Abstract

Background: Soluble ST2 (interleukin 1 receptor-like 1) (sST2) is involved in inflammatory diseases and increased in heart failure (HF). We herein investigated sST2 effects on oxidative stress and inflammation in human cardiac fibroblasts and its pathological role in human aortic stenosis (AS).

Methods and results: Using proteomics and immunodetection approaches, we have identified that sST2 down-regulated mitofusin-1 (MFN-1), a protein involved in mitochondrial fusion, in human cardiac fibroblasts. In parallel, sST2 increased nitrotyrosine, protein oxidation and peroxide production. Moreover, sST2 enhanced the secretion of pro-inflammatory cytokines interleukin (IL)-6, IL-1β and monocyte chemoattractant protein-1 (CCL-2). Pharmacological inhibition of transcriptional factor nuclear factor κB (NFκB) restored MFN-1 levels and improved oxidative status and inflammation in cardiac fibroblasts. Mito-Tempo, a mitochondria-specific superoxide scavenger, as well as Resveratrol, a general antioxidant, attenuated oxidative stress and inflammation induced by sST2. In myocardial biopsies from 26 AS patients, sST2 up-regulation paralleled a decrease in MFN-1. Cardiac sST2 inversely correlated with MFN-1 levels and positively associated with IL-6 and CCL-2 in myocardial biopsies from AS patients.

Conclusions: sST2 affected mitochondrial fusion in human cardiac fibroblasts, increasing oxidative stress production and inflammatory markers secretion. The blockade of NFκB or mitochondrial reactive oxygen species restored MFN-1 expression, improving oxidative stress status and reducing inflammatory markers secretion. In human AS, cardiac sST2 levels associated with oxidative stress and inflammation. The present study reveals a new pathogenic pathway by which sST2 promotes oxidative stress and inflammation contributing to cardiac damage.

Introduction

Inflammation has been recognized to play an important role in the pathogenesis of heart failure (HF) [1] and as a consequence, to be an important therapeutic target for the treatment of HF. Cardiac fibroblasts are the most abundant cell type in the myocardium, and they are essential for the maintenance of the extracellular matrix (ECM) [2]. On cardiac fibroblasts, pro-inflammatory cytokines favor fibrosis [3] and cardiac fibroblasts also act as inflammatory support via their capacity to promote the infiltration of immune cells in the heart [4]. Furthermore, activated fibroblasts promote cardiomyocyte hypertrophy and dysfunction via the release of pro-fibrotic factors, such as transforming growth factor (TGF)-β1, Angiotensin II and fibroblast growth factor [5]. Among the intracellular signals involved in cardiac inflammation, the increase in the production of reactive oxygen species (ROS) and the activation of the transcriptional factor nuclear factor (NF)-κB are the best known [6,7]. One of the factor that contribute to increased ROS generation is an imbalance between mitochondrial fission/fusion processes, leading to detrimental consequences on cardiac function [6]. Mitochondrial fusion is regulated by mitofusins (MFN)-1 and -2 and optic atrophy protein (OPA)-1. Inhibition of mitochondrial fusion leads to fragmented mitochondria and reduced oxygen consumption [8].

The ST2 (interleukin 1 receptor-like 1) gene can encode at least two other isoforms in addition to ST2L by alternative splicing, including a secreted soluble ST2 (sST2) form which can serve as a decoy receptor for Interleukin (IL)-33. IL-33 binds to ST2L and activates mitogen-activated protein kinases and several biochemical pathways. The sum total of these events is the activation of the inhibitor of transcriptional factor nuclear factor κB (NFκB) kinase complex [9]. Before the recognition of a cardiovascular role for sST2, this protein has been found to be involved in inflammatory diseases including asthma, pulmonary fibrosis, rheumatoid arthritis, collagen vascular diseases or sepsis [10]. Moreover, sST2 is produced by both cardiac fibroblasts and cardiomyocytes in response to injury or stress, but nonmyocardial production also occurs [11–13]. sST2 levels have been reported to be increased in aorta from obese rats [11]. Interestingly, the increase in sST2 levels paralleled enhanced oxidative stress as well as an augmentation in inflammatory markers [14]. In line with these findings that suggest an association between sST2 and oxidative stress, serum sST2 levels positively correlated with serum malondialdehyde (MDA) and negatively correlated with the antioxidant superoxide dismutase (SOD) activity in HF patients [15]. In patients with severe aortic stenosis (AS), a condition that may lead to HF, sST2 levels were also associated with AS severity [16].

However, whether sST2 could affect mitochondrial function, ROS production and inflammation in adult cardiac fibroblasts has not been investigated, and a better understanding of the pathophysiology played by sST2 in cardiac fibroblasts is needed.

In the present study, a proteomic approach has been used for the characterization of the proteostasis impairment after sST2 treatment. These results lead us to hypothesize that sST2 impairs mitochondrial function leading to ROS production and inflammation in human cardiac fibroblasts. More precisely, we aimed to evaluate the effects of sST2 on oxidative stress and inflammatory markers in vitro in human cardiac fibroblasts and to study the association of sST2 expression with parameters assessing oxidative stress and inflammation in myocardial biopsies from AS patients.

Methods

Cell culture

Human Cardiac Fibroblasts were obtained from Promocell and maintained in medium Fibroblasts Media 3. Cells were cultured according to the manufacturer’s instructions. Cells were used between passages 4 and 6. Cells were stimulated with sST2 (2 μg/ml, R&D Systems) for 24 h according to our previous studies [11,12]. The NFκB inhibitor BAY-11-7082 (Santa Cruz Biotechnology), the mitochondria-targeted antioxidant Mito-Tempo (Sigma–Aldrich) as well as the general antioxidant resveratrol were added at 10−6 M for 30 min prior to the stimulation with sST2.

Mass spectrometry based-quantitative proteomics

A shotgun comparative proteomic analysis of untreated cardiac fibroblasts and cardiac fibroblasts stimulated with sST2 was performed using isobaric Tags for Relative and Absolute Quantitation (iTRAQ). Global experiments were carried out with three biological replicates in each experimental condition. Peptide labeling, peptide fractionation and mass-spectrometry analysis, were performed as previously described [17–21]. After MS/MS analysis, protein identification and relative quantification were performed with the ProteinPilot™ software (version 4.5; Sciex) using the Paragon™ algorithm as the search engine. Although relative quantification and statistical analysis were provided by the ProteinPilot software, an additional 1.3-fold change cutoff for all iTRAQ ratios (ratio <0.77 or >1.3) and a P-value lower than 0.05 were selected to classify proteins as up- or down-regulated (at least in two of three biological replicates). Proteins with iTRAQ ratios below the low range (0.77) were considered to be under-expressed, whereas those above the high range (1.3) were considered to be overexpressed.

Patient population

Myocardial biopsies were obtained from 26 patients with severe AS (aortic valve area ≤1 cm2 (and/or) transaortic mean pressure gradient >40 mmHg), referred to our center for aortic valve replacement from June 2013 to February 2017. Exclusion criteria were moderate or severe concomitant valvular disease; malignant tumor, chronic inflammatory diseases. All patients were evaluated by echocardiography. As controls, myocardial biopsies from subjects who have died from non-cardiovascular-related diseases were obtained at autopsy (Control, n=13). Informed consent was obtained from each patient and control and the study protocol conforms to the ethical guidelines of the 1975 Declaration of Helsinki as reflected in a priori approval by the institution’s human research committee.

Real-time reverse transcription PCR

Total RNA was extracted with TRIzol Reagent (Qiagen), according to the manufacturer’s instructions. First strand cDNA was synthesized according to the manufacturer’s instructions (Bio-Rad). Quantitative PCR analysis was performed with SYBR green PCR technology (Bio-Rad) (Supplementary Table S1). Relative quantification was achieved with MyiQ software. Data were normalized to hypoxanthine phosphoribosyltransferase (HPRT), GADPH and β-actin levels and expressed as percentage relative to controls. All PCRs were performed at least in triplicate for each experimental condition.

Western blot analysis

Aliquots of 20 µg of total proteins were prepared from cells and electrophoresed on SDS polyacrylamide gels and transferred to Hybond-c Extra nitrocellulose membranes (Bio-Rad). Membranes were incubated with primary antibodies for: MFN-1 (Santa Cruz 1:100), MFN-2 (Santa Cruz 1:100), Nitrotyrosin (Santa Cruz; 1:100), dynamin-related protein 1 (DRP-1; Santa Cruz; 1:100), peroxisome proliferator-activated receptor-γ co-activator ((PGC)1-α; Santa Cruz; 1:100), Prohibitin (PHB−1; Cell Signaling; 1:100; PHB−2; Cell Signaling; 1:100), carboxy-methyl-lysine (CML; Abcam; 1:100), MDA (Abcam; 1:100), peroxiredoxin IV (Prx-IV; Santa Cruz; 1:100), fumarate hydratase (FH; Santa Cruz; 1:100), OPA-1 (Santa Cruz; 1:100), IL-33 (Santa Cruz; 1:100), Myeloid differentiation primary response 88 (MyD88; Santa Cruz; 1:100), Tumor necrosis factor receptor associated factor 6 (TRAF6; Santa Cruz; 1:100), activating protein 1 (AP-1; Santa Cruz; 1:100), interleukin-1 receptor-associated kinase 1/4 (IRAK1/4; Santa Cruz; 1:100), NFκβ (Cell Signaling; 1:100), phospho- NFκβ (Cell Signaling; 1:100), p38 (Cell Signaling; 1:100), phospho-p38 (Cell Signaling; 1:100), p42/44 (Cell Signaling; 1:100) and phospho-p42/44 (Cell Signaling; 1:100). Stain-free detection was used as loading control. After washing, detection was made through incubation with peroxidase-conjugated secondary antibody, and developed using an ECL chemiluminescence kit (Amersham). After densitometric analyses, optical density values were expressed as arbitrary units (AU). All Western Blots were performed at least in triplicate for each experimental condition.

Oxyblot

Immunodetection of carbonyl groups introduced into cardiac cells by oxidative stress was performed using an OxyBlot kit according to the manufacturer’s instructions (Millipore) as previously described [21].

Measurement of intracellular superoxide anion production

The oxidative fluorescent dye dihydroethidium (DHE; Invitrogen) was used to evaluate the production of ion superoxide (O2●−). Cells were incubated for 24 h with either vehicle or sST-2 (2 μg/ml) in the presence or absence of Mito-tempo 10−6 M. Cells were then incubated with 5 × 10−6 mol/l DHE for 30 min in a light-protected humidified chamber at 37°C. Cells were subsequently washed with warm PBS and analyzed with a 40× objective in a fluorescent laser scanning Leica DMI 3000 microscope.

ELISA

Peroxide production levels were measured in cells supernatants following manufacturer’s instructions (Sigma–Aldrich). IL-6, IL-1β and monocyte chemoattractant protein-1 (CCL-2) levels were quantified in cells supernatant following manufacturer’s instructions (R&D Systems). The results were normalized to the control condition. Data were expressed as a fold change relative to control conditions.

Immunohistological evaluation

Histological determinations in cardiac tissue were performed in 5-μm thick sections. Slides were treated with H2O2 for 10 min to block peroxidase activity. All samples were blocked with 5% normal goat serum in PBS for 1 h and incubated for 1 h with ST2 (Sigma; 1:100) and MFN-1 (Santa Cruz; 1:100), washed three times and then incubated for 30 min with the horseradish peroxidase-labeled polymer conjugated to secondary antibodies (Dako Cytomation). The signal was revealed by using DAB Substrate Kit (BD Pharmingen). As negative controls, samples followed the same procedure described above but in the absence of primary antibodies. All measurements and quantifications were performed blind in an automated image analysis system (Nikon).

Statistical analyses

For human studies, continuous variables were expressed as mean ± SD or median (25th to 75th percentile) and compared using unpaired Student’s t test. Spearman’s correlation coefficients were calculated to determine correlations. Categorical variables were expressed as percentages and compared using χ2-test.

For cellular studies, data are expressed as mean ± SEM. Normality of distributions was verified by means of the Kolmogorov–Smirnov test. Data were analyzed using a one-way analysis of variance, followed by a Newman–Keuls to assess specific differences among groups or conditions using GraphPad Software Inc. The pre-determined significance level was P<0.05.

Results

Quantitative Proteomics exploration of sST2 effects on adult human cardiac fibroblasts

In order to obtain a deep insight into the protein content and protein function modulated by sST2 on cardiac fibroblasts, a proteome-wide analysis of total cell extracts was performed using isobaric tags (iTRAQ) coupled to 2D nano-liquid chromatography tandem mass spectrometry. More than 2000 proteins were identified and almost 1800 proteins were quantified in the proteomic analysis. sST2 induced proteome alterations (the expression of 18 proteins have been found to be altered) in human cardiac fibroblasts unveiling MFN-1 as a down-regulated protein upon 24 h of stimulation (Supplementary Table S2).

sST2 modified the expression of mitochondrial MFN-1 and oxidative stress-related molecules in adult human cardiac fibroblasts

sST2 decreased (P<0.05) the expression of MFN-1, validating proteomic results, whereas MFN-2 or OPA-1 expressions were not significantly modified by sST2 treatment (Figure 1A). The reduction in MFN-1 under sST2 treatment was also found at the transcriptional level (Figure 1B). To analyze whether sST2 treatment could affect mitochondrial structure and function, other proteins involved in mitochondrial fission, biogenesis and functional integrity were quantified. sST2 did not change the protein and mRNA expression of DRP-1, PHB-1, PHB-2 or PGC-1α (Figure 1C,D).

sST2 effects on mitochondrial proteins, oxidative stress-related molecules and inflammation in adult human cardiac fibroblasts

Figure 1
sST2 effects on mitochondrial proteins, oxidative stress-related molecules and inflammation in adult human cardiac fibroblasts

sST2 effect on MFN-1 and -2 and OPA-1 protein expressions in human cardiac fibroblasts (A). sST2 effect on MFN-1 and -2 and OPA-1 mRNA levels in human cardiac fibroblasts (B). Effects of sST2 on mitochondrial proteins at protein (C) and mRNA (D) levels. Oxidative stress markers (E) and protein carbonyl quantification in cells treated with sST2 (F). Effect of sST2 treatment on inflammatory markers at protein (G–I) and mRNA (J) levels. All conditions were performed at least in triplicate. Histogram bars represent the mean ± SEM of six assays. For Western blot experiments, protein densitometry was expressed in AU once normalized to stain free for protein. For RT-PCR experiments, data were expressed in AU once normalized to HPRT, β-actin and GADPH. *P<0.05 versus Control. Abbreviation: CCL-2, monocyte chemoattractant protein.

Figure 1
sST2 effects on mitochondrial proteins, oxidative stress-related molecules and inflammation in adult human cardiac fibroblasts

sST2 effect on MFN-1 and -2 and OPA-1 protein expressions in human cardiac fibroblasts (A). sST2 effect on MFN-1 and -2 and OPA-1 mRNA levels in human cardiac fibroblasts (B). Effects of sST2 on mitochondrial proteins at protein (C) and mRNA (D) levels. Oxidative stress markers (E) and protein carbonyl quantification in cells treated with sST2 (F). Effect of sST2 treatment on inflammatory markers at protein (G–I) and mRNA (J) levels. All conditions were performed at least in triplicate. Histogram bars represent the mean ± SEM of six assays. For Western blot experiments, protein densitometry was expressed in AU once normalized to stain free for protein. For RT-PCR experiments, data were expressed in AU once normalized to HPRT, β-actin and GADPH. *P<0.05 versus Control. Abbreviation: CCL-2, monocyte chemoattractant protein.

Concerning oxidative stress markers, the expressions of nitrotyrosine, MDA, CML and peroxide production were also increased (P<0.05) by sST2 treatment (Figure 1E). Moreover, quantitative analysis of the oxyblot revealed a significant enhancement in protein carbonyl levels in sST2-treated cells (Figure 1F).

Concerning antioxidant molecules, stimulation with sST2 did not modify the expression of Prx-IV and FH both at the protein (Supplementary Figure S1A) or the mRNA levels (Supplementary Figure S1B). The expression of the antioxidant manganese-SOD (MnSOD) was unchanged in sST2-treated cells (Supplementary Figure S1C).

In parallel, sST2-treated cells presented higher levels in the secretion (Figure 1G–I) and the mRNA expressions (Figure 1J) of the pro-inflammatory cytokines IL-6, IL-1β and CCL-2, without modifying the anti-inflammatory M2 markers IL-10, cd206, peroxisome proliferator-activated receptor γ (PPARγ) and fatty acid-binding protein 4 (FABP4) (Supplementary Figure S1D).

NFκB mediates the pro-oxidant and pro-inflammatory effects of sST2 in human cardiac fibroblasts

Cells treated with recombinant sST2 presented lower levels of the adaptor protein MyD88 but similar levels of IL-33, TRAF6 and AP-1 (Figure 2A). In order to investigate the intracellular pathways early activated by sST2 in human cardiac fibroblasts, cells were treated for 5, 10, 15, 30 and 60 min with sST2. Stimulation with sST2 induced the phosphorylation of NFκB at 30 and 60 min of stimulation (Figure 2B), without affecting the degree of phosphorylation of IRAK1/4 (Figure 2C), p38 MAPK (Figure 2D) and p42/44 MAPK (Figure 2E).

Intracellular pathways involved in sST2 pro-oxidant and pro-inflammatory effects in adult human cardiac fibroblasts

Figure 2
Intracellular pathways involved in sST2 pro-oxidant and pro-inflammatory effects in adult human cardiac fibroblasts

Expression of the sST2 signaling pathway (A). sST2 effects on phosphorylation of NFκB (p-NFκB Ser536) (B), IRAK 1/4 (C), p38 MAPK (p-p38 Thr180-Tyr182) (D) and p42/44 MAPK (p-p42/44 Thr202-Tyr204) (E). MFN-1 expression in cells treated with sST2 and the NFκB pharmacological inhibitor BAY 11-7082 (F). Effects of NFκB pharmacological inhibition on oxidative stress markers (G) and inflammatory markers (H–J). Histogram bars represent the mean ± SEM of six assays. For Western blot experiments, protein densitometry was expressed in AU once normalized to stain free for protein and to total NFκB, p38 MAPK and p42/44 MAPK protein. *P<0.05 versus Control. $, P<0.05 versus sST2. Abbreviation: CCL-2, monocyte chemoattractant protein.

Figure 2
Intracellular pathways involved in sST2 pro-oxidant and pro-inflammatory effects in adult human cardiac fibroblasts

Expression of the sST2 signaling pathway (A). sST2 effects on phosphorylation of NFκB (p-NFκB Ser536) (B), IRAK 1/4 (C), p38 MAPK (p-p38 Thr180-Tyr182) (D) and p42/44 MAPK (p-p42/44 Thr202-Tyr204) (E). MFN-1 expression in cells treated with sST2 and the NFκB pharmacological inhibitor BAY 11-7082 (F). Effects of NFκB pharmacological inhibition on oxidative stress markers (G) and inflammatory markers (H–J). Histogram bars represent the mean ± SEM of six assays. For Western blot experiments, protein densitometry was expressed in AU once normalized to stain free for protein and to total NFκB, p38 MAPK and p42/44 MAPK protein. *P<0.05 versus Control. $, P<0.05 versus sST2. Abbreviation: CCL-2, monocyte chemoattractant protein.

The specific inhibitor of NFκB, BAY 11-7082, was able to prevent the decrease in MFN-1 protein levels (Figure 2F). Moreover, the pre-treatment with the specific inhibitor of p38 NFκB was able to block sST2 effects on nitrotyrosin, MDA, CML and peroxide (Figure 2G). Interestingly, the blockade of NFκB pathway also abolished sST2-induced secretion of the pro-inflammatory markers Il-6, IL-1β and CCL-2 (Figure 2H–J).

sST2-induced pro-oxidant and pro-inflammatory molecules in adult human cardiac fibroblasts were mediated by mitochondrial ROS

Mito-Tempo is a superoxide scavenger that has the role of eliminating mitochondrial ROS and Resveratrol exerts potent protection of mitochondrial function. For instance, both Mito-Tempo and Resveratrol treatment prevented sST2-induced ROS production in human cardiac fibroblasts (Figure 3A). Moreover, treatment with Mito-Tempo or Resveratrol protected the cells against sST2-induced oxidative stress markers nitrotyrosin, MDA, CML and peroxide (Figure 3B). Pre-incubation with the superoxide scavenger or the antioxidant also decreased the expression of proinflammatory markers IL-6 and CCL-2 induced by sST2 (Figure 3C–E). However, only Mito-Tempo diminished the expression of IL-1β induced by sST2 (Figure 3D).

Effects of mitochondrial ROS scavenger and a general antioxidant on oxidative stress and inflammatory markers induced by sST2 in adult human cardiac fibroblasts

Figure 3
Effects of mitochondrial ROS scavenger and a general antioxidant on oxidative stress and inflammatory markers induced by sST2 in adult human cardiac fibroblasts

Representative microphotographs in cells labeled with DHE (A). Mito-tempo and Resveratrol effects on sST2-induced oxidative stress markers (B) and inflammatory markers (C–E). *P<0.05 versus Control. $,P <0.05 versus sST2. Abbreviation: CCL-2, monocyte chemoattractant protein.

Figure 3
Effects of mitochondrial ROS scavenger and a general antioxidant on oxidative stress and inflammatory markers induced by sST2 in adult human cardiac fibroblasts

Representative microphotographs in cells labeled with DHE (A). Mito-tempo and Resveratrol effects on sST2-induced oxidative stress markers (B) and inflammatory markers (C–E). *P<0.05 versus Control. $,P <0.05 versus sST2. Abbreviation: CCL-2, monocyte chemoattractant protein.

ST2 and oxidative stress in myocardial biopsies from AS patients

The baseline characteristics of the patients and controls are presented in Table 1.

Table 1
Baseline characteristics of patients and controls
 Controls AS patients 
Myocardial biopsies   
13 26 
Age (years) 75 ± 11 74 ± 8 
Male 7 (54%) 12 (46%) 
Hypertension 1 (8%) 18 (69%) 
Hyperlipidemia 3 (23%) 16 (62%) 
Diabetes 1 (8%) 2 (7%) 
Coronary artery disease 1 (8%) 7 (27%) 
Lung disease 4 (30%)  
Cause of death   
-Bronchopneumonia 4 (30%)  
-Sepsis 1 (8%)  
-Cancer 6 (46%)  
-Trauma 1 (8%)  
-Old age 1 (8%)  
LVEF (%)  65 ± 13 
NYHA  I (11.5%) 
  II (57.7%) 
  III (23.1%) 
  IV (7.7%) 
Treatments   
ACEi (n, %)  10 (38%) 
ARBs (n, %)  4 (15%) 
β-blockers (n, %)  4 (15%) 
MRA (n, %)  2 (1%) 
 Controls AS patients 
Myocardial biopsies   
13 26 
Age (years) 75 ± 11 74 ± 8 
Male 7 (54%) 12 (46%) 
Hypertension 1 (8%) 18 (69%) 
Hyperlipidemia 3 (23%) 16 (62%) 
Diabetes 1 (8%) 2 (7%) 
Coronary artery disease 1 (8%) 7 (27%) 
Lung disease 4 (30%)  
Cause of death   
-Bronchopneumonia 4 (30%)  
-Sepsis 1 (8%)  
-Cancer 6 (46%)  
-Trauma 1 (8%)  
-Old age 1 (8%)  
LVEF (%)  65 ± 13 
NYHA  I (11.5%) 
  II (57.7%) 
  III (23.1%) 
  IV (7.7%) 
Treatments   
ACEi (n, %)  10 (38%) 
ARBs (n, %)  4 (15%) 
β-blockers (n, %)  4 (15%) 
MRA (n, %)  2 (1%) 

Values are mean ± SD. Abbreviations: ACEi, angiotensin-converting-enzyme inhibitor; ARB, angiotensin II receptor blocker; LVEF, left ventricular ejection fraction; MRA, mineralocorticoid receptor antagonist; NYHA, New York Heart Association classification of heart failure.

sST2 expression quantified by RT-PCR and immunohistochemistry was higher in myocardium from AS patients as compared with controls (Figure 4A). Representative images for ST2 immunostaining in Controls and AS myocardial biopsies are shown in Figure 4B. Moreover, MFN-1 expression was decreased in myocardium from AS patients as compared with controls at the mRNA levels (Figure 4C). Representative images for MFN-1 immunostaining in controls and AS myocardial biopsies are shown in Figure 4D. In myocardial biopsies, sST2 protein levels inversely correlated with MFN-1 expression (r = −0.468, Figure 4E).

sST2, oxidative stress and inflammation in myocardial biopsies from AS patients

Figure 4
sST2, oxidative stress and inflammation in myocardial biopsies from AS patients

Expression of ST2 mRNA levels and quantification of its immunostaining in Controls and AS hearts (A). Representative images of control and AS sections stained for ST2 (B). Expression of MFN-1 mRNA levels in Controls and AS hearts (C). Representative images of control and AS sections stained for MFN-1 (D). Myocardial protein sST2 expression negatively correlated with the expression of MFN-1 (E). Quantification of mRNA expression of CCL-2, IL-6, IL-1β and TNF-α in myocardial biopsies from controls and AS patients (F). Myocardial protein sST2 expression positively correlated with the expression of CCL-2 (G) and IL-6 (H). Magnification of the microphotographs 40×. Histogram bars represent the mean ± SEM of each group of subjects (Control n=13 and patients with AS, n=26) in AU normalized to HPRT, β-actin and GADPH for cDNA. *P<0.05 versus control group. Abbreviation: CCL-2, monocyte chemoattractant protein.

Figure 4
sST2, oxidative stress and inflammation in myocardial biopsies from AS patients

Expression of ST2 mRNA levels and quantification of its immunostaining in Controls and AS hearts (A). Representative images of control and AS sections stained for ST2 (B). Expression of MFN-1 mRNA levels in Controls and AS hearts (C). Representative images of control and AS sections stained for MFN-1 (D). Myocardial protein sST2 expression negatively correlated with the expression of MFN-1 (E). Quantification of mRNA expression of CCL-2, IL-6, IL-1β and TNF-α in myocardial biopsies from controls and AS patients (F). Myocardial protein sST2 expression positively correlated with the expression of CCL-2 (G) and IL-6 (H). Magnification of the microphotographs 40×. Histogram bars represent the mean ± SEM of each group of subjects (Control n=13 and patients with AS, n=26) in AU normalized to HPRT, β-actin and GADPH for cDNA. *P<0.05 versus control group. Abbreviation: CCL-2, monocyte chemoattractant protein.

Regarding the expression of the inflammatory markers, myocardial biopsies from AS patients exhibited higher CCL-2, IL-6, IL-1β and TNF-α mRNA levels (Figure 4F). sST2 protein levels positively correlated with CCL-2 mRNA levels (r = 0.597, Figure 4G) and with IL-6 mRNA levels (r = 0.555, Figure 4H) in all patients.

Discussion

The purpose of the present study was to investigate the effects of sST2 on oxidative stress and inflammation in human cardiac fibroblasts. Using a proteomic approach, MFN-1 has been identified as a protein down-regulated by sST2. Moreover, sST2 induced an increase in ROS markers such as nitrotyrosine and MDA, in CML (an advanced glycation end product) and a consequent increase in peroxide production and protein oxidation in cardiac fibroblasts. sST2 also induced the expression and secretion of pro-inflammatory markers in human cardiac fibroblasts. Interestingly, Mito-Tempo and Resveratrol attenuated oxidative stress and inflammation. Moreover, cardiac sST2 was increased in AS patients and associated with decreased MFN-1 and with increased inflammatory markers. Thus, the present study reveals a new pathogenic pathway by which sST2 promotes oxidative stress and inflammation contributing to cardiac damage.

The studies analyzing the effects of sST2 in cardiac cells are scarce. In cardiomyocytes, recombinant sST2 seems to be deleterious by blocking antihypertrophic effects of IL-33 [22], whereas IL-33 has been reported to exert antioxidant effects in adult cardiomyocytes [15]. Our study shows for the first time that sST2 also exerts deleterious effects in cardiac fibroblasts, decreasing MFN-1 expression without modifying MFN-2 or OPA-1 levels, and increasing ROS production leading to inflammatory molecules production. In fact, either MFN-1 or MFN-2 can functionally replace each other. In conditions where MFN-1 expression is decreased, mitochondrial fusion can be released by MFN-2 overexpression and vice versa [6,23,24]. However, in sST2-treated cells, MFN-2 levels did not increase, suggesting that mitochondrial fusion could be impaired by sST2. Disrupting mitochondrial fusion via MFN-1 ablation could alter the distribution and morphology of mitochondria and precipitates mitochondrial dysfunction [25]. Overall there is a direct correlation between mitochondrial fusion and the oxidative phosphorylation capacity [26]. Controversially, it has been described that MFN-1 KO myocytes are protected against ROS-induced cell death [27]. MFN-1 expression is down-regulated in experimental models of cardiac damage such as pulmonary arterial hypertension [28] and ischemia-reperfusion [29]. In line with these evidences, sST2 increment paralleled MFN-1 decrease in human AS myocardium, suggesting that the reduction of mitochondrial fusion could be a deleterious mechanism that facilitates myocardial protein oxidation and inflammation.

Of special interest, sST2 is able to activate NFκB pathway in human cardiac fibroblasts, leading to increased oxidative stress and inflammatory markers. Accordingly, it has been described that IL-33 can suppress ROS generation [22], that could be implicated in NFκB activation [30]. Consistent with these findings, pathways involving MyD88 such as IL-33 may actually limit the inflammatory responses [31]. Interestingly, sST2-induced the expression of inflammatory molecules is mediated by mitochondrial oxidative stress, since the use of Resveratrol or Mito-Tempo abolished sST2 effects on oxidative stress and inflammatory molecules secretion in cardiac fibroblasts.

The inflammatory response to conditions of oxidative stress could lead to early organ damage and dysfunction. In our study, we show for the first time that sST2 exerts pro-inflammatory effects in human cardiac fibroblasts, without modifying the expression of the anti-inflammatory molecules. This result is reinforced in human myocardial biopsies, where sST2 levels positively correlated with the expression of inflammatory markers. According to this result, sST2 serum levels have been found to be associated with C-reactive protein levels in AS patients [32] and in acute ischemic stroke patients [33]. However, and in conflict with our results, sST2 levels were not increased in 12 myocardial biopsies from AS patients as compared with controls [32]. Thus, understanding the pathways leading to the initial activation of inflammatory pathways in oxidative stress is essential to devising strategies to limit the detrimental consequences of the inflammatory response to injury.

In summary, the present study shows that sST2 could exert a deleterious effect by affecting mitochondrial fusion, leading to increase oxidative stress and inflammation in human cardiac fibroblasts (Figure 5). Given the importance of the dynamics of mitochondria in HF, as well as the essential role of cardiac fibroblasts in the development of cardiac inflammation, studies focusing on the interactions between new signaling molecules such as sST2 and oxidative stress in human cardiac fibroblasts are warranted.

General scheme summarizing sST2 effects on human cardiac fibroblasts

Figure 5
General scheme summarizing sST2 effects on human cardiac fibroblasts

sST2 decreases MFN-1 levels through NFκB activation leading to an induction of oxidative stress and finally to an increase in inflammatory markers secretion.

Figure 5
General scheme summarizing sST2 effects on human cardiac fibroblasts

sST2 decreases MFN-1 levels through NFκB activation leading to an induction of oxidative stress and finally to an increase in inflammatory markers secretion.

Limitations

Several limitations of our study must be acknowledged. First, the present study involves relatively small amounts of tissue obtained from LV from AS patients (there was no material available for Western blot studies), making it difficult to undertake an extensive range of analyses. Second, the greatest potential limitation to endomyocardial biopsy evaluation is sampling error. In addition, at the time of valve replacement patients were under polymedication, which may influence the results.

Clinical perspectives

  • sST2 is produced by cardiac fibroblasts and its expression increases in inflammatory diseases and in HF.

  • sST2 exert a deleterious effect by affecting mitochondrial fusion, leading to increase oxidative stress and inflammation in human cardiac fibroblasts, contributing to cardiac damage.

  • In human AS, cardiac sST2 levels associated with oxidative stress and inflammation.

Competing Interests

The authors declare that there are no competing interests associated with the manuscript.

Funding

This work was supported by the Miguel Servet contract CP13/00221 from the ‘Instituto de Salud Carlos III-FEDER’; the Fondo de Investigaciones Sanitarias [grant number PI18/01875]; the Proteomics Unit of Navarrabiomed is a member of ProteoRed and PRB3- ISCIII and is supported by grant PT17/0019, of the PE I+D+i 2013- 2016, funded by ISCIII and ERDF; FIGHT-HF [grant number ANR-15-RHU-0004 (to P.R.)]; and the CAM (Atracción de talento) (to E.M.-M.).

Author Contribution

Natalia López-Andrés conceived and designed the study; Lara Matilla, Jaime Ibarrola, Vanessa Arrieta, Ernesto Martinez-Martinez, Virginia Alvarez, Rafael Sádaba, Enrique Santamaría, Joaquín Fernández, Amaia Garcia-Peña, Adela Navarro, Alicia Gainza and Amaya Fernández-Celis performed the data. Lara Matilla, Ernesto Martinez-Martinez, Jaime Ibarrola, Amaya Fernández-Celis, Patrick Rossignol, Antoni Bayés-Genís and Natalia López-Andrés analyzed and interpreted the data. Lara Matilla, Jaime Ibarrola, Antoni Bayés-Genís, Patrick Rossignol and Natalia López-Andrés led the design and drafted the paper.

Abbreviations

     
  • AP-1

    activating protein 1

  •  
  • AS

    aortic stenosis

  •  
  • CCL2

    monocyte chemoattractant protein

  •  
  • CD206

    cluster of differentiation 206

  •  
  • CML

    carboxy-methyl-lysine

  •  
  • DHE

    dihydroethidium

  •  
  • DRP-1

    dynamin-related protein-1

  •  
  • ECM

    extracellular matrix

  •  
  • FABP4

    fatty acid-binding protein 4

  •  
  • FH

    fumarate hydratase

  •  
  • GADPH

    glyceraldehyde-3-phosphate dehydrogenase

  •  
  • HF

    heart failure

  •  
  • HPRT

    hypoxanthine phosphoribosyltransferase

  •  
  • IL

    interleukin

  •  
  • IRAK1/4

    interleukin-1 receptor-associated kinase 1/4

  •  
  • iTRAQ

    isobaric Tags for Relative and Absolute Quantitation

  •  
  • LV

    left ventricle

  •  
  • MAPK

    mitogen-activated protein kinase

  •  
  • MDA

    malondialdehyde

  •  
  • MFN-1

    mitofusin-1

  •  
  • MnSOD

    manganese-SOD

  •  
  • MyD88

    myeloid differentiation primary response 88

  •  
  • NFκB

    transcriptional factor nuclear factor κB

  •  
  • OPA-1

    optic atrophy protein 1

  •  
  • PGC-1α

    peroxisome proliferator-activated receptor-γ co-activator 1 α

  •  
  • PHB

    prohibitin

  •  
  • PPARγ

    peroxisome proliferator-activated receptor γ

  •  
  • PRX-IV

    peroxiredoxin IV

  •  
  • ROS

    reactive oxygen species

  •  
  • RT-PCR

    real time polymerase chain reaction

  •  
  • SOD

    superoxide dismutase

  •  
  • ST2

    interleukin 1 receptor-like 1

  •  
  • sST2

    soluble ST2

  •  
  • ST2L

    ST2 transmembrane receptor

  •  
  • TGF-β1

    transforming growth factor β1

  •  
  • TNF-α

    tumor necrosis factor α

  •  
  • TRAF6

    tumor necrosis factor receptor associated factor 6

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Author notes

*

These authors contributed equally to this work.