2228 FEBS Journal 280 (2013) ª 2013 The Authors Journal compilation ª 2013 FEBS

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1 Innate immune signaling induces expression and shedding of the heparan sulfate proteoglycan syndecan-4 in cardiac fibroblasts and myocytes, affecting inflammation in the pressure-overloaded heart Mari E. Strand 1,2, Kate M. Herum 1,2, Zaheer A. Rana 1,2, Biljana Skrbic 1,2,3, Erik T. Askevold 2,4,5, Christen P. Dahl 2,4,5, Maria Vistnes 1,2, Almira Hasic 1,2, Heidi Kvaløy 1,2, Ivar Sjaastad 1,2,6, Cathrine R. Carlson 1,2, Theis Tønnessen 2,3, Lars Gullestad 2,5,7, Geir Christensen 1,2 and Ida G. Lunde 1,2 1 Institute for Experimental Medical Research, Oslo University Hospital Ulleval and University of Oslo, Norway 2 KG Jebsen Cardiac Research Center and Center for Heart Failure Research, University of Oslo, Norway 3 Department of Cardiothoracic Surgery, Oslo University Hospital Ulleval, Norway 4 Research Institute of Internal Medicine, Oslo University Hospital Rikshospitalet, Norway 5 Department of Cardiology, Oslo University Hospital Rikshospitalet, Norway 6 Department of Cardiology, Oslo University Hospital Ulleval, Norway 7 Institute of Clinical Medicine, University of Oslo, Norway Keywords heart failure; heparan sulfate; inflammation; proteoglycan; shedding Correspondence Ida G. Lunde, Institute for Experimental Medical Research, Oslo University Hospital Ulleval, Building 7, 4th floor, Kirkeveien 166, N-0407 Oslo, Norway Fax: Tel: i.g.lunde@medisin.uio.no (Received 2 December 2012, revised 21 January 2013, accepted 28 January 2013) doi: /febs Sustained pressure overload induces heart failure, the main cause of mortality in the Western world. Increased understanding of the underlying molecular mechanisms is essential to improve heart failure treatment. Despite important functions in other tissues, cardiac proteoglycans have received little attention. Syndecan-4, a transmembrane heparan sulfate proteoglycan, is essential for pathological remodeling, and we here investigated its expression and shedding during heart failure. Pressure overload induced by aortic banding for 24 h and 1 week in mice increased syndecan-4 mrna, which correlated with mrna of inflammatory cytokines. In cardiac myocytes and fibroblasts, tumor necrosis factor-a, interleukin-1b and lipopolysaccharide through the toll-like receptor-4, induced syndecan-4 mrna. Bioinformatical and mutational analyses in HEK293 cells identified a functional site for the proinflammatory nuclear factor-jb transcription factor in the syndecan-4 promoter, and nuclear factor-jb regulated syndecan-4 mrna in cardiac cells. Interestingly, tumor necrosis factor-a, interleukin-1b and lipopolysaccharide induced nuclear factor-jb-dependent shedding of the syndecan-4 ectodomain from cardiac cells. Overexpression of syndecan-4 with mutated enzyme-interacting domains suggested enzymedependent heparan sulfate chains to regulate shedding. In cardiac fibroblasts, lipopolysaccharide reduced focal adhesion assembly, shown by immunohistochemistry, suggesting that inflammation-induced shedding affects function. After aortic banding, a time-dependent cardiac recruitment of T lymphocytes was observed by measuring CD3, CD4 and CD8 Abbreviations AB, aortic banding; ADAMTS, a disintegrin and metalloproteinase domain with thrombospondin motifs; CsA, cyclosporin A. ECM, extracellular matrix; EGFP, enhanced green fluorescent protein; GAG, glycosaminoglycan; HA, hemagglutinin; HEK, human embryonic kidney cells; HSPGs, heparan sulfate proteoglycans; IL, interleukin; LPS, lipopolysaccharide; LV, left ventricle; LVW, left venticle weight; LW, lung weight; NFAT, nuclear factor of activated T cells; NF-jB, nuclear factor jb; TGF, transforming growth factor; TLR4, toll-like receptor 4; TNF, tumor necrosis factor FEBS Journal 280 (2013) ª 2013 The Authors Journal compilation ª 2013 FEBS

2 M. E. Strand et al. Syndecan-4 shedding in the failing heart mrna, which was reduced in syndecan-4 knockout hearts. Finally, syndecan-4 mrna and shedding were upregulated in failing human hearts. Conclusively, our data suggest that syndecan-4 plays an important role in the immune response of the heart to increased pressure, influencing cardiac remodeling and failure progression. Introduction In response to sustained pressure overload, the myocardium undergoes intra- and extracellular alterations affecting the size, shape and function of the heart [1]. This remodeling process drives the progression to heart failure, a syndrome affecting as many as 23 million people worldwide [2,3]. Even when treated according to current recommendations, heart failure remains the main cause of morbidity and mortality in the Western world, reflecting that the underlying mechanisms are poorly understood. Hence, increased knowledge regarding the molecular mechanisms involved is highly relevant to improve current heart failure treatment strategies. Proteoglycans are macromolecules composed of a specific core protein substituted with covalently linked glycosaminoglycan (GAG) chains and are major constituents of the extracellular matrix (ECM). Surprisingly, despite their important functional roles in other tissues, the role of proteoglycans in the heart has received little attention. Thus, research in this field opens a potential for obtaining new and important information about the pathophysiology of cardiac disease. Using microarray, our group found that the four members of the syndecan family of transmembrane heparan sulfate proteoglycans (HSPGs) were upregulated in the noninfarcted, hypertrophic left ventricle following myocardial infarction, suggesting a role for this proteoglycan family in cardiac remodeling [4]. In cardiac cells, one member of the syndecan family, namely syndecan-4, is of particular interest because of its localization to cellular adhesion sites which are important for signaling across the membrane, i.e. focal adhesions in fibroblasts [5] and costameres in cardiomyocytes [6]. Interestingly, we recently showed that syndecan-4 is essential for pathological myocardial remodeling in response to pressure overload [7,8]. Furthermore, we showed increased syndecan-4 levels in the hypertrophic myocardium of patients with aortic stenosis [7], and we [4] and others [9,10] have shown increased syndecan-4 levels in the post-infarcted heart, indicating that this particular proteoglycan is important during heart failure. Despite this, little is known about cardiac regulation of syndecan-4, and in this study, we investigated mechanisms responsible for elevated syndecan-4 in the pressure-overloaded, failing murine and human myocardium. Cardiac inflammation is believed to be central during heart failure progression. Interestingly, heparan sulfate GAG chains, such as those attached to syndecan-4, bind cytokines and immune cells [11,12]. Leukocyte recruitment to the inflamed tissue is an important step in inflammation believed to be dependent on HSPGs, and thus, HSPGs may play important roles in regulating tissue inflammation. However, this process has not previously been linked to syndecan-4 in the pressure-overloaded heart. Furthermore, shedding of the HSPG ectodomain, containing the heparan sulfate GAG chains, is a mechanism in which cell-bound heparan sulfate is turned into a soluble effector, a process that may affect the inflammatory process. Currently, this important aspect of syndecan-4 function has not been investigated in cardiac cells. Thus, we examined whether syndecan-4 shedding from cardiac myocytes and fibroblasts is part of the inflammatory response of the failing heart. Because T cells are believed to be important for the cardiac remodeling process, we also examined whether syndecan-4 affected lymphocyte recruitment to the pressure-overloaded myocardium [13 15]. Results Increased expression of syndecan-4 correlated with inflammatory cytokines in the myocardium of mice following AB To study in vivo regulation of syndecan-4 expression in the pressure overloaded heart, syndecan-4 mrna was measured in left ventricles (LVs) of wild-type (WT) mice following 24 h, 1 and 3 weeks of aortic banding (AB). Post-mortem organ weights confirmed increased LV weight (LVW) and lung weight (LW) at all time points (Table 1). Echocardiographic assessment of LV posterior wall thickness in diastole at 1 and 3 weeks confirmed hypertrophy at both time points. Impaired cardiac function was shown by reduced fractional shortening at both 1 and 3 weeks after AB, and increased left atrial diameter, together with increased FEBS Journal 280 (2013) ª 2013 The Authors Journal compilation ª 2013 FEBS 2229

3 Syndecan-4 shedding in the failing heart M. E. Strand et al. Table 1. Baseline characteristics of aortic banded WT mice. Post-mortem and echocardiographic data (mean SEM) of mice 24 h, 1 week and 3 weeks after aortic banding (AB), and in respective sham-operated controls (n = 58). TL, tibia length; LAD, left atrial diameter; LVPWd, left ventricular posterior wall thickness in diastole; LVIDd, left ventricular internal diameter in diastole; FS, fractional shortening. *P < 0.05; **P < 0.01; ***P < versus respective sham-operated controls. Sham AB24 h AB24 h Sham AB1w AB1w Sham AB3w AB3w N Body weight (g) * LVW/TL (mgmm 1 ) * *** *** LW/TL (mgmm 1 ) *** *** *** Echocardiography N LAD (mm) NA NA *** *** LVPWd (mm) NA NA *** *** LVIDd (mm) NA NA * FS (%) NA NA * * LW, suggested congestive heart failure. Both LVW and LW were increased at 1 and 3 weeks after AB, compared with the 24 h time point (P < for LVW and LW at both time points), with LVW being significantly higher at 3 weeks compared with 1 week of AB (P < 0.01), indicating progressive remodeling. Importantly, LV internal diameter in diastole was significantly reduced after 3 weeks of AB, suggesting progressive concentric remodeling, as expected after AB, over time. Cardiac syndecan-4 mrna levels were increased 24 h (3.6-fold) and 1 week (1.4-fold) after AB compared with respective sham-operated animals, but were not different from sham levels at 3 weeks (Fig. 1A). Furthermore, cardiac syndecan-4 mrna was positively correlated with mrna levels of tumor necrosis factor (TNF)a (Fig. 1B), interleukin (IL)-1b (Fig. 1C), IL-6 (Fig. 1D) and transforming growth factor (TGF)b (Fig. 1E), indicating that inflammatory cytokines may stimulate syndecan-4 expression in the pressure overloaded myocardium. To investigate whether pressure overload would also induce expression of the other three members of the syndecan family, cardiac syndecan-1 3 mrna levels were assessed. Increased mrna of syndecan-1, -2 and -3 were found 24 h after AB (Fig. S1A C), suggesting that pressure overload of the heart induced increased expression levels across the four-membered syndecan HSPG family. A B P < R 2 = 0.47 C P < R 2 = 0.93 NS Sham 24 h AB 24 h Sham AB 1 week AB 1 week Sham AB 3 weeks AB 3 weeks D P < R 2 = 0.88 E P < R 2 = 0.45 Fig. 1. Increased syndecan-4 expression correlated with inflammatory cytokines in the myocardium of mice following AB. Relative mrna levels of syndecan-4 in myocardium of WT mice following 24 h, 1 or 3 weeks of AB and in respective sham-operated controls, n = 9 10 (A). Correlations of cardiac syndecan-4 and TNFa (B), IL-1b (C), IL-6 (D) and TGFb (E) mrna levels in sham- and AB-operated mice at 24 h, 1 and 3 weeks (n = 58). For baseline characteristics of mice, see Table 1. **P < 0.01; ***P < 0.001; ns, nonsignificant; AU, arbitrary units FEBS Journal 280 (2013) ª 2013 The Authors Journal compilation ª 2013 FEBS

4 M. E. Strand et al. Syndecan-4 shedding in the failing heart Expression of syndecan-4 was regulated by proinflammatory stimuli and cyclic mechanical stretch in cardiac cells To identify the direct regulators of syndecan-4 expression in the heart, isolated cardiac myocytes and fibroblasts were treated with inflammatory stimuli. Syndecan-4 mrna was increased by TNFa, IL-1b and lipopolysaccharide (LPS) in both cardiomyocytes (Fig. 2A) and cardiac fibroblasts (Fig. 2C). Of notice, on average, LPS increased syndecan-4 expression sixfold (ranging from 1.5-fold to 13-fold) in cardiac fibroblasts. IL-6, TGFb, IL-18 and CXCL16 had no direct effects on syndecan-4 mrna levels, nor did angiotensin II and norepinephrine, the two central neurohumoral players of heart failure. To investigate pressure overload as a stimulus per se, cells were subjected to cyclic mechanical stretch for 24 h. Syndecan-4 mrna was increased 1.5-fold in response to mechanical stretch of cardiomyocytes (Fig. 2B), whereas no change was seen in cardiac fibroblasts (Fig. 2D). Cardiac expression of both cytokines regulating syndecan- 4 in cells was upregulated at 24 h (TNFa and IL-1b) and 1 week (IL-1b) following AB, but not at 3 weeks (Fig. 2E F), showing a similar expression pattern to that of syndecan-4 (see Fig. 1A) and suggesting that these two cytokines regulate syndecan-4 mrna in the heart. The innate immunity receptor TLR4 regulated syndecan-4 expression in cardiac cells Interestingly, the induction of syndecan-4 expression by LPS suggested involvement of the innate immune system and the LPS-specific toll-like receptor (TLR)4 in the heart [16,17]. To examine the direct role of TLR4 in regulating syndecan-4 mrna, cardiac myocytes and fibroblasts were cotreated with TLR4 inhibitors. Blocking TLR4 attenuated LPS-induced syndecan-4 expression in both cardiac cell types (Fig. 2G H), indicating the innate immunity receptor TLR4 as a regulator of syndecan-4 expression in the heart. The syndecan-4 promoter contained a functional nuclear factor-jb transcription factor site Next, we investigated which transcription factors mediated syndecan-4 expression. Bioinformatical analysis of the first 1000 bp of the mouse syndecan-4 promoter was performed to identify possible sites for the proinflammatory transcription factor nuclear factor jb (NF-jB) and A B C D NS E F G H NS NS NS Sham 24 h AB 24 h Sham AB 1 week AB 1 week Sham AB 3 weeks AB 3 weeks Sham 24 h AB 24 h Sham AB 1 week AB 1 week Sham AB 3 weeks AB 3 weeks Fig. 2. Syndecan-4 expression was regulated by mediators of innate immunity and mechanical stretch in cardiac myocytes and fibroblasts. Relative expression of syndecan-4 mrna in cardiomyocytes (A) and cardiac fibroblasts (C) following 24 h stimulation with TNFa, IL-1b, LPS, angiotensin II, norepinephrine, CXCL16, IL-18, TGFb and IL-6, and in cardiomyocytes (B) and cardiac fibroblasts (D) subjected to cyclic mechanical stretch (MS) for 24 h. Relative mrna levels of TNFa (E) and IL-1b (F) in myocardium of WT mice following 24 h, 1 or 3 weeks of AB and in respective sham-operated controls. Expression of syndecan-4 mrna in cardiac myocytes (G) and fibroblasts (H) treated with LPS alone or LPS combined with TLR4 inhibitors CLI-095 and LPS-RS. *P < 0.05; **P < 0.01; ***P < 0.001; ns, nonsignificant. FEBS Journal 280 (2013) ª 2013 The Authors Journal compilation ª 2013 FEBS 2231

5 Syndecan-4 shedding in the failing heart M. E. Strand et al. the pro-hypertrophic nuclear factor of activated T cells (NFAT). An NF-jB site (GGGGAATTCC) at -106/ -97 bp was confirmed, as well as four NFAT binding sites (TTTCC) identified at -557/-553, -500/-495, -331/ -327 and -151/-147 bp (Fig. 3A). To investigate the contribution of NF-jB and NFAT in regulation of syndecan-4 expression, enhanced green fluorescent protein (EGFP) reporter constructs were designed containing the WT syndecan-4 promoter (-1010/-1), the syndecan-4 promoter with inactive NF-jB site and syndecan-4 promoter where NF-jB and the four NFAT sites were inactive (Fig. 3B). Because of the low transfection efficiency of primary culture cardiac cells [18], these syndecan-4 promoter constructs were transfected into human embryonic kidney (HEK293) cells. A Immunoblotting revealed that in these noncardiac cells, TNFa increased EGFP in cells transfected with WT syndecan-4 promoter (Fig. 3C,D). The increase in EGFP in response to TNFa was significantly reduced when the NF-jB site alone was mutated, and not different from when both NF-jB and NFAT sites were mutated (Fig. 3C,D). Supporting that NF-jB is responsible for syndecan-4 transcription, TNFa, but not IL-1b nor LPS, activated NF-jB in this noncardiac cell type, shown by reduced levels of the NF-jB inhibitory protein IjBa (Fig. 3C,E). TNFa also activated NFAT in this cell type, shown by reduced levels of phosphorylated NFATc4 (Fig. 3C,F). To examine the role of NFAT alone, HEK293 cells transfected with the WT syndecan-4 promoter were cotreated with TNFa and the B C D E F G H I Fig. 3. The syndecan-4 promoter contained a functional NF-jB transcription factor site. One NF-jB and NFAT binding sites were found in the syndecan-4 promoter by bioinformatical analyses, and are indicated in the schematic relative to the mouse syndecan-4 start codon (arrow) (A). Schematic of EGFP reporter constructs expressing the syndecan-4 WT promoter, syndecan-4 promoter with NF-jB mut or syndecan-4 promoter with NFAT and NF-jB sites mutated (NFAT + NF-jB mut) used to transfect HEK293 cells (B). Representative immunoblots and quantitative data of EGFP in HEK293 cells transfected with syndecan-4 reporter constructs and treated with TNFa, IL-1b or LPS (C,D). IjBa protein (C,E) and NFATc4 phosphorylation (C,F) were used to confirm NF-jB and NFAT activation, respectively. Representative immunoblot of EGFP in HEK293 cells transfected with the WT syndecan-4 promoter and treated with TNFa alone or in combination with the calcineurin-nfat inhibitor CsA (G,H). Alignment of mouse, rat and human syndecan-4 promoter with conserved NF-jB and NFAT binding sites highlighted (I). GAPDH was used for loading control. *P < 0.05; **P < 0.01; ***P < 0.001; ns, nonsignificant FEBS Journal 280 (2013) ª 2013 The Authors Journal compilation ª 2013 FEBS

6 M. E. Strand et al. Syndecan-4 shedding in the failing heart calcineurin-nfat inhibitor cyclosporin A (CsA). EGFP levels were not different in cells stimulated with TNFa alone and those costimulated (Fig. 3G,H), excluding transcriptional regulation of syndecan-4 by NFAT in this noncardiac cell type. Finally, alignment of the syndecan-4 promoter in mouse, rat and human revealed that the NF-jB site and the NFAT sites at -331/-327 and -151/ -147 were conserved among the three species (Fig. 3I). Proinflammatory NF-jB signaling regulated syndecan-4 transcription in cardiac cells To determine if syndecan-4 expression in the heart was dependent on NF-jB, cardiac myocytes and fibroblasts were treated with the NF-jB inhibitor SM7368. Basal and TNFa-, IL-1b- and LPS-induced syndecan-4 expression were inhibited by blocking NF-jB signaling, showing that NF-jB regulates transcription of syndecan-4 in both cardiac cell types (Fig. 4A B). Increased levels of phospho-p65 NF-jB, p65 NF-jB and reduced levels of IjBa were found after TNFa, IL-1b and LPS treatment of both cardiac myocytes and fibroblasts, confirming NF-jB activation by all three proinflammatory stimuli which regulated syndecan-4 expression in these cardiac cell types (Fig. 4C J). Calcineurin NFAT signaling is a central mediator of hypertrophy in cardiomyocytes [19,20]. Thus, we investigated whether NFAT signaling was important for syndecan-4 expression in this particular cardiac cell type. NFAT activity in response to TNFa, IL-1b and LPS was assessed in cardiomyocytes harboring an NFAT luciferase reporter system [21]. TNFa increased NFAT activation in cardiomyocytes, and TNFainduced NFAT activity was blocked by CsA, confirming the specificity of this blocker (Fig. 4K). However, although CsA blocked NFAT activation, CsA did not cause any change in TNFa-induced syndecan-4 expression in cardiomyocytes (Fig. 4L), showing that NFAT activity does not regulate syndecan-4 expression in cardiomyocytes. These results suggest that NF- jb, but not NFAT, regulates transcription of syndecan-4 in cardiac myocytes and fibroblasts, similar to what we found in the noncardiac HEK293 cells (see Fig. 3). TNFa, IL-1b and LPS promoted syndecan-4 shedding from cardiac cells To study whether the proinflammatory stimuli regulating syndecan-4 mrna affected syndecan-4 protein levels, we performed immunoblotting of TNFa-, IL-1band LPS-treated cardiac myocytes and fibroblasts. Interestingly, increased levels of a ~ 10 kda syndecan-4 fragment was observed after TNFa, IL-1b and LPS treatment of cardiomyocytes (Fig. 5A,B) and cardiac fibroblasts (Fig. 5C,D). TNFa induced less of this fragment than IL-1b and LPS. Importantly, the fragment was detected using an antibody recognizing a cytoplasmic epitope, but not with an antibody recognizing an extracellular epitope (Fig. 5E) [7], suggesting that the fragment constituted the cellular part of syndecan-4 remaining after shedding of the extracellular part. The same ~ 10 kda cellular syndecan-4 cleavage product was observed when overexpressing full-length syndecan- 4 in HEK293 cells, suggesting constitutive shedding when syndecan-4 levels were elevated, and the amount of the cellular shedding fragment was increased by TNFa (Fig. 5F,G), which also stimulated syndecan-4 expression in this cell type (see Fig. 3C,D). Cellular syndecan-4 shedding was confirmed by elevated syndecan-4 levels in the culture medium following TNFa treatment, measured by ELISA (Fig. 5H). Constitutive shedding of syndecan-4 was also found in HL-1 cardiomyocytes (Fig. 5I) and HT1080 fibroblasts (Fig. 5J) following syndecan-4 overexpression. Finally, we performed HEK293 experiments to confirm that the ~ 10 kda syndecan-4 fragment represented the cellular cleavage product of syndecan-4 after shedding of the extracellular part. Membrane protein fractions of HEK293 cells overexpressing syndecan-4 confirmed that the cellular fragment was found in the cell membrane (Fig. 5K). This suggested the presence of the syndecan- 4 transmembrane domain in the cleavage product remaining in cells after shedding of the extracellular part, and that this cleavage product does not immediately translocate to other cellular compartments nor is it degraded. Furthermore, HEK293 overexpressing fulllength syndecan-4 and syndecan-4 with the 28-amino acid cytoplasmic domain exchanged with an intracellular flag-tag were analysed for presence of the fragment. An anti-flag IgG confirmed the presence of a cellular fragment slightly smaller than the fragment from syndecan-4 (Fig. 5L), suggesting again that the fragment represents the cellular part of syndecan-4 after shedding. Finally, as predicted, only full-length, and not the ~ 10 kda syndecan-4 fragment, was detected using an antibody towards the N-terminal hemagglutinin (HA) tag (Fig. 5M). To study whether the proinflammatory stimuli also affected full-length syndecan-4 levels, TNFa-, IL-1b- and LPS-stimulated cardiac myocytes and fibroblasts were treated with heparan sulfatedegrading enzymes. TNFa, IL-1b and LPS increased full-length syndecan-4 in cardiomyocytes (Fig. 5N,O), whereas in cardiac fibroblasts, full-length syndecan-4 was only increased by IL-1b (Fig. 5P,Q). Collectively, our data indicate that proinflammatory stimuli induce syndecan-4 expression and subsequent FEBS Journal 280 (2013) ª 2013 The Authors Journal compilation ª 2013 FEBS 2233

7 Syndecan-4 shedding in the failing heart M. E. Strand et al. A B C D E F G H I J K L Fig. 4. Syndecan-4 expression was regulated by NF-jB in cardiac cells. Relative expression of syndecan-4 mrna in cardiomyocytes (A) and cardiac fibroblasts (B) following 24 h stimulation with TNFa, IL-1b and LPS compared with respective costimulation with the NF-jB inhibitor SM7368. Representative immunoblots (C, G) and quantitative data of p65 NF-jB phosphorylation (D, H), p65 NF-jB (E, I) and IjBa protein (F, J) showing NF-jB activation by 24 h TNFa, IL-1b and LPS treatment of cardiac myocytes and fibroblasts, respectively. GAPDH was used for loading control. Relative NFAT luciferase (NFAT luc) activity was measured in cardiomyocytes from NFAT luc reporter mice after 24 h treatment with TNFa, IL-1b, LPS, the calcineurin-nfat inhibitor CsA or TNFa and CsA in combination (K). Relative levels of syndecan-4 mrna were measured after treatment with TNFa, CsA or TNFa and CsA in combination (L). *P < 0.05; **P < 0.01; ***P < 0.001; ns, nonsignificant. shedding of the extracellular part of this proteoglycan from cardiac myocytes and fibroblasts. Syndecan-4 shedding from cardiac cells was regulated by proinflammatory NF-jB signaling and enzymes depending on its heparan sulfate glycosaminoglycan chains As cardiac syndecan-4 expression was regulated by NF-jB signaling, we investigated whether syndecan-4 shedding from cardiac cells also was a result of activation of this proinflammatory intracellular pathway. Indeed, when NF-jB was blocked using the SM7368 inhibitor, the cellular cleavage product of syndecan-4 was barely detectable in cardiac myocytes (Fig. 6A) and fibroblasts (Fig. 6B), suggesting NF-jB to regulate both increased expression and shedding in cardiac cells. Shedding of syndecan-4 is believed to be mediated by enzymatic cleavage at the juxtamembrane domain (amino acid sequence SNIFERTE) or close to the heparan sulfate attachments sites (S44/S62/S64) [22] (Fig. 6C). To study the cleavage site resulting in 2234 FEBS Journal 280 (2013) ª 2013 The Authors Journal compilation ª 2013 FEBS

8 M. E. Strand et al. Syndecan-4 shedding in the failing heart A C E F G H B D I J K N P L O Q M Fig. 5. TNFa, IL-1b and LPS induced syndecan-4 shedding from cardiac cells. Representative immunoblots and quantitative data of syndecan-4 protein in cardiomyocytes (A, B) and cardiac fibroblasts (C, D) stimulated with TNFa, IL-1b and LPS using antibodies against a cytoplasmic epitope in syndecan-4 (A, C; upper) and an extracellular epitope (middle). The ~ 10 kda cellular syndecan-4 fragment was quantified. Schematic of the two antibodies used to detect syndecan-4 (E). Levels of cellular syndecan-4 protein fragment and IjBa protein following stimulation for 24 h with TNFa in HEK293 cells overexpressing full-length human influenza HA-tagged syndecan-4 (HA syn-4), shown as representative immunoblots (F) and quantitative data (G). Relative syndecan-4 levels (pgml 1 ) in cell culture medium were measured by ELISA (H). Representative immunoblots of syndecan-4 protein in HL-1 cardiomyocytes (I) and HT1080 fibroblasts (J) overexpressing HA syn-4. Syndecan-4 in membrane fractions of HA syn-4-overexpressing HEK293 cells (K). Representative immunoblots showing levels of syndecan-4 and flag-tagged protein in HEK293 cells transfected with HA syn-4 and syndecan-4 with the cytoplasmic domain exchanged with an intracellular flag-tag [syn-4 (ext + mem)-5g-flag] (L) and levels of HA-tagged protein in HEK293 cells transfected with HA syn-4 (M). Arrow indicates the ~ 10 kda cellular syndecan-4 fragment resulting from shedding of the ectodomain. GAPDH, EGFR and actin were used for loading control. Representative immunoblots and quantitative data of syndecan-4 protein in TNFa, IL-1b and LPSstimulated cardiomyocytes (N,O) and cardiac fibroblasts (P, Q) after treatment with HS-degrading enzymes. *P < 0.05; **P < 0.01; ***P < FEBS Journal 280 (2013) ª 2013 The Authors Journal compilation ª 2013 FEBS 2235

9 Syndecan-4 shedding in the failing heart M. E. Strand et al. A B C D E Fig. 6. Shedding of syndecan-4 from cardiac cells was regulated by NF-jB and enzymes depending on its heparan sulfate glycosaminoglycan chains. Representative immunoblots of syndecan-4 protein shown for TNFa, IL-1b and LPS-stimulated cardiomyocytes (A) and cardiac fibroblasts (B) cotreated with the NF-jB inhibitor SM7368 (A). Arrow indicates the ~10 kda cellular syndecan-4 fragment resulting from shedding of the ectodomain. Schematic of the two known enzymatic cleavage domains in the syndecan-4 ectodomain [22] (C). Syndecan-4 in HEK293 cells transfected with human influenza HA-tagged syndecan-4 (HA syn-4), HA syn-4 D and HA syn-4 S44A/S62A/S64A, shown as representative immunoblots (D) and quantitative data (E). GAPDH and vinculin were used for loading control. *P < 0.05; ***P < the cellular syndecan-4 fragment observed in our studies, syndecan-4, syndecan-4 without the SNIFERTE domain and syndecan-4 with nonfunctioning heparan sulfate GAG chain attachment sites (S44A/S62A/ S64A) were overexpressed in HEK293 cells. Immunoblotting showed that removal of the juxtamembrane shedding domain only slightly reduced the amount of constitutive shedding (by 18%) (Fig. 6D,E). Interestingly, mutation of the heparan sulfate GAG attachment sites reduced the amount of cellular syndecan-4 fragment by 66%. Thus, although mutating GAG attachment sites could affect the accessibility of enzymes to the core protein [23], our data suggest that syndecan-4 shedding primarily is regulated by enzymes dependent on interacting with heparan sulfate GAG chains. Shedding of syndecan-4 altered cardiac fibroblast adhesion properties One of the established, important functions of syndecan-4 is participation in focal adhesion assembly and actin stress fiber formation [24,25]. Thus, we investigated whether inflammation-induced syndecan-4 shedding affected this process in cardiac fibroblasts. A positive correlation between syndecan-4 mrna and shedding indicated that the majority of syndecan-4 induced by LPS is shed from cardiac fibroblasts (Fig. 7A). As previously shown [8,26], isolated syndecan-4 knockout fibroblasts have a reduced number of focal adhesions compared with WT cardiac fibroblasts (Fig. 7B). Interestingly, when WT cardiac fibroblasts were treated with LPS, the number of focal adhesions was reduced compared with controls, resembling genetic ablation of this proteoglycan. Thus, these results suggest that increased syndecan-4 shedding reduces cardiac fibroblast adhesion properties. Reduced T-lymphocyte recruitment and cardiac dysfunction after AB in mice lacking syndecan-4 Heparan sulfate GAG chains have been shown to participate in essentially every stage of leukocyte transmigration through the blood vessel wall, thereby regulating T-cell recruitment into the tissue [11,12]. Altered syndecan-4 levels could therefore affect recruitment of T cells into cardiac tissue following pressure overload, and thereby affect inflammation. To investigate whether syndecan-4 is important for this process, mrna levels of T-cell markers were assessed in the LVs of WT and syndecan-4 knockout mice after AB. In WT mice there was a approximately fourfold 2236 FEBS Journal 280 (2013) ª 2013 The Authors Journal compilation ª 2013 FEBS

10 M. E. Strand et al. Syndecan-4 shedding in the failing heart A B Fig. 7. LPS stimulation of cardiac fibroblasts reduced focal adhesion properties. Correlation of syndecan-4 mrna and shedding fragment in cardiac fibroblasts (two samples from four cell cultures) (A). Representative confocal immunofluorescence images showing staining of focal adhesion components F-actin (phalloidin in red) and vinculin (green), and as merged pictures, in nonstimulated syndecan-4 knockout (upper) and WT (middle) cardiac mouse fibroblasts cultured on fibronectin-coated glass cover slips (B). Cardiac fibroblasts after 24 h of LPS stimulation are shown in lower panels. Scale bars 50 and 10 lm as indicated. Omission of primary antibodies was used as negative control. increase in expression of the pan-t-cell receptor CD3 3 weeks after AB (Fig. 8A). Expression of CD4, representing T-helper cells, was significantly increased at both 1 and 3 weeks of AB (Fig. 8B), whereas expression of CD8, representing cytotoxic T cells, was significantly induced at the 3-week time point (Fig. 8C). Thus, infiltration of T lymphocytes seemed to be part of the late, rather than the early, response to pressure overload. Interestingly, expression of CD3 (Fig. 8D), CD4 (Fig. 8E) and CD8 (Fig. 8F) was significantly reduced in syndecan-4 knockout compared with WT hearts following 3 weeks of AB, suggesting that syndecan-4 is important for T-cell recruitment to the pressure-overloaded heart. Moreover, we have previously shown that syndecan-4 knockout mice develop premature heart failure and cardiac dilatation following AB [7]. Increased dilatation and reduced heart function compared with WT hearts following AB, as shown by increased LV internal diameter in diastole (Fig. 8G) and reduced fractional shortening (Fig. 8H), was confirmed also in the present data set. Thus, following 3 weeks of pressure overload, syndecan-4 knockout mice displayed reduced cardiac T-cell recruitment and reduced function compared with WT mice. Furthermore, syndecan-4 mrna levels correlated positively to LW following AB, suggesting that syndecan-4 induction is higher in animals with a more severe response to pressure overload (Fig. 8I). To investigate whether reduced T-lymphocyte recruitment in syndecan-4 knockout hearts following AB was due to a general impairment of inflammation in these mice, cardiac expression levels of various inflammatory cytokines were assessed. The results showed that syndecan-4 knockout mice produced an adequate immune response as indicated by elevated expression levels of the cytokines TNFa (Fig. 9A), IL-1b (Fig. 9B), IL-6 (Fig. 9C), IL-18 (Fig. 9D) and TGFb (Fig. 9E) after AB, suggesting that the reduced T-cell recruitment was not due to an impaired cytokine response. Of note, syndecan-4 knockout mice expressed higher levels of cardiac TNFa, IL-6, IL-18 and TFGb compared with WT following AB, suggesting an increased level of general inflammation to accompany exacerbated dysfunction when syndecan-4 was not present in the heart. Increased inflammation in syndecan-4 knockout hearts following AB was finally demonstrated by a higher expression level of a marker found on all leukocytes, i.e. CD45R, compared with WT hearts (Fig. 9F). In contrast to a study showing that syndecan-4 is important for macrophage recruitment to the heart in response to myocardial infarction [9,10], we found no significant differences in the macrophage marker F4/80 between syndecan-4 knockout and WT in response to AB (Fig. S2). Syndecan-4 expression and shedding were increased in failing human myocardium Finally, we investigated syndecan-4 expression and shedding in the failing human myocardium. Syndecan-4 FEBS Journal 280 (2013) ª 2013 The Authors Journal compilation ª 2013 FEBS 2237

11 Syndecan-4 shedding in the failing heart M. E. Strand et al. A B C Sham 24 h AB 24 h Sham AB 1 week AB 1 week Sham AB 3 weeks AB 3 weeks Sham 24 h AB 24 h Sham AB 1 week AB 1 week Sham AB 3 weeks AB 3 weeks D E F G H I Sham 24 h AB 24 h Sham AB 1 week AB 1 week Sham AB 3 weeks AB 3 weeks Lung weight/tibia (mg mm 1 ) P = R 2 = 0.46 Fig. 8. Reduced T-cell recruitment and cardiac dysfunction after AB in syndecan-4 KO mice. Relative mrna levels of T-lymphocyte markers CD3 (A), CD4 (B) and CD8 (C) in the myocardium of WT mice following 24 h, 1 or 3 weeks of AB compared with respective sham-operated controls, n = Relative mrna levels of CD3 (D), CD4 (E) and CD8 (F) in the myocardium of WT and syndecan-4 KO mice following 3 weeks of AB relative to respective sham-operated controls. Left ventricular internal diameter in diastole (LVIDd) (G) and fractional shortening (FS) (H) of WT and syndecan-4 knockout mice following 3 weeks of AB. Cardiac syndecan-4 mrna correlated to lung weight/ tibia length of WT mice following AB (I). *P < 0.05; **P < mrna and protein were assessed in myocardial samples from patients with end-stage heart failure undergoing cardiac transplantation, and compared with samples from nondiseased hearts considered unsuitable for transplantation. Patients were years of age and 4 of 20 were females (Table 2). On average, ejection fraction in patients was %, and 40% and 60% were in New York Heart Association (NYHA) classes III and IV, respectively, confirming severe heart failure. Controls were years of age and 3 of 10 were females. Syndecan-4 mrna levels were increased 1.24-fold in the myocardium of heart failure patients (Fig. 10A), with no significant differences in syndecan-4 mrna between patients in NYHA classes III and IV (P = 0.19). Interestingly, the ~ 10 kda cellular syndecan-4 fragment was detected in all five patients tested (Fig. 10B). Increased NF-jB signaling in these failing hearts was confirmed by increased p65 NF-jB phosphorylation and protein levels (Fig. 10C). Hence, our data suggest that syndecan-4 expression as well as shedding was increased in the myocardium of heart failure patients. Discussion This study investigates mechanisms for increased syndecan-4 expression in the pressure-overloaded myocardium and a role for this proteoglycan and its shedding during heart failure progression through modulation of cardiac inflammation. Our study demonstrates that syndecan-4 expression in cardiac myocytes and fibroblasts is regulated by the proinflammatory cytokines TNFa and IL-1b, and by LPS through TLR4, via the central, proinflammatory transcription factor NF-jB. Interestingly, we found that increased syndecan-4 expression leads to NF-jB-dependent shedding of the ectodomain of syndecan-4 from cardiac cells, a process we confirmed to occur in end-stage, failing human myocardium. Syndecan-4 cleavage resulting in shedding of the ectodomain seemed mainly to be dependent on enzymes binding to heparan sulfate GAG chains. Importantly, our finding that LPS-induced syndecan-4 expression and shedding resulted in reduced focal adhesion assembly in cardiac fibroblasts, combined with the finding of reduced T-lymphocyte 2238 FEBS Journal 280 (2013) ª 2013 The Authors Journal compilation ª 2013 FEBS

12 M. E. Strand et al. Syndecan-4 shedding in the failing heart P = 0.13 A B C P = 0.11 WT AB 24 h Syn-4KO AB 24 h WT AB 1 week Syn-4KO AB 1 week WT AB 24 h Syn-4KO AB 24 h WT AB 1 week Syn-4KO AB 1 week WT AB 24 h Syn-4KO AB 24 h WT AB 1 week Syn-4KO AB 1 week D E F WT AB 24 h Syn-4KO AB 24 h WT AB 1 week Syn-4KO AB 1 week WT AB 24 h Syn-4KO AB 24 h WT AB 1 week Syn-4KO AB 1 week WT AB 24 h Syn-4KO AB 24 h WT AB 1 week Syn-4KO AB 1 week Fig. 9. Inflammation was increased in syndecan-4 KO hearts after AB. Relative mrna expression of TNFa (A), IL-1b (B), IL-6 (C), IL-18 (D), TGFb (E) and the pan leukocyte marker CD45R (F) in the myocardium of WT and syndecan-4 knockout mice following 24 h, 1 or 3 weeks of AB relative to respective sham-operated controls, n = *P < 0.05; **P < 0.01; ***P < recruitment to the pressure-overloaded syndecan-4 knockout myocardium, suggested that syndecan-4 mediated aspects of cardiac remodeling through inflammation. A proposed mechanism for modulation of cardiac inflammation by syndecan-4 is illustrated in Fig. 11. The transmembrane proteoglycans, including the four-membered syndecan family, constitute a group of fewer than 10 proteins, of which all animal cells express at least one [27]. Although syndecans have been implicated in a variety of biological functions related to cellular stress, their function in the heart has received little attention. Based on previous studies indicating a role for syndecan-4 in cardiac disease, this study focused particularly on this HSPG. However, as we here also show that the other three members of the syndecan family were upregulated in the heart following AB, an importance for the whole syndecan HSPG family in response to pressure overload is indicated. Syndecan-4 is upregulated in murine hearts after myocardial infarction [4,9] and in human hearts in response to pressure overload [7]. Importantly, recent data from our group [7,8] suggest that syndecan-4 is essential for the development of pathological hypertrophy and remodeling in response to pressure overload, and we found that lack of syndecan-4 induces cardiac dilatation and premature heart failure in mice. In this study, we expand this knowledge placing syndecan-4 among the important players of cardiac remodeling, showing that proinflammatory signals in the heart stimulate increased syndecan-4 expression in cardiac myocytes and fibroblasts. Specifically, our finding that cardiac syndecan-4 expression is induced by TNFa, IL-1b and LPS through NF-jB and the TLR4, opens up the possibility that syndecan-4 is part of an innate immune response of the heart to pressure overload. Furthermore, these results indicate that syndecan-4 may be important in cardiac diseases such as myocarditis or during sepsis. The evolutionary old innate immune system confers the first line of defense, and comprises the cells and mechanisms that defend the host from infection by other organisms in a nonspecific manner. This immune system detects highly conserved structural motifs of pathogens (pathogen-associated molecular patterns, e.g. LPS) or host-derived molecules (danger-associated FEBS Journal 280 (2013) ª 2013 The Authors Journal compilation ª 2013 FEBS 2239

13 Syndecan-4 shedding in the failing heart M. E. Strand et al. Table 2. Clinical and echocardiographic data (mean SEM) of heart failure (HF) patients. Nondiseased hearts considered for transplantation but deemed unsuitable were used as control samples (age years, 30% female gender, n = 10). BMI, body mass index; BNP, brain natriuretic peptide; LVEF, left ventricular ejection fraction; WMSI, wall motion score index [53]; LVEDV, LV end diastolic volume; LVESV, LV end systolic volume; SV, stroke volume; LVOT, left ventricular outflow tract; IVSd, interventricular septal diameter in diastole; LVIDd, LV internal diameter in diastole; LVPWd, LV posterior wall thickness in diastole. HF (n = 20) Age (years) Female gender 20% Prior smoking (%) 75% NYHA class (40% in NYHA III, 60%, in NYHA IV) BMI (kgm 2 ) Pro-BNP (pmoll 1 ) LVEF (%) (all < 35%) WMSI LVEDV (ml) LVESV (ml) SV (ml) LVOT (cm) IVSd (cm) LVIDd (cm) LVPWd (cm) molecular patterns, e.g. damaged tissue). Myocardial stress such as pressure overload is associated with inflammation, which is required for adequate tissue healing and scar formation, but also causes damage, maladaptive remodeling, dysfunction and if chronic, heart failure [28,29]. Increasing evidence supports the involvement of the innate immune system in heart failure, with signaling through TLRs playing a central role [30]. Activation of these receptors leads to enhanced synthesis of inflammatory cytokines, inducing adaptive immunity, but has also been linked to pressure overload-induced remodeling and failure [31,32]. Herein, we suggest that syndecan-4 is a possible effector of innate immune activation in the pressure-overloaded heart. An essential feature of any inflammatory response is the rapid recruitment of leukocytes from the blood to the site of inflammation. Interestingly, heparan sulfate is believed to be a key player in leukocyte transmigration through the blood vessel wall, affecting attachment and rolling, activation by chemokines, stable adherence and migration into the tissue [11,12], and syndecans constitute the major source of cell surface heparan sulfate on all cell types [5,11,33]. Our observation of reduced recruitment of T cells to the pressureoverloaded myocardium lacking syndecan-4 suggests that syndecan-4 is not only differentially regulated by proinflammatory, innate immunity signaling pathways, but is also a functional effector of this system. Interestingly, relatively recent evidence indicates that T lymphocytes mediate important aspects of cardiac remodeling [13 15]. T cells have been shown to be crucial regulators of ECM structure and composition. Thus, we speculate that syndecan-4 could serve as a link between inflammation and T-cell-mediated changes in the ECM. Inflammation is closely linked to wound healing. Syndecans are believed to maintain the proteolytic balance of wound fluids, and the soluble ectodomains resulting from shedding appear to be involved in A B C Fig. 10. Syndecan-4 expression and shedding were increased in failing human myocardium. Relative mrna levels of syndecan-4 in myocardial samples from explanted hearts of patients with end-stage heart failure compared with nondiseased controls (left) and comparing patients in NYHA classes III and IV (right) (A). Ribosomal protein L32 (Rpl32) was used for normalization. Syndecan-4 protein levels in failing human myocardium compared with controls, shown as representative immunoblot of n = 5 (B). Arrow indicates the ~ 10 kda syndecan-4 fragment resulting from shedding of the ectodomain. Calsequestrin was used for loading control. Representative immunoblots of p65 and phosphorylated p65 NF-jB (C). For patient details, see Table FEBS Journal 280 (2013) ª 2013 The Authors Journal compilation ª 2013 FEBS

14 M. E. Strand et al. Syndecan-4 shedding in the failing heart A B C D Fig. 11. Schematic illustration of proposed mechanism for modulation of inflammation by syndecan-4 in the pressure-overloaded heart. Pressure overload of the left ventricle induces cardiac remodeling and eventually, heart failure (A). The box in (A) is magnified to show cardiac fibroblasts and myocytes surrounded by extracellular matrix in the ventricular wall (B). Boxes in (B) are magnified in (C) showing that in pressure-overloaded cardiac fibroblasts and myocytes, the transmembrane proteoglycan syndecan-4 is upregulated by proinflammatory stimuli (TNFa, IL-1b and LPS) through their respective receptors, e.g. the TLR4 for LPS, and the downstream NF-jB transcription factor. Syndecan-4 is subsequently shed from the surface of cardiac cells through the action of shedding enzymes (indicated by the scissors) (C). Heparan sulfate glycosaminoglycan chains on syndecan-4 bind leukocytes (D). Thus, we propose a mechanism whereby syndecan-4 regulates cardiac inflammation in the pressure-overloaded heart. CM, cardiomyocyte; FB, fibroblast. wound healing as well as in host defense mechanisms [33]. Interestingly, we now show that proinflammatory stimuli induce both increased syndecan-4 expression and shedding of its ectodomain in cardiac cells, and importantly, that shedding of syndecan-4 occurs in failing human hearts. Ectodomain shedding of syndecans is believed to be a highly regulated process that requires the direct action of sheddase enzymes [22], e.g. matrix metalloproteinases at the highly conserved juxtamembrane domain and a disintegrin and metalloproteinase domain with thrombospondin motifs (ADAMTS) enzymes close to the heparan sulfate GAG attachment sites. Our results suggest that syndecan-4 shedding predominantly was regulated by enzymes dependent on intact GAG attachment sites, and not those dependent on an intact juxtamembrane domain, suggesting ADAMTS enzymes to regulate shedding of this HSPG. Indeed, ADAMTS-1 and -4 cleave syndecan-4 near the first heparan sulfate chain [22], thus representing promising candidate regulators of syndecan-4 shedding in the heart. In the future, identification of the specific enzymes responsible for syndecan-4 shedding in the heart, in combination with an improved understanding of the cardiac effects of shedding, might underlie novel therapeutic strategies aiming at modulating syndecan-4 shedding. Although syndecan-4 knockout mice are useful for studying the functional role of this proteoglycan, they cannot be used to dissect the roles of cell surface syndecan-4 from those of soluble syndecan-4 in the heart. For instance, the delayed wound repair [34], the high LPS-induced mortality due to severe endotoxin shock [35,36], the increased susceptibility to cardiac rupture following myocardial infarction [9] and the premature failure and inhibited hypertrophic and remodeling response to pressure overload [7,8] in syndecan-4 knockout mice could reflect the lack of this proteoglycan per se, but could also, in theory, reflect inhibited syndecan-4 shedding. Although syndecan-4 shedding has not been extensively studied in vivo, it has been shown in one study that the shed form of syndecan-4 when overexpressed in the heart, increases mortality in mice following myocardial infarction [9]. Taken together with a recent study showing that overexpres- FEBS Journal 280 (2013) ª 2013 The Authors Journal compilation ª 2013 FEBS 2241

15 Syndecan-4 shedding in the failing heart M. E. Strand et al. sion of full-length syndecan-4 in the heart preserves cardiac function and reduces mortality following myocardial infarction [37], we speculate that reducing syndecan-4 shedding during remodeling and development of heart failure may contribute to a beneficial outcome. Studying the role of syndecan-4 shedding in the heart can also be approached directly at the cellular level. Syndecan-4 is the only family member identified in focal adhesions, stable point of interactions between cells and the ECM [5], and it is essential for assembly of these contact points in fibroblasts. In this study, we show that LPS-induced shedding of syndecan-4 in isolated cardiac fibroblasts resembles syndecan-4 knockout cells having impaired focal adhesion assembly, suggesting that inflammation-induced shedding affects cardiac fibroblast function. Indeed, cleavage of syndecan-4 by ADAMTS1 in HEK293 cells has been shown to induce functional loss of focal adhesions and at the same time, gain of migratory capacities [38]. Furthermore, syndecan shedding is well known from cancer research to be particularly active in proliferating, invasive and highly mobile cells [22,39,40]. Therefore, although this requires further research, altered migratory properties of cardiac fibroblasts through inflammation-induced syndecan-4 shedding may ultimately affect the properties of the ECM and fibrotic processes of the failing heart. Serum levels of syndecan-4 are increased in patients with acute myocardial infarction [41] and with chronic heart failure, and correlate with severity of cardiac remodeling [42]. Based on our findings of increased syndecan-4 shedding in the failing human myocardium, it is tempting to speculate that the soluble ectodomains found in the blood of heart failure patients represent a spill-over from the failing heart. However, syndecan-4 is expressed in several cell types relevant to inflammation [11,43,44] and proinflammatory syndecan-4 shedding was recently shown in smooth muscle cells of the airways [45], suggesting a conserved mechanism. Thus, syndecan-4 in the circulation of heart failure patients could also come from tissues other than the heart. Using the syndecan-4 knockout mouse, we have previously found that syndecan-4 is essential for cardiac remodeling in response to pressure overload [7,8]. In addition to an established role in inflammatory signaling, NF-jB is required for cardiac hypertrophy in vivo [46]. Thus, NF-jB is in a position to integrate proinflammatory and pro-hypertrophic signaling, two important processes pushing failure progression. Interestingly, the identification of syndecan-4 being a NF-jB target gene combined with our previous findings that syndecan-4 is essential for hypertrophy [7], indicates a role for this heparan sulfate proteoglycan in integrating these processes in the heart. Our finding that syndecan-4 in the heart is a downstream mediator of TNFa and IL-1b suggests that potential therapies aimed at modulating syndecan-4 could be based on pharmacological TNFa and IL-1b inhibition. Although clinical anti-inflammatory therapies generally have failed to show benefit for heart failure patients, this is a field of ongoing research and is yet to be discarded [47]. In summary, mechanical stress and proinflammatory signals increase the expression of syndecan-4, a transmembrane proteoglycan of importance to heart failure progression, in myocytes and fibroblasts of the pressure-overloaded heart. Importantly, proinflammatory stimuli induced cleavage and shedding of the heparan sulfate GAG-containing ectodomain of syndecan-4 from cardiac cells, a process known to modulate cellular adhesion properties and the inflammatory response. We show that syndecan-4 is upregulated and shed in the myocardium of heart failure patients. Using syndecan-4 knockout mice, we show that this proteoglycan is important for T-cell recruitment to the pressure-overloaded heart. Taken together, our data suggest that syndecan-4 plays a role in the immune response of the heart to increased pressure, possibly influencing subsequent cardiac remodeling. Materials and methods Mouse model of pressure overload Animal experiments were approved by the Norwegian National Animal Research Committee (ID 2845 and 4325) and conformed to the Guide for the Care and Use of Laboratory Animals (NIH publication No , revised 1996, US). Banding of the ascending aorta was performed on 8-week old male WT (C57BL/6JBomTac; Taconic, Skensved, Denmark) and syndecan-4 knockout mice [34] as previously described [7]. Sham-operated animals underwent the same procedure without tightening of the suture around the aorta. During surgery, animals were intubated and ventilated with a mixture of 98% oxygen and 2% isoflurane. Animals received postoperative analgesia by subcutaneous injection of 0.02 ml buprenorphine (0.3 mgml 1 ). Echocardiographic examinations of mice, breathing 1.75% isoflurane on a mask, were performed using a VEVO 2100 system (VisualSonics, Toronto, ON, Canada) 24 h after AB to confirm the presence of AB and degree of stenosis, and 1 or 3 weeks after AB to evaluate cardiac geometry and 2242 FEBS Journal 280 (2013) ª 2013 The Authors Journal compilation ª 2013 FEBS