CO2). Cell growth was observed 2–4 days after initial plating. Fibroblasts were identified by positive staining for fibronectin and used in experiments between passages 2 and 6. At a 70 to 80%, confluency cardiac fibroblasts were serum starved for 24 h and then infected with CVB3 at a multiplicity of infection (MOI) of 10 for 6 h. HeLa cells, which were cultured under standard conditions, were infected by an MOI of 10 for 1 h, washed with phosphate-buffered saline (PBS) and probed for the expression of CTGF 6 h after infection. Of note, the experiments with fibroblasts and HeLa cells were also performed using 0.01 MOI CVB3, demonstrating the same results regarding CTGF expression after 24 h (data not shown). DNA microarray hybridization and analysis In situ hybridization CVB3 positive-strand genomic RNA in tissues was detected using single-stranded 35S-labeled RNA probes, which were synthesized from the dual-promoter plasmid pCVB3-R1 Control RNA probes were obtained from the vector pSPT18 [4]. For the detection of CTGF mRNA, 35S-labeled antisense RNA probes were transcribed from the plasmid pGEMT-CTGF (a gift of Heike Heuer, IMB Jena), which contain the nucleotides 3,081 to 3,907 of the gene coding for murine CTGF. Control hybridization experiments were performed using single-strand 35S-labeled sense RNA probes from the same plasmid. TGF-β mRNA in myocardial tissue sections was detected using 35S-labeled antisense RNA probes from the plasmid pBluescript II KS+mTH//Qβ1, which contains 599 bp of the gene coding for TGF-β1 (a gift of siRNA transfection HeLa cells (5×105), grown in 35-mm dishes (Nunc, Roskilde, Denmark), underwent siRNA transfection upon reaching 60 to 70% confluency. Thereafter, cells were washed by PBS and overlaid with the transfection mixture containing Lipofectamine reagent (5 μl; Invitrogen), and siRNA against CTGF or CVB3 (400 nM final) in a final volume of 1.25 ml of DMEM for 2 h. After a PBS washing step, cells were cultured in complete growth medium and infected by CVB3 for 1 h at a MOI of 0.01. Twenty-four 52 hours pi, the mRNA/protein levels of CTGF and CVB3 genomes/capsid protein levels were evaluated. For the treatment studies using siRNA molecules the approach with an infection dose of 0.01 MOI CVB3 and an infection time of 24 h was applied, as the effects of infection on cell morphology of treated cells can be monitored more precisely within 24 h than within only 6 h. The siRNA (Eurogentec SA, Belgium) against CTGF targets the region 879–897 (5′-GACCUGGAAGAGAA CAUUA-3′); siRNA against the CVB3 genome (3,541–3,559 5′-GAGGTCCAAGAGAGTGAAT-3′) is directed against the region coding for the viral proteinase 2A (2Apro) (siRNA3541). As control, a siRNA directed against the luciferase mRNA was used (sense siGL2 5′-CGUACGCGGAAUACUUCG A-3′). The siGL2, conjugated to fluorescein, was also used to evaluate transfection efficiency, following the protocol above reported. The amount of fluorescein positive cells was evaluated by flow cytometry (FACScalibur, Becton Dickinson, Mountain View, CA). Quantitative real-time RT-PCR RNA of frozen heart tissue was isolated by using the RNeasy Mini Kit (Qiagen, Hilden, Germany). For the isolation of RNA from cultured cells the High Pure RNA Isolation Kit (Roche, Mannheim, Germany) was used. RNA (0.5 to 1.0 μg) was used for cDNA synthesis by Transcriptor Reverse Transcriptase (Roche). CTGF, procollagen type Iα2, and TGF-β1 mRNA were quantified using the LightCycler FastStart DNA Master SybrGreen I (Roche Diagnostics GmbH, Roche Applied Science). The primers specific for murine CTGF were purchased from MWG-Biotech AG (Ebersberg, Germany) and those for procollagen type Iα2 and TGF-β1 from Invitrogen. Primers used were: CTGF sense 5′-ACG AGC CCA AGG ACC GCA-3′, CTGF antisense 5′-TTG TAA TGG CAG GCA CAG-3′, procollagen sense 5′-CAC CCC GGT CTT GCT GGT GC-3′, procollagen antisense 5′-CCA CTC TCA CCC GGG GTA CC-3′, TGFβ1 sense 5′-GAC TCT CCA CCT GCA AGA CCA-3′, and TGF-β1 antisense 5′-GGG ACT GGC GAG CCT TAG TT-3′. The transcript levels of the housekeeping gene glyceraldehyde-phosphate dehydrogenase (GAPDH) were also determined for each sample using a commercial primer kit (Search LC, Heidelberg, Germany). Amplification of the target DNA was performed during 35 cycles, each 10 s at 95°C, 10 s at 68°C, and 16 s at 72°C. Data analysis was performed as relative quantification with external standards using the LightCycler software 3.5. and calculated as a ratio of the target vs housekeeping gene transcripts. CVB3 copy numbers were quantified using real-time TaqMan quantitative PCR according to the method described by Nijhuis et al. [19]. Data analysis of viral genomes was performed using the method of absolute quantification with external virus standards. J Mol Med (2008) 86:49–60 Western blot analysis Parts of frozen cardiac tissue or cultivated heart fibroblasts were homogenized in lysis buffer containing 50 mM Tris–HCl, pH 7.4,100 mM NaCl, 1 mM ethylenediamine tetraacetic acid, 1 mM ethylene glycol tetraacetic acid, 50 mM sodium fluoride, 5 mM sodium pyrophosphate, 1 mM sodiumorthovanadate, 1% Triton X-100, 1% sodium deoxycholate, 1% sodium dodecyl sulfate SDS, and protease cocktail inhibitor (Roche). The homogenates were centrifuged at 10,000 rpm at 4°C for 15 min; the supernatant was removed and used for Western blotting. Whole cell lysates (50 μg) and heart homogenates (70 μg) were separated by SDS–polyacrylamide gel electrophoresis (PAGE) using a 10% polyacrylamide gel. Western blots were performed as previously described [11] using a goat polyclonal CTGF primary antibody (diluted 1:400 in blocking buffer, Santa Cruz, Heidelberg, Germany) and a horseradish peroxidase-conjugated anti-goat secondary antibody (SantaCruz). Akt/p-Akt was detected using rabbit antibodies (anti-Akt IgG, anti-phospho-Akt (Ser473), both from Cell Signaling, Boston, MA) at a dilution of 1:1,000. The PI3K inhibitor LY294002 was obtained from Merck, Darmstadt, Germany, and applied at a concentration of 50 μM. Visualization was done with enhanced chemiluminescence (ECL) according to manufacturer’s instructions (Amersham, Freiburg, Germany). Membranes were also probed with a primary horseradish peroxidase-conjugated GAPDH (Santa Cruz, Heidelberg, Germany) antibody for control. Densitometric analysis of CTGF was performed using Scion Image (Scion, Maryland, USA) and normalized to GAPDH. Polyclonal antibodies against CVB3 capsid protein VP1 [20] were chosen to detect CVB3 proteins in cultured cells. Cell monolayers were washed with PBS and were lysed in SDS-PAGE sample buffer and subjected to SDS-PAGE on 12.5% acrylamide gels, incubated with antiCVB3 VP1 antibodies (1:300, vol/vol of 1% bovine serum albumin [BSA] in PBS), followed by a goat anti-rabbit IgG secondary antibody (Vector, Burlingame, CA; 1:5,000, v/v of 1% BSA in PBS) and developed with ECL reagents as described above. LDH test Cell death or cytotoxicity was assessed by determination of lactate dehydrogenase (LDH) release from the cytosol of injured cells into the supernatant. LDH was quantified by a photometrical assay (BioVision, Mountain View, CA) after 24 h pi. Statistics Data are provided as means±SEM, and n represents the number of independent experiments. All data were tested J Mol Med (2008) 86:49–60 for significance with Student’s t test with Welch’s correction when indicated. P values less than 0.05 were considered statistically significant. Results Fibrosis as end stage of CVB3-induced inflammatory heart disease At later stages of enteroviral myocarditis, the deposition of matrix proteins induces in susceptible mice severe cardiac interstitial fibrosis and, finally, alterations of the myocardial architecture. The formation of fibrotic lesions was determined by evaluating the amount of collagen by staining heart tissue of ABY/SnJ mice using picrosirius-red (Fig. 1a–d). As shown in Fig. 1a and b, tissue sections of infected mouse hearts obtained 28 days pi revealed significant fibrosis compared to noninfected hearts (Fig. 1c and d). The area fractions of fibrosis were determined in the course of myocarditis by using a digital image analysis system. An increase in collagen formation was not observed before 12 days pi. At day 28 pi, when inflammation declined and infected myocytes have been eliminated from the myocardium, a significant part (22% at day 28 pi) of the heart of susceptible ABY/SnJ mice was composed of fibrous tissue (P<0.01, day 28 pi vs noninfected heart tissue; Fig. 1e). 53 Densitometric analysis of CTGF protein levels in the course of infection revealed significant expression of CTGF protein from day 4 pi (P<0.01 for 4, 8, 12, 28 days pi vs noninfected mouse hearts, Fig. 2b and c). Immunohistochemical stainings of CTGF expression in noninfected mouse hearts exhibited CTGF protein in endothelial cells (arrows) and in a few interstitial cells (arrowheads; Fig. 2d, left image). In CVB3-infected hearts, the localization of CTGF proteins was found to be confined to endothelial cells (arrows) but also to numerous interstitial cells (arrowheads) as exemplarily shown in a heart obtained 12 days pi (Fig. 2d, right image). CTGF upregulation in CVB3-infected mouse hearts To define molecules that are implicated in virus–host interrelationships during acute and chronic enteroviral myocarditis, microarray analysis (Affymetrix) from hearts of two resistant and two susceptible mouse strains in the course of CVB3 infection was performed [18]. Preferentially, in CVB3-infected hearts of mouse strains susceptible for development of chronic myocarditis and cardiac fibrosis (ABY/SnJ and SWR/J mice), we observed a considerable overexpression of different molecules of the extracellular matrix with CTGF mRNA, being upregulated more than threefold compared to noninfected heart tissue probes at 4 days pi. To investigate the induction of CTGF mRNA at different stages of myocarditis in more detail, we performed quantitative RT-PCR from CVB3-infected ABY/SnJ mouse hearts that exhibit the most extensive fibrosis at later phases of myocarditis. As soon as 4 days pi, a significant increase (P<0.01) of CTGF mRNA in cardiac tissue compared to controls was observed (Fig. 2a). The maximum of CTGF mRNA expression was reached 8 days pi but was still present at high levels 12 days pi, which parallels the maximum of cardiac inflammation (P<0.01) [4]. A strong increase in CTGF in the myocardium after CVB3 infection was also observed at protein levels. Fig. 1 a, b Collagen deposition in CVB3-infected hearts 28 days pi with CVB3 and c, d noninfected control hearts. Picrosirius-red stainings (a, c) and also picrosirius-red stainings after polarization (b, d) reveal significant differences of cardiac fibrosis in infected hearts compared to noninfected hearts. To quantify the fibrous tissue in the course of myocarditis, the area fractions of fibrosis were determined by a digital image analysis system. e At day 28 pi, when inflammation declined and infected myocytes have been eliminated from the myocardium, a significant part (22%) of the heart of susceptible ABY/SnJ mice was composed of collagen deposits (asterisk, P < 0.01 day 28 pi vs noninfected heart tissue). In noninfected hearts of susceptible mice, the percentage of fibrous tissue was less than 3% 54 Localization of CTGF mRNA in hearts of CVB3-infected mice To examine the expression patterns of cardiac CTGF mRNA and to determine the spatial relationship to CVB3infected cardiac cells in the course of myocarditis, we performed in situ hybridization experiments from heart tissue of ABY/SnJ mice. Radioactive in situ hybridization for the detection of CTGF mRNA (Fig. 3a,c,e,g) and CVB3 RNA (Fig. 3b,d,f,h) was done on consecutive paraffinembedded tissue sections taken from hearts at different time points pi. As shown in Fig. 3a, in noninfected hearts (Fig. 3b), low levels of CTGF mRNA were only expressed in some endothelial cells and a few perivascular interstitial cells. In contrast, in hearts obtained 4 days after CVB3 infection (Fig. 3d), a significant expression of CTGF mRNA was observed in endothelial cells but additionally in numerous interstitial cells within the myocardium (Fig. 3c). As at day 4 pi, inflammatory cells, such as macrophages or T cells, are not yet present in CVB3infected mouse hearts [4, 21], it is most likely that CTGFpositive cells in the interstitium represent fibroblasts. At days 8 and 12 pi (Fig. 3e), CTGF mRNA expression patterns were comparable to those observed at day 4 pi and did not colocalize with CVB3-positive myocytes (Fig. 3f). The presence of CTGF mRNA expression in areas where no viral genomes are detectable point to a possible paracrine effect of CTGF regulation (compare encircled areas in Fig. 3c,d and e,f). At later stages of myocarditis (28 days pi), CTGF mRNA-positive interstitial cells (Fig. 3g) were only observed in regions with persistently infected cardiac cells (Fig. 3h). Myocytes were not found to express CTGF mRNA at any time of infection. J Mol Med (2008) 86:49–60 (P>0.05). In situ hybridization revealed that TGF-β is mainly expressed in interstitial cells within areas of inflammation of infected hearts (Fig. 4b, arrow). As CTGF is well known to induce the synthesis of procollagen type I [8], we also performed quantitative RTPCR from the cardiac tissue to determine the levels of Coordinate cardiac expression of CTGF, TGF-β, and procollagen in the course of CVB3 myocarditis TGF-β has been implicated as an initiating cytokine in numerous fibrotic disorders and is well known as a potent stimulator of CTGF and collagen synthesis [6, 22]. To evaluate whether there is coordinate expression of TGF-β and CTGF mRNA in the course of CVB3 myocarditis, quantitative RT-PCR assays were performed from ABY/SnJ mouse heart tissue for the detection of TGF-β1, which is described to be the most relevant TGF-β in the induction of fibrosis (Fig. 4a). A basal level of TGF-β mRNA expression could already be detected in the myocardium of uninfected mice. Comparable to CTGF, TGF-β mRNA was upregulated upon CVB3 infection as soon as day 4 pi, and the peak of TGF-β expression was noted at day 8 pi. Compared to uninfected control hearts, cardiac TGF-β transcript levels were still elevated after the acute phase of myocarditis but did not reach statistical significance Fig. 2 Upregulation of CTGF mRNA and protein in CVB3-infected mouse hearts. The expression levels of CTGF mRNA were determined by quantitative RT-PCR and normalized against those of GAPDH mRNA. a Values are the mean of seven mice in each group. CTGF mRNA in CVB3-infected mouse hearts was found to be significantly upregulated at days 4, 8, and 12 pi (asterisk, P<0.01 infected vs. noninfected hearts). b This finding is paralleled by enhanced CTGF protein expression in CVB3-infected hearts as shown by Western blot analysis. c The densitometric analysis of CTGF protein expression proves a significant upregulation at any time of infection (asterisk, P< 0.01 infected (days 4, 8, 12, 28 pi vs. noninfected hearts). d Immunohistochemical stainings reveal CTGF expression in noninfected (non-inf.) mouse hearts in endothelial cells (arrows) and a few interstitial cells (arrowheads). In infected hearts the localization of CTGF protein was found to be confined to endothelial cells (arrows) and numerous interstitial cells (arrowheads point exemplarily to some positive interstitial cells) as shown in a heart obtained 12 days pi J Mol Med (2008) 86:49–60 55 target for CVB3 [21]. To investigate in detail whether the interstitial cells expressing CTGF mRNA in the heart may represent fibroblasts, we performed experiments on isolated primary heart fibroblasts of ABY/SnJ mice. Infectivity of these cells was proven by quantitative RT-PCR, demonstrating a threefold increase in CVB3 copy numbers between 3 and 6 h after infection with CVB3, showing that CVB3 can replicate in primary mouse heart fibroblasts. Moreover, cardiac fibroblasts expressed increased transcript levels of CTGF upon CVB3 infection (P<0.01) as shown in Fig. 5a. To determine whether this cell type may further contribute to the development of CVB3-induced cardiac fibrosis, we have also determined the levels of TGF-β1 mRNA and procollagen type Iα2 mRNA in CVB3-infected Fig. 3. a, c, e, g Visualization of CTGF transcripts and b, d, f, h genomic CVB3 RNA in consecutive heart tissue sections by in situ hybridization in the course of CVB3-infection. a, b In non-infected mice myocardial CTGF mRNA is confined to endothelial cells and a few perivascular cells. c, e, g In the course of infection CTGF mRNA is detected in endothelial cells and also in numerous interstitial cells within the myocardium, most likely representing fibroblasts. d, f, h Expression of CTGF mRNA was not found to be spatially correlated with CVB3-infected myocytes. The detection of CTGF mRNA positive cells in areas where no CVB3 genomes are detectable point to a possible paracrine effect of CTGF regulation (compare encircled areas in c, d and e, f). (d=days pi) procollagen type I transcripts upon CVB3 infection. As shown in Fig. 4c, the course of procollagen type Iα2 mRNA expression in infected hearts paralleled the expression patterns of CTGF and TGF-β. Procollagen type I mRNA expression reached a maximum 12 days pi and declined slowly in the course of myocarditis (P<0.01 for 4, 8, 12, 28 days pi vs noninfected hearts). Expression of CTGF, TGF-β, and procollagen in CVB3infected primary fibroblasts From our results of electron microscopic in situ hybridization experiments performed from acutely and persistently infected mice, we know that heart fibroblasts represent a Fig. 4 a Detection of TGF-β mRNA in CVB3-infected hearts by quantitative RT-PCR in the course of myocarditis. The expression levels of TGF-β mRNA were normalized against GAPDH mRNA expression levels. Values are the mean of n=7 mice in each group. The maximum of TGF-β mRNA is reached during acute myocarditis and slowly declines at later stages of the disease. b In situ hybridization reveals TGF-β expression mainly in cells within inflammatory lesions (arrow) as exemplarily shown in a mouse heart 12 days pi (left image). Only background levels of TGF-β mRNA are observed in noninfected hearts (right image). c Quantitative RT-PCR for the detection of procollagen type Iα2 mRNA in the course of infection reveals significant expression levels at 4, 8, 12, and 28 days pi (asterisk, P<0.01) compared to noninfected hearts 56 J Mol Med (2008) 86:49–60 Relative CTGF mRNA expression a have incubated murine cardiac fibroblasts with LY294002 (50 μM) and infected cells with CVB3 at a MOI of 10. Treatment of fibroblasts with LY294002 resulted in a significant decrease in the ratio of phosphorylated Akt (p-Akt)/Akt (Fig. 6a, P<0.05) and in a reduction in CTGF mRNA expression by 50% in treated compared to nontreated cells (Fig. 6b, P<0.05). P<0.01 3,5 3 2,5 2 1,5 1 0,5 0 a non-inf. P-Akt 6 h pi CVB3 Akt P<0.05 3 0 µM 50µM Ly 294002 + CVB3 6 h pi 2,5 2 3 1,5 2,5 pAkt/Akt Relative TGF-β mRNA expression b 1 0,5 0 non-inf. 6 h pi CVB3 1,5 1 0 P<0.05 50µM 0 µM LY294002 + CVB3 6 h pi 2 1,5 1 b 0,5 P<0.05 3,5 0 non-inf. 6 h pi CVB3 Fig. 5 Quantitative real-time RT-PCR experiments from cultivated murine primary heart fibroblasts reveal significant expression of a CTGF mRNA (P<0.01), b TGF-β mRNA (P<0.05), and c procollagen type Iα2 mRNA (P<0.05) in CVB3-infected cells vs noninfected cells. The expression levels of CTGF, TGF-β1, and procollagen mRNAs are normalized against those of GAPDH mRNA. Values are the mean from triplicate experiments. Error bars indicate SEM primary heart fibroblasts and compared them with those of noninfected cells. As demonstrated in Fig. 5b and c, both types of mRNA were significantly upregulated in infected fibroblasts (P<0.05), underlining the relevance of this cell type in the pathogenesis of virus-induced cardiac fibrosis. From studies in HeLa cells, it is known that CVB3 leads to phosphorylation of PKB/Akt through a PI3K-dependent mechanism and that treatment with the specific PI3Kinhibitor LY294002 significantly suppresses viral RNA expression [23]. To investigate whether these mechanisms may also play a role in CVB3-mediated cardiac fibrosis, we Relative CTGF mRNA expression Relative procollagen mRNA expression 2 0,5 c 2,5 P<0.05 3 2,5 2 1,5 1 0,5 0 0 µM 50µM LY294002 + CVB3 6 h pi Fig. 6 Cultivated murine primary heart fibroblasts were treated with the PI3 kinase inhibitor LY294002 (50 μM) or left untreated before CVB3 infection for 6 h. a Treatment of fibroblasts with LY294002 resulted in a significant decrease in the ratio of phosphorylated Akt (p-Akt)/Akt (P<0.05) b Quantitative real time RT-PCR experiments reveal a significant decrease in CTGF mRNA in treated cells compared to nontreated cells (P<0.05) Values are the mean from triplicate experiments. Error bars indicate SEM J Mol Med (2008) 86:49–60 Reduced CTGF expression by downregulation of CVB3 replication using siRNA As siRNA molecules were found to effectively inhibit CVB3 replication in vitro in HeLa cells and also in vivo, we wanted to elucidate whether these molecules also represent a potential treatment option for preventing CVB3-induced fibrosis. For this purpose, we used for our experiments with siRNA molecules CVB3-infected HeLa cells, a widely accepted in vitro model to investigate cellular processes relevant in CVB3-induced diseases. To determine the efficacy for the siRNA transfection into HeLa cells, we used in a first approach siGL2–fluorescein-conjugated molecules, which resulted in 87±5% fluorescein-positive cells, thus ensuring an excellent delivery efficacy. Consistently, with our findings in primary cardiac mouse fibroblasts, CVB3 was found to trigger significantly CTGF expression in HeLa cells. At the protein (P<0.05) and mRNA (P<0.05) levels, CTGF amounts increased of about 2.5 and 3-folds, respectively, in infected HeLa cells compared to uninfected cells (Fig. 7a,b,c). Treatment of HeLa cells by a specific siRNA (siRNA3541) directed against the viral 2Apro RNA before CVB3 infection was found to reduce CVB3 replication as demonstrated by reduced levels of CVB3 genomes (Fig. 7d) (quantitative RT-PCR, P<0.01), viral capsid protein VP1 (Western blotting), infectious virions (plaque assay, P<0.05), and of cell cytotoxicity (LDH test, P<0.05), compared to that of CVB3-infected and siRNAGL2-treated infected HeLa cells. Importantly, siRNA3541 also significantly reduced CTGF mRNA levels (Fig. 7e, P<0.05) compared to infected and also to siRNAGL2-treated infected HeLa cells. In contrast, the pretreatment of HeLa cells by siRNA molecules targeted against CTGF, although significantly (P<0.05) reducing CTGF mRNA (Fig. 7e), did not significantly reduce CVB3 loads, as determined by quantitative evaluation of viral genomes (Fig. 7d, P>0.05) and capsid protein VP1 (data not shown), indicating that CTGF is not essential for the CVB3 life cycle. Finally, our data confirm that the chosen siRNA molecule targeting CTGF reduces CTGF mRNA levels also in noninfected HeLa cells, confirming that this siRNA molecule is adequate for the downregulation of CTGF transcripts. (Fig. 7e, P<0.05). Discussion In this study, we took advantage of the murine model of ongoing CVB3 myocarditis to examine the interrelationships between the immune cell response and the development of cardiac fibrosis, a key determinant in heart dysfunction and failure [2]. It has been shown by different groups that accumulation of interstitial matrix proteins 57 during later stages of murine CVB3 myocarditis depends on the balance between stimulation of collagen synthesis and degradation by matrix metalloproteinases (MMPs). There is evidence that accumulation of MMPs and downregulation of tissue inhibitor of MMPs (TIMP) may lead to a functional defect of the collagen turnover, thus influencing cardiac remodeling processes [24]. Moreover, dysregulation of the myocardial MMP/TIMP systems in CVB3 myocarditis was found to be mediated by the inflammatory response via induction of specific cytokines [25]. It was reported that in the myocardium of susceptible mice, developing chronic myocarditis, numerous profibrotic cytokines, comprising TGF-β, tumor necrosis factor-α, interleukin-1, and interleukin-4 [24, 25] are released already in early stages of CVB3 myocarditis, all being known to mediate proliferation of fibroblasts and the synthesis of collagen. Among these cytokines, TGF-β has been suggested to be the most relevant factor for the stimulation of matrix protein production in vivo [14, 22], thus promoting myocardial remodeling and left ventricular dysfunction. Five different isoforms of TGF-β have been identified, with TGF-β1 as the most relevant isoform in cardiac tissue. Activation of latent TGF-β1 has been linked to the fibrotic complications associated with chronic disorders in many organs and its secretion has also been documented in cardiac fibroblasts, which in turn are differentiated into more active connective tissue cells, called myofibroblasts [22]. As TGF-β can mediate a wide range of biological activities, other mechanisms besides interference with the MMP/TIMP system might play a role in the development of cardiac fibrosis. TGF-β is known to induce accumulation of collagen via a post-TGF-β receptor mechanism mediated by CTGF [8]. Grotendorst et al. [22] have shown that TGF-β stimulates fibroblast proliferation and collagen synthesis and can be effectively blocked by application of anti-CTGF antibodies or by inhibition of CTGF synthesis, indicating CTGF as a secondary cytokine for TGF-β-induced biological effects on fibroblasts. In a mouse fibrosis model, it was reported that CTGF maintains TGF-β-induced fibrosis by sustaining the promoter activity of proα2(I) collagen [26]. The hypothesis that CTGF might also be involved in cardiac remodeling processes was supported by the relevance of CTGF in deoxycorticosterone acetate-induced cardiac fibrosis [11]. Enhanced expression of CTGF was also detected in cardiac tissue of patients with cardiac ischemia supporting the relevance of this molecule in cardiac repair and remodeling processes [15]. Our results in this study add new insights into the pathogenesis of virus-induced cardiac fibrosis, substantiating the finding that fibroblasts function as the main CTGF-producing cell type in the damaged heart. Hereby, it is important to mention that the vast majority of nonmyocyte cells in the myocardium represent endothelial cells and fibroblasts. As demonstrated 58 CTGF protein levels a b CTGF protein levels Relative CTGF expression P< 0.05 CTGF (38 kDa) GAPDH (37 kDa) non-inf. CVB-inf. 3 2 1 0 non-inf. d CTGF mRNA levels 4 Relative CVB3 genome equivalents P< 0.05 3 2 1 0 non-inf. e P<0.01 1 0,5 0 si3541treated CVB3-inf. CVB3-inf. P< 0.05 2,5 2 1,5 1 0,5 0 siCTGFtreated CVB3-inf. by in situ hybridization and immunhistochemistry, in addition to endothelial cells, numerous interstitial cells express CTGF already early in infection at day 4 pi. Inflammatory cells in the interstitium such as macrophages or T cells could be excluded as CTGF-synthesizing cells as they are not yet present in hearts at day 4 pi with CVB3 [4]. P>0.05 1,5 Effects of si3541 and siCTGF on CTGF mRNA levels in infected cells si3541treated CVB3-inf. CVB3-inf. Effects of si3541 and siCTGF on levels of CVB3 genomes f Relative CTGF mRNA expression Relative CTGF mRNA expression c Relative CTGF mRNA expression Fig. 7 Relationship between CTGF expression and CVB3 infection in HeLa cells. HeLa cells were infected by CVB3 and the protein levels of CTGF were evaluated 6 h pi. A representative Western blot is shown in a, and the quantitative results are reported in b (P<0.05). c Comparable to the elevated protein levels, the CTGF mRNA levels are significantly upregulated in CVB3-infected HeLa cells (P<0.05). d HeLa cells transfected with siRNA 3541, directed against the viral proteinase 2A (2Apro) (P<0.01) but not those transfected with siRNA against CTGF were found to have significantly reduced amounts of CVB3 genomes compared to infected and siGL2-treated infected cells. e It is interesting to note that transfection with siRNA against CTGF and also with siRNA directed against CVB3 2Apro before infection was found to reduce CTGF mRNA levels significantly (P<0.05). f The siRNA directed against CTGF (siCTGF) reduced the amount of CTGF mRNA also in noninfected HeLa cells and in noninfected siGL2-transfected HeLa cells (P<0.05), confirming the efficacy of the chosen siRNA molecule for the downregulation of CTGF transcripts J Mol Med (2008) 86:49–60 siGL2treated CVB3-inf. siCTGFtreated CVB3-inf. siGL2treated CVB3-inf. Effects of siCTGF on CTGF mRNA levels in non-infected cells 2,5 P< 0.05 2 1,5 1 0,5 0 siGL2transfected siCTGFtransfected To provide further insights into the regulation of CTGF expression, we have also performed appropriate investigations in resistant C57BL/6 mice, which do not develop a significant fibrosis in the course of myocarditis. We found that these mice develop upon infection with CVB3 only a mild cardiac infection and inflammation and reveal com- J Mol Med (2008) 86:49–60 parably low levels of CTGF within relatively few interstitial cells at any stage of the disease compared to susceptible mice. These data further substantiate the paracrine effects of CTGF regulation on the development of virus-induced cardiac fibrosis. According to in vitro experiments, expression of CTGF in cardiac fibroblasts is significantly correlated with an increase in collagen synthesis [15]. Our findings in CVB3-infected cultivated primary heart fibroblasts to produce simultaneously TGF-β, CTGF, and procollagen type I mRNA substantiates the notion that this cell type is decisive in the development of enteroviral cardiac fibrosis. To delineate regulatory mechanisms of CTGF expression in response to TGF-β and other cytokines, signaling processes have been analyzed in different cultured cell types [14]. Several signaling pathways influence CTGF expression, thereby influencing processes such as synthesis of extracellular matrix proteins. Besides Smad molecules, the Ras/MEK/ERK cascade as well as the JNK-dependent pathway was found to contribute to TGF-β-dependent stimulation of CTGF expression in fibroblasts [14, 27, 28]. In addition, it was shown by Crean et al. [7] that treatment of mesangial cells with CTGF leads to phosphorylation of Akt/PKB and that this phosphorylation can be abrogated by the use of the specific PI3 kinase inhibitor LY294002. It was further reported that VEGF induces CTGF expression via a selectively activated PI3-kinase-Akt pathway [29]. Regarding the molecular mechanisms linking coxsackievirus infection with CTGF expression, it is noteworthy to mention that coxsackieviruses can theoretically mediate CTGF synthesis via different signaling pathways. Coxsackieviruses have not only been shown to phosphorylate ERK [30], but they can also activate JNK [31], thereby activating cyr61, another member of the CCN family [32]. It is interesting to note that CVB3 is also reported to activate the PI3 kinase with subsequent activation of protein kinase B PKB/Akt [23]. The PI3 kinase/PKB/Akt pathway was found to enhance type I collagen expression [33], an effect at least partially resulting from increased response to TGFβ [34] and enhanced formation of CTGF [29]. So far, it is not known to what extent the different pathways may influence CTGF expression in the CVB3-infected cardiac fibroblasts. However, we have evidence that at least PI3 kinase/PKB/Akt signaling is relevant in CVB3-induced cardiac CTGF expression as application of the PI3 kinase inhibitor LY294002 was found to significantly reduce the ratio of pAkt/Akt and also of CTGF mRNA expression in cultivated CVB3-infected heart fibroblasts. Scarring of the heart as a consequence of virus-induced inflammatory heart disease is likely to be the major driving force in the development of cardiac dysfunction. However, at present, there are no treatment options or agents available 59 that may prevent virus replication and cardiac damage and thus cardiac fibrosis. To exploit the possibility of using RNA interference as a therapeutic approach to fight viral heart disease, siRNAs were generated to target the CVB3 genome. In our study, the application of siRNAs against the viral 2Apro was found to downregulate the generation of viral RNA and proteins, thus reducing virus-induced cytopathic effects and the generation of infectious virions in agreement with previous observations [35]. More interestingly, the siRNA directed against the viral 2Apro (siRNA3541) used in this study was found to significantly reduce expression of CTGF mRNA in infected cells. Notably, a siRNA directed against CTGF did not significantly inhibit CVB3 replication, suggesting that CTGF is not necessary for the CVB3 life cycle. Thus, the mechanisms underlying this specific cellular reaction to CVB3 remains to be elucidated. Despite this observation, the design of new intervention strategies aimed to target specific interference with CTGF expression at the sites of progressive fibrosis is now a major topic, as this approach may reinforce the prevention of pathological fibrotic processes [36]. Our results support the notion that the use of siRNAs against CVB3 and CTGF is not only useful to reduce viral load and spread in acute viral heart disease but may also prevent simultaneously the consequences of chronic myocarditis, the development of cardiac fibrosis, and eventual cardiac dysfunction. Acknowledgments This study was supported by the Deutsche Forschungsgemeinschaft, SFB/TR 19-04, the fortüne program of the Medical Faculty of Tübingen (1271-0), and in part by the “Fondazione Cassa di Risparmio of Trieste,” by the “Fondazione Benefica KatleenForeman Casali of Trieste.” G. Grassi is supported by the program “Rientro cervelli” art. 1 DM n.13, MIUR (Ministero dell’Istruzione, dell’Università e della Ricerca Scientifica.” The support of S. Berchtold, Institute for Medical Microbiology and of Sandra Bundschuh, Department of Molecular Pathology, University Hospital Tübingen, is acknowledged. There are no potential conflicts of interest anywhere, relating to this article. 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