Distinct structural and molecular features of the myocardial ... - Core

IJC Heart & Vessels 4 (2014) 145–160

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IJC Heart & Vessels journal homepage: http://www.journals.elsevier.com/ijc-heart-and-vessels

Distinct structural and molecular features of the myocardial extracellular matrix remodeling in compensated and decompensated cardiac hypertrophy due to aortic stenosis Victoria Polyakova a,b,1, Manfred Richter a,1, Natalia Ganceva c, Hans-Jürgen Lautze c, Sokichi Kamata a, Jochen Pöling d, Andres Beiras-Fernandez e, Stefan Hein a, Zoltan Szalay a, Thomas Braun b, Thomas Walther a, Sawa Kostin b,⁎ a

Department of Cardiac Surgery, Kerckhoff-Clinic, Bad Nauheim, Germany Max-Planck-Institute for Heart and Lung Research, Bad Nauheim, Germany c Department of Anesthesiology and Intensive Care, Kerckoff-Clinic, Bad Nauheim, Germany d Department of Cardiac Surgery, Schüchtermann-Clinic, Bad Rothenfelde, Germany e Department of Thoracic and Cardiac Surgery, Johann-Wolfgang-Goethe University, Frankfurt am Main, Germany b

a r t i c l e

i n f o

Article history: Received 28 April 2014 Accepted 12 May 2014 Available online 22 May 2014 Keywords: Extracellular matrix Collagen Fibrosis Aortic stenosis

a b s t r a c t Objectives: We used immuhistochemistry and Western blot to study fibrillar and non-fibrillar collagens, collagen metabolism, matricellular proteins and regulatory factors of the ECM remodeling in left ventricular (LV) septum biopsies from 3 groups of patients with aortic valve stenosis (AS): (AS-1,n = 9): ejection fraction (EF) N 50%; AS-2,(n = 12): EF 30%–50%; AS-3,(n = 9): EF b 30%). Samples from 8 hearts with normal LV function served as controls. Results: In comparison with controls, fibrillar collagens I and III were progressively upregulated from compensated (AS-1) toward decompensated hypertrophy (AS-3). The collagenIII/collagen I ratio decreased 2-fold in the AS2 and AS-3 groups as compared with AS-1 and controls. Non-fibrillar collagen IV was upregulated only in AS-3 patients, whereas collagen VI progressively increased from AS-1 to AS-3 group. Collagen synthesis in AS-3 was shifted to collagen I, while the maturation/degradation level was shifted to collagen III. RECK was downregulated only in AS-3 patients. Matricellular proteins tenascin and osteopontin were increased in all AS patients. However, thrombospondin 1, 4 and CTGF were increased only in AS-3. Only AS-3 patients were characterized by increased levels of TGFβ1 and downregulation of TGFβ3, TGFβ-activated kinase1 and Smad7. In contrast, Smad3 gradually increased from AS-1 toward AS-3. Similar trend of changes was observed for TNFα-R1 and TNFα-R2, whereas TNFα was diminished only in AS-2 and AS-3. Conclusions: Distinct changes in fibrillar collagen turnover, non-fibrillar collagens, matricellular proteins and the key regulatory profibrotic and anti-fibrotic factors of the myocardial ECM remodeling are involved in the transition from compensated to decompensated LV hypertrophy and HF in human patients with AS. © 2014 The Authors. Published by Elsevier Ireland Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

Abbreviations: AS, aortic valve stenosis; BMP1, bone morphogenetic protein 1; CNN, Cyr61, connective tissue growth factor, Nov; CTGF, connective tissue growth factor; EF, ejection fraction; ECM, extracellular matrix; HF, heart failure; IHC, immunohistochemistry; ICTP, carboxyterminal telopeptide (degradation product of cross-linked collagen I); IIINP, aminoterminal telopeptide (cross-linked mature collagen III); MMP, matrix metalloproteinase; NYHA, New York Heart Association; LV, left ventricle; LVEDP, left ventricular enddiastolic pressure; PCP, procollagen c-proteinase; PINP, aminoterminal propeptide of type-I procollagen (newly synthesized collagen I); PIIINP, N-terminal type III collagen peptide (newly synthesized collagen III); QIF, quantitative immunofluorescence; RECK, reversion-inducing cysteine-rich protein with Kazal morifs; Smad, small mother against decapentaplegic; TAK1, transforming growth factor β activated kinase 1; TBST, Tris buffer saline Tween-20; TGFβ, transforming growth factor β; TIMP, tissue inhibitor of matrix metalloproteinases; TNFα, Tumor necrosis factor α; TNFα-R1, Tumor necrosis factor α receptor 1; TNFα-R2, Tumor necrosis factor α receptor 2; TSP, thrombospondin; WB, Western blot. ⁎ Corresponding author at: Core Lab for Molecular and Structural Biology, Max-Planck-Institute for Heart and Lung Research, Ludwigstrasse 43, 61231, Bad Nauheim, Germany. E-mail address: [email protected] (S. Kostin). 1 Both authors contributed equally.

http://dx.doi.org/10.1016/j.ijchv.2014.05.001 2214-7632/© 2014 The Authors. Published by Elsevier Ireland Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

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Table 1 Clinical data.

Age, years Men/women NYHA (classes III–IV) EF, % LV mass/m2 IVS (mm) ΔP mean (mm Hg) LVEDP LVESP LVEDD LVESD Diuretics, (n/%) Digitals, (n/%) ACE inhibitors, (n/%) β-Blockers, (n/%)

Table 2 List of antibodies. Control (n = 8)

AS-1 (n = 9)

AS-2 (n = 12)

AS-3 (n = 9)

55.8 ± 9.8 6/2 0 N60% 92.7 ± 12.2 11 ± 0.8 – 8±1 130 ± 12 49 ± 2 33 ± 3 – – – –

64.3 ± 8.5 5/4 0 59.5 ± 4.8 130.5 ± 20.2 15.1 ± 4.2 65.2 ± 11.5 14 ± 4 171 ± 17 47 ± 6 30 ± 7 0 0 2 (22.2%) 1

66.1 ± 8.2 5/7 3 40.6 ± 6.4 138.3 ± 11.7 17.4 ± 4.6 54.3 ± 14.9 18 ± 5 165 ± 14 48 ± 6 36 ± 6 3 (25%) 1 (8.3%) 2 (16.7%) 3 (25%)

67.2 ± 9.9 6/3 7 (77.8%) 24.3 ± 4.8 145.2 ± 14.4 19.8 ± 6.2 42.8 ± 17.7 23 ± 7 152 ± 15 56 ± 6 42 ± 10 7 (77.8%) 7 (77.8%) 2 (22.2%) 3 (33.3%)

LVEDP, LV end-diastolic pressure; LVESP, LV end-systolic pressure; IVS, interventricular septum; LVEDD, LV end-diastolic diamter; LVESD, LV end-systolic diamter; ΔP mean, mean ventricular-aortic pressure gradient.

1. Introduction Aortic valve stenosis (AS) is often associated with heart failure and high mortality [1–3]. Myocardial remodeling is a determinant stage of heart failure (HF), which is characterized by changes of ventricular size, shape and function [4]. The process of left ventricular (LV) remodeling is accompanied by cardiomyocyte hypertrophy and loss, extracellular matrix (ECM) reorganization and the increased ECM elements [5–8]. Despite numerous studies, the exact cellular and molecular mechanisms of cardiac remodeling in AS remain mostly unknown and are limited mainly to experimental animal data [9–14]. We and others have previously shown that the extent of ECM remodeling and myocardial fibrosis may not be reversible after delayed AS surgery [3,5,15–17]. Therefore, detection of the structural and molecular components involved in myocardial ECM remodeling could help to find promising therapeutic targets and prospective biomarkers to optimize the time of aortic valve replacement. The collagens are the most abundant proteins of the ECM and wellaccepted tissue markers of cardiac remodeling [18]. The adult myocardium consists of fibrillar collagen type I and collagen type III [19]. Collagen type V is another fibrillar collagen that in humans is encoded by the COL5A1 gene [20]. The distribution and expression of collagen V in the normal or pathological heart are almost unknown. Although collagen type I and III are the most prevalent in myocardial ECM and their role in myocardial remodeling is increasingly studied, the role of non-fibrillar collagens such as collagen type IV and VI is less studied. Recently it was demonstrated that deletion of collagen VI in mice has a protective effect after myocardial infarction by reducing fibrosis and pathological remodeling [21]. It is reasonable therefore to hypothesize that collagen VI is also involved in ECM remodeling in the hypertrophied heart. The fibrillar collagens are secreted into the ECM as N-terminal propeptides PINP and PIIINP, carboxyterminal (ICTP) and aminoterminal telopeptides. PINP and PIIINP represent the newly synthesized collagen I and collagen III, respectively. ICTP detects degraded collagen I, whereas IIINTP is a marker of cross-linked mature collagen III [15, 22–25]. Collagen turnover is essential in ECM remodeling. Removal of cterminal propeptides of fibrillar procollagens is a crucial event in

Antibody

Company

Host

WB/IHC

Collagen I Collagen III Collagen IV Collagen V Collagen VI BMP1 RECK Osteonectin Osteopontin Tenascin Tenascin C TSP1 TSP2 TSP4 CTGF CTGF TGFβ1 TGFβ2 TGFβ3 TAK1 Smad1 Smad2 Smad3 Smad 7 TAK1 TNFα TNFα-R1 TNFα-R2 Actin (HHF35)

Rockland Rockland Rockland Rockland Rockland Oncogene BD Biosciences Santa Cruz Acris Sigma R&D Calbiochem Santa Cruz Santa Cruz Santa Cruz Abcam Santa Cruz Abcam Abcam Cell Signaling Cell Signaling Cell Signaling Cell Signaling Santa Cruz Cell Signaling Sigma Biovision Abcam Sigma

Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit Mouse Rabbit Rabbit Mouse Rat Mouse Rabbit Mouse Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit Mouse Rabbit Rabbit Rabbit Mouse Mouse Rabbit Mouse

IHC IHC IHC/WB IHC/WB IHC/WB WB WB WB WB IHC IHC IHC/WB WB WB IHC WB WB WB WB WB WB WB WB WB WB WB WB WB WB

WB, Western blot, IHC, immunohistochemistry.

collagen fibril-formation and is accomplished by procollagen cproteinases which are identical to bone morphogenetic protein-1 (BMP1), a member of the tolloid family of Zn-dependent astacin-like metalloproteinases [26]. Collagen turnover is regulated basically by matrix metalloproteinases and their tissue inhibitors. A newly discovered alternative inhibitor of matrix metalloproteinases is RECK (reversion-inducing cysteine-rich protein with Kazal morifs) [27]. However, neither BMP1 nor RECK has been studied in relation to compensated or decompensated myocardial hypertrophy. Matricellular proteins have been classified as a family of nonstructural family of extracellular proteins that do not play primarily a structural role in the ECM but influence collagen fibril assembly [28]. The role of this protein family in myocardial remodeling in the hypertrophied and/or failing myocardium still remains obscure. As an example, tenascin, thrombospondin-1 and 2, osteonectin and osteopontin are highly expressed during embryogenesis and are very low or absent in adult normal hearts. In contrast, during HF progression these proteins appear to be up-regulated [29]. In addition, the role of connective tissue growth factor in the heart which also belongs to matricellular proteins is limited to myocardial infarction [30]. Myocardial ECM remodeling is essentially regulated by profibrotic and anti-fibrotic factors. Among them, an important role has TGF-β, consisting of three highly conserved isoforms, designated TGFβ-1, TGFβ-2, and TGFβ-3. From all these 3 isoforms, only TGFβ-1 has intensively been studied in the pathological heart. Tumor necrosis factoralpha (TNFα) is another factor which is believed to be involved in the regulation of fibrosis [31]. However, more studies are needed to substantiate its role in fibrosis, especially in the transition from compensated to decompensated cardiac hypertrophy.

Fig. 1.Representative confocal micrographs of collagen type I (green) and type III (red) in controls and in patients with AS demonstrating a gradual increase accumulation of these fibrillar collagens from controls to AS-1, AS-2 and AS-3. Merged images (right panels) show the prevalence of collagen III over that of collagen I in control and partially in AS-1 patients, whereas in the AS-2 and AS-3 groups, the quantity of both collagens is apparently equal. Nuclei are shown in white after staining with DAPI and F-actin is shown in blue after labeling with Alexa633phalloidin. Lower diagrams represent quantitative data of collagen I, collagen III, and the collagen III/collagen I ratio in control and in AS groups. Notice that the AS-2 and AS-3 groups exhibit significantly lower collagen III/collagen I ratio in comparison with the AS-1 group and controls.


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From this background, we have hypothesized and show that changes in fibrillar collagen turnover, non-fibrillar collagens, matricellular proteins and the key regulatory profibrotic and anti-fibrotic factors of the ECM remodeling are involved in the transition from compensated to decompensated LV hypertrophy and HF in human patients with AS. 2. Material and Methods 2.1. Patients Thirty patients with isolated aortic valve stenosis were subdivided into three different groups on the basis of (LV) determined

echocardiographically at the time of admission: group I (AS-1) EF N 50% (n = 9), group II (AS-2), EF 50%–30% (n = 12) and group III (AS-3), EF b 30% (n = 9). All patients underwent surgical aortic valve replacement. As previously described [5,15,32], based on clinical and hemodynamic data (Table 1), the myocardium from the AS-1 group was regarded as representing compensated LV hypertrophy, the AS-2 as the transitional stage and the AS-3 group as representing decompensated hypertrophy and the failing myocardium. Controls were LV biopsies from 8 hearts with normal LV function. The institutional Ethical Committee approved the study. All patients gave informed consent. The investigation conforms with the principles outlined in the Declaration of Helsinki.

Fig. 2. Typical confocal images of collagen V (green) distribution in control and AS groups. Nuclei are stained blue with DAPI. Lower left panels are representative WB for collagen V and actin (HHF-35). For WB quantification, collagen VI is normalized to sarcomeric actin and expressed as percent of control values. Notice that the amount of collagen V is significantly increased only in decompensated hypertrophy (AS-3) in comparison with controls.


2.2. Immunofluorescent labeling and confocal microscopy During aortic valve replacement, when a subaortic myocardial resection was performed, these samples from the LV septum were immediately frozen in liquid nitrogen, and stored at − 80 °C. Before immunolabeling, tissue characterization and orientation were recorded by hematoxylin and eosin staining. Frozen sections were fixed for 15 min with 4% paraformaldehyde and then incubated with the primary antibodies which are listed in Table 2. Anti-mouse or anti-rabbit IgGconjugated with Cy3 or Cy2 (Biotrend) served as detection systems in single or double immunolabelings. The nuclei were stained with 1 μg/ml 4,6-diamidino-2-phenylindole (DAPI) (Molecular Probes). F-actin was

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fluorescently stained using TRITC-conjugated (Sigma) or Alexa633conjugated phalloidin (Molecular Probes). Negative controls were obtained by omitting the primary antibody, in an otherwise similar protocol. The samples were examined by confocal laser microscopy (Leica TCS-2). Digital images were further processed for 3D-reconstruction using software “Imaris” (Bitplane AG, Zürich, Switzerland). 2.3. Quantitative immunofluorescent microscopy Measurements of immunofluorescence were done using × 40 Planapo objective (Leica) and a Leica (Leitz DMRB) fluorescent microscope equipped with a Leica DC380 digital camera. Cryosections from

Fig. 3. Confocal images of collagen IV (green) distribution in control and AS groups. Nuclei are stained blue with DAPI. The left lower diagram shows the quantitative data of collagen IV determined by QIF and expressed as percent of the signal per tissue area. Lower right panels are representative WB for collagen IV quantitative data of collagen IV normalized to actin in control and AS groups.

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at least two different tissue blocks in each case were used. For quantitative analysis all sections were immunolabeled simultaneously using identical dilutions of primary and secondary antibodies and other reagents. Immunofluorescent images were obtained under identical parameters of imaging, zoom, pinholes, objectives, and fluorescence power. Sections exposed to PBS instead of primary antibodies served as negative controls. One channel with the format of 1024 × 1024 pixels and appropriate filters were used. The image acquisition settings were standardized for all groups to ensure that the image collected demonstrated a full range of fluorescence intensity from 0 to 255 pixel intensity level and were kept constant during all measurements. For each patient at least 10 random fields of vision were analyzed for the quantity of the investigated markers using images analysis software (Leica) and Image J program. The area occupied by different markers was calculated as percent of the signal per tissue area. 2.4. Western Blot Samples were processed for Western blot analysis as previously described [15,24]. In brief, frozen tissue was homogenized in RIPA buffer (containing 20 mmol/l Tris–HCl at pH 7.4, 100 mmol/l NaCl, 5 mmol/l thylene-diamine tetraacetic acid, 1% Triton X-100, 10% glycerol, 0.1% sodium dodecylsulfate, 1% deoxycholate, 50 mmol/l NaF, 10 mmol/l Na4P2O7, 1 mmol/l Na3VO4, 1 mmol/l phenylmethylsulfonylflouride) and mammalian protease inhibitor cocktail (Sigma) at pH 7.4 and centrifuged at 2000 ×g at 4 °C for 10 min. LV myocardial extracts were loaded onto 12% polyacrylamide gel and separated under the reducing conditions. Proteins were electrotransferred onto nitrocellulose membrane (Invitrogen) and blocked with 5% non-fat dry milk in Trisbuffered saline Tween-20 (TBST) at 4 °C. After washing with TBST,

proteins were exposed overnight at 4 °C to antibodies against MMP2 and MMP9 (Biotrend) diluted in TBS with 5% powdered milk. Bound antibodies were detected by peroxidase-conjugated anti-mouse IgG horseradish peroxidase-conjugated and SuperSignal WestFemto (Pierce) detection system and exposed to X-ray film. Quantification of immunoblots was done by scanning on a STORM 860 (Amersham Pharmacia Biotech) using ImageQuant software. The immunoblotting values for the investigated proteins were normalized per actin (clone HHF-35, Sigma). The control values were set at 100% (see Fig. 2, as an example). 2.5. Statistical analysis All data are presented as means ± SD. For multiple comparisons we used ANOVA, followed by analysis with the Bonferroni t-test. Associations between variables were assessed using Spearman’s rank correlation. Differences between groups were considered significant at p b 0.05. 3. Results 3.1. Fibrillar collagens The typical localization, distribution and the amount of collagen I and collagen III are shown in Fig. 1. In comparison with controls, both collagens gradually increased in AS patients reaching the highest amount in decompensated hypertrophy (AS-3). Quantitative immunofluorescence (QIF) confirmed these observations and demonstrated significant upregulation of both, collagen I and collagen III in AS groups, especially in the AS-2 and AS-3 groups (Fig. 1). The collagen III/collagen

Fig. 4. Expression of collagen VI (green) in control and in patients with AS. Cardiomyocytes are stained red with phalloidin. Nuclei are stained blue with DAPI.


I ratio was 3.35 ± 0.48 in control tissue and 3.1 ± 0.63 in compensated hypertrophy (the AS-1 group). This ratio was dramatically decreased in the transition phase (AS-2) and reached 1.69 ± 0.46 in decompensated hypertrophy and HF (AS-3). Collagen type V is another fibrillar collagen. Representative confocal pictures of collagen V localization and distribution are shown in Fig. 2 and depict that fibers formed by collagen V surround individual cardiomyocytes and vessels. The most remarkable increase in collagen V was observed in decompensated hypertrophy (AS-3). QIF showed that collagen V occupied 5.1% + 2.1% area of the control tissue and increased modestly by 25% and 34% in the AS-1 and AS-2 groups, respectively. In the AS-3 group, collagen V was 14.7% + 2.6% and differed significantly from AS-1, AS-2 and control values. Quantitative WB showed that the quantity of collagen V was increased only in the AS-3 group (Fig. 2). 3.2. Non-fibrillar collagens Collagen IV is a typical non-fibrillar collagen that forms a fine network surrounding individual cardiomyocytes and blood vessels. Fig. 3 compares the distribution and the amount of collagen IV in control and AS groups. In the AS-2 and AS-3 groups, collagen IV showed a strong tendency to form thick septa isolating individual cardiomyocytes. QIF showed that collagen IV was significantly increased in the AS-2 and AS-3 groups. WB showed that collagen IV was significantly increased only in decompensated hypertrophy (AS-3 group). Collagen VI is another non-fibrillar collagen and representative confocal images of collagen VI in LV myocardial tissue in control and in AS patients are shown in Fig. 4 and demonstrate that collagen VI is

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obviously increased in all AS groups. These observations were confirmed by QIF (Fig. 5A). Typical WB for collagen VI discriminates between total and destructed collagen VI (Fig. 5B). Quantification of the total collagen VI showed significant increases in the AS-2 and AS-3 groups (Fig. 5C). However, the most remarkable differences between groups were observed in the amount of the destructed collagen VI (Fig. 5D). 3.3. Collagen metabolism The level of newly synthesized collagen I as observed by PINP immunolabeling was significantly enhanced in the AS-2 and AS-3 groups as compared to control (Fig. 6). The level of cross-linked but partly destructed collagen I as detected by ICTP immunolabeling was the highest in the AS-1 group. In comparison with control, the level of newly synthesized collagen III as indicated by PIIINP staining was the highest in compensated hypertrophy (AS-1 group). In comparison with the latter group, the levels of PIIINP showed a tendency to be diminished in the transition phase (AS-2 group) and in decompensated hypertrophy (AS-3 group). The mature form of collagen III (IIINTP) gradually increased from AS-1 to AS-3. Calculation of collagen markers ratios presented in the Table 3 demonstrated a significant prevalence of collagen I synthesis over its degradation (PINP/ICTP ratio) in the AS-3 group as compared with other groups. In contrast, synthesis of collagen III was much lower than mature collagen III (PIIINP/IIINTP ratio) in the AS-3 group. These changes led to the prevalence of collagen I synthesis over that of collagen III (PINP/PIIINP ratio) which was more pronounced in decompensated (AS-3) than in compensated hypertrophy (AS-1) or in control.

Fig. 5. Collagen VI expression levels in control and in patients with AS. (A) QIF shows a gradual increase in collagen VI per tissue area (expressed in %) from AS-1 to AS-3 patients as compared to controls. (B) A typical WB for collagen VI showing the total (upper band) and destructed collagen VI (lower band). (C, D) Quantitative data of the total and destructed collagen VI in control and AS patients.

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Fig. 6. Markers of collagen metabolism. Representative immunofluorescent images of PINP (green) demonstrating low expression in a control (upper left panel) and marked expression in a AS-3 patient (upper right panel). Cardiomyocytes are stained red with phalloidin. Nuclei are stained blue with DAPI. Diagrams showing QIF analysis of markers of collagen I (PINP and ICTP) and of collagen III (PIIINP, IIINTP).

Removal of C-terminal propeptides of fibrillar procollagens is a crucial event in collagen fibril-formation and is accomplished by procollagen C-proteinases which are identical to bone morphogenetic Table 3 Ratios of collagen metabolism markers in control and in patients with AS. Ratios

Control (n = 8)

AS-1 (n = 9)

AS-2 (n = 12)

PINP/ICTP PIIINP/IIINTP PINP/PIIINP ICTP/IIINTP

1.12 3.26 0.76 2.76

0.79 3.01 0.57 1.87

1.25 1.51 0.98 1.14

a b c

Versus control. Versus AS-1. Versus AS-2.

± ± ± ±

0.56 1.31 0.27 0.88

± ± ± ±

0.39 1.06 0.22 0.77

± ± ± ±

0.41b 0.53a,b 0.23 0.39a,b

AS-3 (n = 9) 1.85 0.62 1.57 0.52

± ± ± ±

0.57a,b,c 0.17aa,b,c 0.33a,b,c 0.14a,b,c

protein-1 (BMP1), a member of the tolloid family of Zn-dependent astacin-like metalloproteinases. The level of BMP1 was significantly upregulated in all AS groups as revealed by WB (Fig. 7A). The expression of RECK, an alternative inhibitor of MMPs, was recently described in adult human hearts [24]. RECK is known as a protective agent of ECM. In comparison with controls, the AS-3 group of patients with AS was characterized by significant downregulation of RECK, whereas the AS-1 and AS-2 groups showed only a negligible upregulation of RECK (Fig. 7B). 3.4. Matricellular proteins Among ECM proteins there are a number of proteins, collectively called matricellular proteins, that do not have a structural role but


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Fig. 7. Representative WB and quantitative data of BMP1 and RECK.

influence collagen fibril assembly and contribute to ECM remodeling. Tenascins belong to matricellular proteins. In the present study, by performing immunostaining of serial sections we observed that total tenascins co-localizes with tenascin C (Fig. 8A and B) in both, control and AS myocardial tissues. QIF showed that both, total tenascins and tenascin C, gradually increased from compensated to decompensated hypertrophy (Fig. 8C and D). Osteonectin and osteopontin are also typical matricellular proteins. WB for osteonectin showed no differences between groups in the expression levels (Fig. 8E). Osteopontin-positive staining was found in endothelial cells and some fibroblast-like cells of human myocardium (data not shown). By WB, both levels of osteopontin and osteopontin cleaved by MMP were increased in all patients with LV hypertrophy due to AS (Fig. 8F and G). Matricellular protein thrombospondin 1 (TSP1) was immunohistochemically absent or present in minor quantities in controls, whereas in patients with AS it was dramatically increased (Fig. 9). WB analysis showed that TSP1 levels were elevated in patients with AS, especially in patients with decompensated hypertrophy (AS-3). The level of TSP2 was significantly enhanced in the AS-2 and AS-3 groups (Fig. 9). We analyzed also TSP4, although it doesn't belong to matricellular proteins, but it is a member of the thrombospondins family of the ECM glycorproteins. It was upregulated only in the AS-3 group indicating that TSP4 is a reliable marker of ECM remodeling in decompensated hypertrophy. Connective tissue growth factor (CTGF) is a secreted multifunctional protein that belongs to the CNN-family and matricellular proteins. By immunohistochemistry we observed in control samples that CTGF is robustly expressed as small dot-like structures, whereas in AS patients it is abundantly expressed in the ECM (Fig. 10). Both, QIF and WB showed a significant upregulation of CTGF in the AS-3 group (Fig. 10).

expression level of TGFβ-2. TGFβ-3 was mostly downregulated in the AS-3 group. TGFβ acts mainly via activation of Smads signaling proteins or TGFβ activated kinase (TAK1). There were no differences between groups in the expression level of Smad1. As compared with controls, Smad2 was significantly upregulated in the AS-2 and AS-3 groups. Smad3 gradually was increased from compensated (AS-1) to decompensated hypertrophy (AS-3). The inhibitory Smad protein - Smad7 was found to be downregulated only in patients with decompensated hypertrophy (AS-3). Similarly, TAK1 was mostly downregulated in the AS-3 group (Fig. 11). TNF-α was found significantly decreased in the AS-2 and AS-3 groups as compared with the AS-1 and control groups (Fig. 12). TNF-α exerts its effects by binding to specific receptors TNFα-R1 and TNFαR2. In comparison with controls, the expression levels of TNFα-R1 gradually increased from compensated (AS-1) to decompensated hypertrophy (AS-3). TNFα-R2 was mostly elevated in myocardial tissue of patients with decompensated hypertrophy (Fig. 12). 4. Discussion 4.1. Major findings In our previous studies we have established that the transition from compensated to decompensated hypertrophy and HF is accompanied by MMP/TIMP imbalance and progressive fibrosis [5,15,25]. In the present study we have further analyzed the factors contributing to the pathogenesis of fibrosis. The summary of our results and the major differences between compensated and decompensated hypertrophy are listed in Table 4. 4.2. Collagens and collagen metabolism

3.5. Profibrotic and anti-fibrotic regulatory factors The myocardial remodeling is essentially regulated by equilibration of profibrotic and anti-fibrotic factors. An important significance in ECM remodeling and especially in fibrosis has TGFβ, consisting in three highly conserved isoforms, designated TGFβ-1, TGFβ-2, and TGFβ-3. We analysed all these isoform by WB and the results are shown in Fig. 11. TGFβ-1 was found to be upregulated only in decompensated hypertrophy (AS-3). There were no differences between groups in the

Synthesis of collagen I as revealed by PINP expression was 3-fold increased in patients with decompensated hypertrophy in comparison with controls. In contrast, synthesis of collagen III in decompensated hypertrophy did not differ significantly from control hearts. These changes in collagen I and III synthesis observed in decompensated hypertrophy were in marked contrast to the corresponding markers of collagen I and III degradation, ICTP and IIINTP, respectively. Therefore, increases in collagen I synthesis and collagen III degradation are a hallmark of decompensated hypertrophy. Our immunohistochemical data showing a

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Fig. 8. Matricelullar proteins, tenascin, osteonectin and osteopontin in the myocardium of patients with AS and controls. (A, B) Representative immunofluorescent serial images of total tenascin (green) and tenascin C (red) demonstrating their co-localization (arrows). (C, D) QIF data of tenascin and tenascin C expressed as percent per tissue area. (E) Representative WB and quantitative data of osteonectin. (F, G) Representative WB for osteopontin showing the native (arrow) and cleaved by MMP (arrowhead) form of osteopontin and their quantification.

decrease in the collagen III/collagen I ratio concur with the recently published observations of genes encoding these forms of collagen in patients with AS [33]. Such a cumulative shift to collagen I in myocardial content may underlie a higher myocardial stiffness and its functional deteriorations which are typical features of decompensated

hypertrophy and heart failure in the pressure-overloaded hearts [11, 13,16,18,33,34]. Collagen V is a fibrillar collagen that is required for the normal formation of collagen I fibrils [35]. In line with such a function of collagen V, the upregulation of collagen V in our AS patients, especially in those


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Fig. 9. Thrombospondins (TSP) in the myocardium of patients with AS and controls. Immunofluorescent images of TSP1 myocardial expression (green) in a control (upper left panel) and in a AS-3 patient (middle left panel). Cardiomyocytes are stained red with phalloidin. Representative WB for TSP1, TSP2 and TSP4 and their quantification.

with decompensated hypertrophy, would predict its role in abundant deposition of collagen I that is a typical feature of HF [25]. Non-fibrillar collagens IV and VI were found in enhanced levels in our AS patients. Collagen IV is a main component of the basement membrane. Type VI collagen interacts with type IV creating an anchoring bridge between the basement membrane and the interstitial matrix. Collagen VI acts as an adhesive structure and interacts with other ECM components including type I collagen and fibronectin. It has recently been demonstrated that ablation of the collagen VI gene has a protective effect after myocardial infarction. Collagen VI disruption in these experimental models reduced aberrant ECM remodeling and fibrosis leading to preservation of cardiac function [21]. Therefore, the upregulation of collagen VI observed in our AS patients may reflect an activation of compensatory cardioprotective mechanisms. BMP1 is the enzyme that cleaves the C-terminal propeptides from procollagen type I, III and V and is regarded therefore a determinant event in collagen deposition. An elevation of BMP1 level in AS patients shows that HF progression is characterized by an intensive ECM remodelling and collagen deposition.

Observed downregulation of RECK in patients with decompensated hypertrophy can be of negative importance, because RECK acting as a MMPs inhibitor protects fibrillar collagens from degradation and therefore maintains myocardial fibrosis [36]. 4.3. Matricellular proteins In the present study, we found that tenascins showed a low expression in the control, whereas in all AS patients were significantly increased and therefore can be recommended as early tissue markers of cardiac remodelling. Thrombospondins (TSPs) are ECM proteins that influence collagen fibril assembly and contribute to regulation of fibril formation, diameter and uniformity [37]. In addition, TSPs bind to collagens and affect collagen turnover and collagen fibril organization [38,39]. The family of TSPs consists of five members, where TSP1 and TSP2 are the most studied and belong to matricellular proteins. TSP1 can modulate a variety of cellular events in ECM and namely TSP1 is a crucial activator of TGFβ1, inhibits angiogenesis and modulates MMP expression and activity,

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Fig. 10. Matricelullar CTGF in cardiac remodeling. Representative immunofluorescent pictures of CTGF (green) in a control patient (upper left panel) and in a patient with AS-III (upper right panel). Cardiomyocytes are stained red with phalloidin, nuclei are stained blue with DAPI. QIF analysis of CTGF expressed as percent per tissue area in the study groups (lower left panel). Representative WB and quantitative data of CTGF (lower right panel). All WB data are normalized to actin.

myofibroblast transdifferentiation and collagen synthesis [40]. TSP1 was found to be induced in patients after myocardial infarction [41] or in pressure-overloaded myocardium [42]. In our study the expression of TSP1 was almost absent in control and was extremely increased in AS patients, especially in those with decompensated hypertrophy. In contrast to TSP1, TSP2 does not activate TGFβ1 and is more important for controlling angiogenesis and integrity of newly formed matrix by means of induction of increased collagen synthesis and inhibition of its degradation [29]. An enhanced level of TSP2 mRNA was identified in rats and humans with LV hypertrophy [42]. We observed an upregulation of TSP2 during the transition from compensated (the AS-2 group) to decompensated hypertrophy (the AS-3 group) suggesting the role of TSP2 in promoting fibrosis by increasing collagen synthesis. TSP4 is a pentameric protein of the TSPs family that does not contain procollagen homology domain. TSP4 modulates the proliferation of endothelial and smooth muscle cells [43] and is highly expressed in the adult human heart [38]. TSP4 appears to inhibit fibrotic response given that TSP4 null mice had increased collagen deposition in the pressure-overloaded heart [10]. In addition, mRNA levels and gene expression of TSP4 were found to be markedly upregulated in experimental pressure overload and myocardial infarction in rats [44,45]. Our data demonstrating remarkably increased expression levels of the TSP4 in decompensated hypertrophy due to AS are in contrast with such observations and suggest that TSP4 might serve as a compensatory mechanism to overwhelm myocardial fibrosis in human patients. Osteopontin can modulate a variety of cellular activities associated with fibrotic responses [46]. It has been shown that osteopontin is increased in cardiac hypertrophy and modulates cardiac fibrosis probably through the modulation of cellular adhesion and proliferation [47]. According to experimental data, osteopontin may be cleaved by MMPs

and functioning thus as a cytokine being more active than the native osteopontin [41]. Osteopontin has been shown to interact with collagen types I, III, V and VI, and it is supposed to play a crucial role in atherosclerosis, valvular stenosis, hypertrophy, myocardial infarction and HF [48]. Increased levels of osteopontin were associated with HF after myocardial infarction. In our study both, intact osteopontin and osteopontin cleaved by MMP were upregulated in all AS patients indicating that these proteins are reliable markers of the ECM remodeling and AS severity. Osteonectin is a matricellular protein taking part in collagen production and deposition [49]. Although there are some indications to suggest the role of osteonectin in procollagen processing after myocardial infarction or in pressure-overload mice models [50], we did not find any significant differences between the AS groups in the osteonectin expression indicating that in human patients the role of osteonectin in ECM remodeling is negligible. CTGF is another matricellular protein that is abundantly expressed in the neonatal heart [51]. In the adult heart, CTGF expression becomes restricted to the atria and large vessels in the adult heart [52]. Biological functions of CTGF include stimulation of fibrosis and ECM production [53]. As a TGFβ inducible protein, CTGF may contribute to the pathogenesis of fibrosis by accentuating TGFβ-mediating actions. CTGF in turn enhances TGFβ signalling leading thus to detrimental ECM accumulation, myocardial fibrosis and cardiac dysfunction. We have reported enhanced CTGF levels in patients with HF, in myocardial infarction and in myocarditis [25]. Increased CTGF mRNA was demonstrated in a rat pressure overload model [54]. In the present study, we have found increased levels of CTGF only in decompensated hypertrophy. Taken together, our data, as well as recent findings that patients with chronic HF patients have increased plasma levels of CTGF which correlate with BNP, TGFβ


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Fig. 11. Representative WB and quantitative data of TGFβ1, 2, 3 and their signaling proteins Smad1, 2, 3, 7 and TAK1. All WB data are normalized to actin.

and LV stiffness, suggest that CTGF could be used as a novel diagnostic marker for cardiac dysfunction in HF. 4.4. Profibrotic and anti-fibrotic regulatory factors TGFβ, a multifunctional peptide growth factor, exists in three highly conserved isoforms: TGFβ1, TGFβ2, and TGFβ3. TGFβ isoforms show different heart tissue distributions and it is believed to affect different cell types [55,56]. Most of studies have focused on TGFβ1 [7,57]. The data concerning the role of TGFβ2 and TGFβ3 isoforms are contradictory. It was found that TGFβ2 and TGFβ3 possess an anti-fibrotic activity [58] and that TGFβ-3 has a beneficial effect on scar formation [59]. In the present study we showed upregulation of TGFβ1 and downregulation of TGFβ3 only in decompensated hypertrophy suggesting that TGFβ1 and TGFβ3 may counteract and it is also tempting to speculate that

most probably the balance of TGFβ1/TGFβ3 determines the clinical outcome and their ratio has a promising prognostic potential. Activation of Smads by TGFβ is related to detrimental cardiac remodeling and progression to HF. According to experimental data, TGFβ/Smad signaling contributes to an enhancement of cardiac fibrosis [60]. In our recent study we have demonstrated that TGFβ1 and Smad3 play an important role in fibrosis progression in patients with atrial fibrillation [24]. The present study showing upregulations of Smad2 and Smad3 indicates the role of this signaling pathway in AS and confirms previous experimental and clinical data. In addition a profibrotic action of TGFβ1 via Smad2/3 was associated with Smad7 downregulation, indicating a decrease of inhibitory Smads effects. Moreover, TAK1 and Smad1/5/8 signaling pathways seem not to be activated in AS patients, since we did not find any upregulation of TAK1 and Smad 1 (Fig. 11) or Smad 5 and 8 (data not shown). Indeed, our data show that TAK1

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V. Polyakova et al. / IJC Heart & Vessels 4 (2014) 145–160 Table 4 Summary of results. Parameter

Compensated hypertrophy (AS-1)

Transition phase (AS-2)

Decompensated hypertrophy (AS-3)

Collagen I Collagen III Collagen III/Collagen I ratio Collagen V Collagen IV Collagen VI PINP ICTP PIIINP IIINT BMP1 RECK Osteonectin Osteopontin Tenascin Tenascin C TSP1 TSP2 TSP4 CTGF TGFβ1 TGFβ2 TGFβ3 TAK1 Smad 1 Smad 2 Smad 3 Smad 7 TNFα TNFα-R1 TNFα-R2

+ ++ n n n n n ++ ++ n +++ n n + + + n n n n n n n n n n + n n + n

++ ++ −− n n + ++ + + ++ +++ n n + ++ ++ + +++ n n n n n n + + ++ n −− ++ n

+++ ++ −− + + ++ +++ n n +++ +++ − n + +++ +++ +++ +++ +++ ++ + n ++ −− ++ ++ +++ − −− ++ ++

Changes in comparison with control values: n, unchanged; +, mildly increased; +++, moderately increased; +++, severely increased; −, mildly decreased; –, moderately decreased.

The downregulation of TNFα found in our AS patients is most probably connected with a decrease of its anti-fibrotic action, given that TNFα activates MMPs and inhibits the expression of its inhibitors [63,64]. TNFα exerts its effects by binding to the specific receptors TNFα-R1 and TNFα-R2. The pro-fibrotic effects of TNFα signaling in myocardium appear to be due to interactions involving the TNFα-R1. In contrast, TNFα-R2 signaling may reduce fibrosis [31]. In the present study we found an increase in TNFα receptors in AS patients suggesting their protective functions during HF progression, given that TNFα-R1 and TNFα-R2 are able to bind ligands serving thereby as “biological buffers” neutralizing the activities of TNFα [31].

5. Study limitations

Fig. 12. Representative WB and quantitative data of TNFα and TNFα receptors. All WB data are normalized to actin.

expression is decreasing in parallel with the severity of AS and development of HF. Given that TAK1 may exert paradoxical anti-fibrotic effects [61,62], our data suggest that diminished TAK1 expression may sustain progressive development of myocardial fibrosis in AS patients.

The findings of the present study should be interpreted in light of certain limitations. As for ethical reasons only myocardial samples were retrieved from the interventricular septum. Therefore, it cannot be excluded that the observed distinct features of ECM remodelling in different AS groups are representative of the whole ventricle. Nevertheless, many our observations concur well with the results obtained in numerous experimental model of aortic banding which have investigated the entire LV [10–14]. Another limitation pertains to the remarkable changes observed in the AS-3 group (decompensated LV hypertrophy and HF). Due to the small cohort size of this group of patients we cannot fully rule out an overlap between end-stage HF due to aortic valve disease and a primary cardiomyopathy. In addition, virtually little is known whether myocardial ECM remodeling is similar to ECM remodelling of the aortic cusps as previously reported [65,66] and reviewed in [9,67].


6. Clinical perspective Aortic valve replacement remains the most effective treatment of chronic pressure overload secondary to aortic valve stenosis. Detection of the structural and molecular components involved in myocardial ECM remodeling could therefore help to find promising therapeutic targets and prospective biomarkers to optimize the time of aortic valve replacement. The results of the present study may further stimulate studies to identify serum levels of fibrosis regulators in LV hypertrophy. Whereas circulating biomarkers related to collagen synthesis and degradation have increasingly been used to monitor myocardial ECM remodelling in diverse cardiac diseases (reviewed in [68,69]), more ECM biomarkers are needed to explore. For example, myocardial expression levels of TGFβ1 and BMP1 which were investigated in the present study were quantified in the serum of patients with aortic stenosis or in patients with HF and have demonstrated their plausibility as non-invasive markers of fibrosis [70,71]. In addition, our findings of increased levels of myocardial tenascin C and its correlation with the severity of AS and HF progression are in good accordance with quantifications of circulating tenascin C in HF patients [72]. Based on our study, CTGF represents a promising new circulating ECM biomarker which was recently documented in patients with atrial fibrillation [73] or in patients with liver diseases developing fibrosis [74]. 7. Conclusions Our findings indicate distinct changes in collagen deposition and metabolism, differences in the expression of matricellular proteins, TGF-β signaling, TNF-α and its receptors which are involved in the transition from compensated to decompensated LV hypertrophy and HF in human patients with AS. Conflict of interest The authors report no relationships that could be construed as a conflict of interest Acknowledgments The authors thank Brigitte Matzke and Beate Grohmann for excellent technical assistance. This work was supported by the Stiftung William G. Kerckhoff-Herz- und Rheumazentrum Bad Nauheim (in support of M.R. and V.P.). References [1] Ross Jr J, Braunwald E. Aortic stenosis. Circulation 1968;38:61–7. [2] Vahanian A, Alfieri O. Guidelines on valvular heart disease in clinical practice. EuroIntervention 2013;9(Suppl.):S11–3. [3] Yarbrough WM, Mukherjee R, Ikonomidis JS, Zile MR, Spinale FG. Myocardial remodeling with aortic stenosis and after aortic valve replacement: mechanisms and future prognostic implications. J Thorac Cardiovasc Surg 2012;143:656–64. [4] Cohn JN, Ferrari R, Sharpe N. Cardiac remodeling—concepts and clinical implications: a consensus paper from an international forum on cardiac remodeling Behalf of an International Forum on Cardiac Remodeling. J Am Coll Cardiol 2000;35:569–82. [5] Hein S, Arnon E, Kostin S, Schonburg M, Elsasser A, Polyakova V, et al. Progression from compensated hypertrophy to failure in the pressure-overloaded human heart: structural deterioration and compensatory mechanisms. Circulation 2003;107:984–91. [6] Hein S, Schaper J. The extracellular matrix in normal and diseased myocardium. J Nucl Cardiol 2001;8:188–96. [7] Fielitz J, Hein S, Mitrovic V, Pregla R, Zurbrugg HR, Warnecke C, et al. Activation of the cardiac renin–angiotensin system and increased myocardial collagen expression in human aortic valve disease. J Am Coll Cardiol 2001;37:1443–9. [8] Schaper J, Kostin S, Hein S, Elsasser A, Arnon E, Zimmermann R. Structural remodelling in heart failure. Exp Clin Cardiol 2002;7:64–8. [9] Yetkin E, Waltenberger J. Molecular and cellular mechanisms of aortic stenosis. Int J Cardiol 2009;135:4–13. [10] Frolova EG, Sopko N, Blech L, Popovic ZB, Li J, Vasanji A, et al. Thrombospondin-4 regulates fibrosis and remodeling of the myocardium in response to pressure overload. FASEB J 2012;26:2363–73.

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