Blood-based protein profiling identifies serum protein c-KIT as a novel biomarker for hypertrophic cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is one of the most common hereditary heart diseases and can be classified into an obstructive (HOCM) and non-obstructive (HNCM) form. Major characteristics for HCM are the hypertrophy of cardiomyocytes and development of cardiac fibrosis. Patients with HCM have a higher risk for sudden cardiac death compared to a healthy population. In the present study, we investigated the abundancy of selected proteins as potential biomarkers in patients with HCM. We included 60 patients with HCM and 28 healthy controls and quantitatively measured the rate of a set of 92 proteins already known to be associated with cardiometabolic processes via protein screening using the proximity extension assay technology in a subgroup of these patients (20 HCM and 10 healthy controls). After validation of four hits in the whole cohort of patients consisting of 88 individuals (60 HCM patients, 28 healthy controls) we found only one candidate, c-KIT, which was regulated significantly different between HCM patients and healthy controls and thus was chosen for further analyses. c-KIT is a tyrosine-protein kinase acting as receptor for the stem cell factor and activating several pathways essential for cell proliferation and survival, hematopoiesis, gametogenesis and melanogenesis. Serum protein levels of c-KIT were significantly lower in patients with HCM than in healthy controls, even after adjusting for confounding factors age and sex. In addition, c-KIT levels in human cardiac tissue of patients with HOCM were significant higher compared to controls indicating high levels of c-KIT in fibrotic myocardium. Furthermore, c-KIT concentration in serum significantly correlated with left ventricular end-diastolic diameter in HOCM, but not HCM patients. The present data suggest c-KIT as a novel biomarker differentiating between patients with HCM and healthy population and might provide further functional insights into fibrosis-related processes of HOCM.

Scientific Reports | (2021) 11:1755 | https://doi.org/10.1038/s41598-020-80868-z www.nature.com/scientificreports/ ventricular outflow tract causing an increased systolic flow rate ultimately leading to a higher degree of mitral valve insufficiency. Therefore further interventional or surgical therapeutic options besides drug therapy are available for HOCM patients. Despite the high prevalence of HCM with 1:500 it is often under-diagnosed and therefore biomarkers are needed to detect this detrimental disease at an early stage and manage the clinical symptoms of patients with unidentified HCM. Several strategies to explore biomarkers have been pursued in the last years and provided information about circulating long non-coding RNAs 3-5 , microRNAs 6,7 , positron emission tomography (PET) 8 or cardiac MRI parameters 9 as well as circulating natriuretic peptides 10 involved into pathophysiology of HCM. First insights into proteomic data from myocardial hypertrophic tissues have been recently provided and several dysregulated proteins in septal myectomies in patients with HCM were described 11 . Nevertheless, detailed correlations to the circulating proteins in blood of HCM patients are still rare.
Reduction in the efficiency of cardiometabolic processes in HCM has been recently characterized 12,13 leading to impaired sarcomere energetics including altered sarcomere activity with lowered cardiac contractility 14 . In the present study we analyzed a series of circulating proteins known to be involved in cardiometabolic processes such as cell adhesion, immune response, complement activation and cellular metabolism, to identify circulating target proteins which were then correlated with tissue levels in human septal myectomies.
c-KIT, also known as tyrosine-protein kinase KIT, mast/stem cell growth factor receptor (SCFR) or CD117, is a plasma protein and receptor for the stem cell factor (SCF). It is mainly localized to the plasma membrane of different cell types, e.g. endothelial cells, fibroblasts or hematopoietic cells, in tissues as well as in body fluids. One of the possible mechanisms for the generation of soluble form of c-KIT is a metalloproteolytic octodomain shedding of c-KIT and it was already described in fibroblasts and mast cells 15 . Binding of SCF to c-KIT leads to an activation of several pathways (e.g. JAK/STAT3 or PI3K/AKT pathways) regulating amongst others cell proliferation and survival, hematopoiesis, gametogenesis and melanogenesis 16 . c-KIT mutations are associated with gastrointestinal, dermatological and hematological tumors [17][18][19][20][21] and c-KIT acts as a biomarker in different cancer types 22,23 . c-KIT expression has also been described in the detection and assessment of the prognosis of patients with tumors 24,25 . Recently the role of c-KIT in the development of bleomycin-induced pulmonary fibrosis was described by Ding et al. 26 . Investigating the importance of cardiac c-KIT has become the aim of the present study. Here, we identified serum protein c-KIT as a potential biomarker for HCM distinguishing between patients with hypertrophic cardiomyopathy and healthy subjects.

Results
We included 60 age-and sex-matched patients with HCM and 28 healthy control individuals to the present study. 33 patients among the HCM cohort had an obstruction (HOCM) and 27 were without obstruction (HNCM) in the left ventricular outflow tract. Detailed patients' characteristics are shown in Table 1. Patients were chosen according to the diagnostic criteria based on the recent European guidelines for the diagnosis and management of hypertrophic cardiomyopathies 2 .
The patients in the HNCM and HOCM group did not differ in clinical symptoms, NYHA classification and co-morbidities. Differences were found in the medication with higher use of AT1 receptor antagonists in HNCM patients, more severe grade mitral regurgitation in HOCM group and in the echocardiographic parameters according to the size of left atrium with larger atrium in the HOCM group. There were no differences in the dimensions of the septum thickness between HNCM and HOCM as well as in the left ventricular end-diastolic diameters. Due to the pathological consequences of the obstructive form of HCM the maximum gradient in the left ventricular outflow tract was higher in the HOCM group.
We applied a proteomic profiling approach by proximity extension assay (PEA) technology to find potential protein biomarkers in the serum of total 30 patients (10 patients with HOCM, 10 patients with HNCM and 10 healthy individuals) (Fig. 1A). We screened for 92 proteins known to be involved in several cardiometabolic processes including cell regulation, angiogenesis, apoptosis, immune response and heart development and came down to four significantly regulated candidates (c-KIT, IL7R, PCPE-1 and IGLC2), which were then subsequently validated via ELISA assay in a larger cohort of patients' sera (60 HCM, 28 healthy). The protein level of tyrosineprotein kinase c-KIT was significantly lower in patients with HCM than in healthy controls, whereby a significant difference occurred between HOCM and HNCM patients (Fig. 1B). Adjustment for age and sex did not affect this association between c-KIT and HCM showing that c-KIT is independently associated with HCM (Table 2). c-KIT was inversely associated with the presence of HCM. Interestingly, the area under the curve (AUC) for c-KIT alone was higher than for age and sex together (AUC 0.695 vs 0.538) (Fig. 2).
Furthermore, we performed correlation analyses of a potential association of HCM with echocardiographic parameters. There were no correlations between c-KIT and echocardiographic parameters in the whole HCM cohort. However, we found a direct significant correlation between c-KIT and left end-diastolic diameter (LVEDD) in HOCM but not HNCM patients (Fig. 3).
To investigate the correlation of c-KIT levels directly in the myocardium, we performed c-KIT analysis via qPCR in human myectomies of patients with HOCM and healthy controls (Fig. 4A). Here, a reverse relationship in comparison to serum was detected suggesting a reduced secretion of soluble c-Kit by HOCM myocardium. The significantly higher c-KIT levels in left ventricle biopsies of patients with HOCM may indicate a potential involvement in pro-fibrotic or pro-hypertrophic signaling pathways. To follow this hypothesis, we next measured several established fibrosis markers (collagen 1A1, collagen 3, CTGF and MMP2) in human biopsies with qPCR ( Fig. 4B-E). The expression of collagen 1A1 and MMP2 were significantly increased in myectomies of HOCM patients compared to healthy controls, whereas collagen 3 and CTGF expression was not different in both groups (Fig. 4B-E).      www.nature.com/scientificreports/ Next, we investigated which genes may be associated with c-KIT deregulation in cardiac cells. Therefore we silenced c-KIT expression in human cardiac fibroblasts (HCFs) via siRNA targeting c-KIT or a control siRNA (Suppl. Figure 1) followed by global RNA sequencing. As shown in Fig. 5A we found 1043 deregulated genes after c-KIT silencing. This data set was used for further functional enrichment analysis using David ontology server (Fig. 5B), as described 27 . Several biological processes associated with fibrosis as well as with cardiomyopathies in particular with HCM could be determined. Appropriate to the known data on the cardiometabolic changes

Discussion
The present study identified c-KIT as a novel protein biomarker for patients with hypertrophic cardiomyopathy and revealed a novel role for c-KIT to be directly involved in cardiac remodeling processes in HCM patients. Serum c-KIT concentrations are negatively correlated with the presence of HCM and positively correlated with echocardiographic parameter LVEDD in the HOCM cohort providing a further distinguishing feature between the patient groups of HOCM and HNCM. Additionally, in fibrotic tissues of human myectomies increased c-KIT levels correlated with higher markers of cardiac fibrosis in the HCM group compared to controls suggesting a possible link to a cardiac fibrosis already known to be associated with HCM. These data were also supported by global RNA sequencing results from human cardiac fibroblasts after c-KIT silencing revealing the involvement of fibrotic and cardiometabolic pathways. The RNA-Seq data give insights into cellular processes and identify strongly regulated pathways, e.g. inositol phosphate pathway involved into the modulation of cellular calcium release known to be affected in HCM as well as sphingolipid signaling pathway regulating several signal transduction processes including cell proliferation, differentiation and apoptosis. Additionally, silencing of c-KIT had a significant effect on the down-regulation of genes of hypertrophic, dilated and arrhythmogenic right ventricular cardiomyopathies. c-KIT and it's ligand hematopoietic cytokine stem cell factor (SCF) are essential during the embryonic development and their absence leads to intrauterine or perinatal death due to a severe macrocytic anemia 17 . Binding of SCF to c-KIT leads, via dimerization and phosphorylation, to the activation of the receptor. Activated c-KIT serves as binding site for multiple kinases which in turn activate further pathways. c-KIT tyrosine kinase signaling is involved in several cellular functions such as cell proliferation and survival and plays a decisive role inter alia in hematopoiesis and melanogenesis. c-KIT acts as proto-oncogene in tumors and pharmacological inhibitors of the c-KIT axis are used for therapeutic interventions 28 . Besides the oncologic importance of c-KIT signaling, few studies exist on the importance of c-KIT in the pulmonary and cardiovascular field. Ding et al. 26 reported about the role of SCF-c-KIT axis in bleomycin induced pulmonary fibrosis (IPF) and showed that expression of c-KIT in fibroblasts from patients with IPF was significantly higher than in those of control patients and there were more fibrosis markers, e.g. TGF-ß in the IPF group. In addition, the authors showed that fibroblasts were the main source of SCF in IPF mice in comparison to the controls and had a chemotactic effect on migration of bone marrow-derived cells to the injured lungs with subsequent paracrine activation of fibroblasts.
c-KIT signaling was also described to be involved in the overexpression of nerve growth factor, which was shown to exert several beneficial effects during injury and repair after myocardial infarction 29  www.nature.com/scientificreports/ Soluble c-KIT has been known to be released by hematopoietic, mast and endothelial cells [31][32][33] and circulates in human serum in concentrations considerably higher than those of circulating SCF. Wypych and colleagues showed that circulating c-KIT binds SCF and blocks SCF-induced cell proliferation 33 thus acting as an internal inhibitor of SCF and in turn of membrane bound c-KIT in cells. In line with these findings, in our study the levels of c-KIT in serum was significant lower in HCM patients compared to healthy persons without structural heart disease. According to Wypych et al., lower circulating c-KIT levels are associated with more cell proliferation and in case of HCM possibly with more fibrosis. Recently, Sukhacheva et al. examined the resident cardiac stem cells in myectomies of 40 HOCM patients and revealed that in 82,5% of samples c-KIT positive cells could be detected 34 . These myectomies contained increased density of the connective tissue, moderate hypertrophy of the cardiomyocytes and increased markers of heart failure 34 . In the present study we detected c-KIT in myectomies not only of HOCM patients but also in control biopsies and were able to show higher levels of c-KIT in HOCM group than in controls. The extent of cardiac fibrosis in different cardiomyopathies, e.g. HCM, is clinically relevant for the prognosis and risk assessment of ICD implantation due to the potential burden of sudden cardiac death. To correlate our data with a possible role of c-KIT in fibrosis, we measured several fibrosis markers in myectomies and detected elevated levels of pro-fibrotic factors such as Collagen 1A1 and MMP2 in HOCM biopsies compared to healthy samples. In addition to the levels of c-KIT in cardiac tissue, levels of c-KIT in serum of healthy and HCM patients were also measured. Here, we found that c-KIT levels were significant lower in HCM patients than in healthy controls and significantly correlated with echocardiographic parameter of left ventricular end-diastolic diameter in HOCM patients. c-KIT may thus serve as an indicator of HOCM severity in patients. Additionally our in vitro experiments in c-KIT silenced human cardiac fibroblasts and subsequent in-silico pathway analysis of RNA Sequencing data revealed that c-KIT is involved in several biological processes associated with fibrosis and in particular with HCM. As cardiac fibrosis is known to correlate with the risk of sudden cardiac death in HCM patients, effects of myocardial c-kit modulation should be investigated in further in vitro heart models (e.g. myocardial slices) and in vivo studies. Our medium-size study should therefore be supported by further studies with larger patient populations to examine the interaction between c-KIT and clinical severity of HCM and detailed molecular mechanisms to develop new therapeutic strategies for HCM patients and to alleviate clinical decision making in these patients.
In conclusion, the present data identified c-KIT as a novel serum and tissue biomarker differentiating between patients with HCM and healthy population. Such biomarker-assisted approach could be added to the current knowledge understanding fibrotic signaling within HCM.

Materials and methods
Patient data. Patients with HNCM/HOCM were enrolled at the Special Outpatient Clinic for HCM, Department of Cardiology and Angiology (Hannover Medical School). Blood from healthy controls was obtained from the Hannover Medical School blood donation service. Human biopsies were obtained from patients with HOCM who have undergone myocardial surgery at the Hannover Medical School or from non-transplanted donor hearts (accident victims; Sydney Heart Bank, Australia) as controls. Written informed consents were obtained from all patients before surgery. The study was approved by the local ethic committee of Hannover Medical School (No. 507/09).
The diagnosis of HCM was based on the recent European guidelines for the diagnosis and management of hypertrophic cardiomyopathies 2 and included presence of a hypertrophic cardiac septum (≥ 15 mm) or combined presence of a hypertrophic cardiac septum (≥ 13 mm) and positive family history and/or ECG abnormalities. HOCM was defined by a left ventricular outflow tract gradient ≥ 30 mmHg to differentiate between non-obstructive and obstructive form.
All methods were carried out in accordance with relevant guidelines 2 and regulations.

Measurement of biomarkers.
Serum levels of 92 proteins related to cardiometabolic processes were analyzed with multiplex proximity extension assay of the OLINK platform (OLINK Target 96 Cardiometabolic Panel, Olink Proteomics, Uppsala, Sweden), as described before [35][36][37] . Proximity extension assay (PEA) is based on pairs of antibodies linked to oligonucleotides which bind to their target proteins in the sample. The quantity of the target protein is measured by qPCR. The protein levels are expressed with normalized protein expression (NPX) values, OLINK Proteomics' arbitrary unit on log2 scale. Detailed information could be found at manufacturer's website www.olink .com/resou rces-suppo rt/docum ent-downl oad-cente r.

Transfection experiments.
As described before 38  Raw data processing and quality control. Raw data processing was conducted by use of nfcore/rnaseq (version 1.3) which is a bioinformatics best-practice analysis pipeline used for RNA sequencing data at the National Genomics Infrastructure at SciLifeLab Stockholm, Sweden. The pipeline uses Nextflow, a bioinformatics workflow tool. It pre-processes raw data from FastQ inputs, aligns the reads and performs extensive quality-control on the results. The genome reference and annotation data were taken from GENCODE.org (Homo sapiens; GRCh38; release 29).
Normalization and differential expression analysis (without outlier filtering). Normalization and differential expression analysis was performed with DESeq2 (Galaxy Tool Version 2.11.40.2) with default settings except for "Output normalized counts table", "Turn off outliers filtering", and "Turn off independent filtering", all of which were set to "True".
Gene set enrichment analysis. List of the 1043 significant genes (p value < 0.05) were submitted to DAVID functional annotation tool 27 for gene set enrichment analysis. KEGG pathway was used as annotation resource 40,41 .
Statistical analysis. The statistical software package R version 3.5.2 (www.r-proje ct.org) was used for statistical analyses. The characteristics of the studied population were summarized through standard descriptive statistics. Data are presented as the median (25th percentile, P25 -75th percentile, P75) for continuous variables and as frequency (percentage) for categorical variables. The demographic, clinical, echocardiographic and biochemical characteristics were compared between groups Mann-Whitney U test for continuous variables and Chi-squared test or Fisher's exact test for categorical variables. Spearman's rho coefficient was used to assess the correlation between continuous variables. Logistic regression was used to assess the relationship between c-Kit and HCM. To establish whether the associations could be influenced by confounding factors, the association was adjusted by age and sex. The results are presented as odds ratios (OR) and 95% confidence intervals (CI). Receiver operating characteristic (ROC) curves were constructed to estimate the area under the ROC curve. Data are presented as the C-index and 95% CI. The two-tailed significance level was set at < 0.05.

Data availability
All data generated or analyzed during this study are included in this published article and its Supplementary Information files.