Identification of altered serum proteins in rheumatic heart diseases through mitral stenosis and the potential clinical implications

Nancy Bright Arul Joseph Raj; Shanavas Syed Mohamed Puhari; Panneerselvam Gomathi; Andiappan Rathinavel; Govindan Sadasivam Selvam

doi:10.4103/ACCJ.ACCJ_42_20

ORIGINAL ARTICLE

Year : 2021 | Volume : 3 | Issue : 1 | Page : 20-28

Identification of altered serum proteins in rheumatic heart diseases through mitral stenosis and the potential clinical implications

Nancy Bright Arul Joseph Raj¹, Shanavas Syed Mohamed Puhari¹, Panneerselvam Gomathi¹, Andiappan Rathinavel², Govindan Sadasivam Selvam¹
¹ Department of Biochemistry, Molecular Cardiology Unit, Centre for Excellence in Genomic Sciences, School of Biological Sciences, Madurai Kamaraj University, Madurai, Tamil Nadu, India
² Dean Madurai Medical College & Govt Rajaji Hospital, Madurai, Tamil Nadu, India

Date of Submission	29-Nov-2020
Date of Acceptance	04-Mar-2021
Date of Web Publication	26-Jun-2021

Correspondence Address:
Govindan Sadasivam Selvam
Department of Biochemistry, Molecular Cardiology Unit, Center for Excellence in Genomic Sciences, School of Biological Sciences, Madurai Kamaraj University, Madurai - 625 021, Tamil Nadu
India

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ACCJ.ACCJ_42_20

Abstract

Background: Rheumatic heart disease (RHD) results from group A beta-hemolytic streptococcal pharyngeal infection is an autoimmune sequela of acute or recurrent episodes of acute rheumatic fever (ARF). This study is focused on identifying heart tissue-specific proteins implicated in the secondary immunopathogenesis of RHD. Methods: Sera from 49 RHD patients and 32 controls were probed in 2DE to study the differential expression of proteins. After 2DE, the spots were analyzed and identified using ESI-MS. A total of 1082 protein spots were detected in RHD patients and controls. Results: Two protein spots were significantly down-regulated (p≤0.01) and 34 protein spots were significantly up-regulated (p≤0.01) compared to controls. The differentially expressed protein spots were trypsin-digested and identified as hyaluronan-mediated motility receptor (RHAMM), troponin 1, janus kinase and microtubule interacting protein 1 (Jakmip 1), nuclear ubiquitous casein and cyclin-dependent kinase substrate 1, basal body-orientation factor 1 and muscle-related coiled-coil protein. A positive correlation was established with the up-regulated and down-regulated expression of these proteins suggests them as potential biomarker for RHD. Conclusion: This study highlights rheumatic mitral stenosis and regurgitation, an active inflammatory process and provides novel information about the proteins thereby elaborates the knowledge of physiology and etiology of this disease.

Keywords: Biomarkers, hyaluronic acid-mediated motility receptor, rheumatic heart disease, serum

How to cite this article:
Arul Joseph Raj NB, Mohamed Puhari SS, Gomathi P, Rathinavel A, Selvam GS. Identification of altered serum proteins in rheumatic heart diseases through mitral stenosis and the potential clinical implications. Ann Clin Cardiol 2021;3:20-8

How to cite this URL:
Arul Joseph Raj NB, Mohamed Puhari SS, Gomathi P, Rathinavel A, Selvam GS. Identification of altered serum proteins in rheumatic heart diseases through mitral stenosis and the potential clinical implications. Ann Clin Cardiol [serial online] 2021 [cited 2023 Jun 4];3:20-8. Available from: http://www.onlineacc.org/text.asp?2021/3/1/20/319520

Introduction

Rheumatic heart disease (RHD) is primarily an autoimmune sequela of acute rheumatic fever (ARF)^[1],[2] which follows Group A beta-hemolytic streptococcal pharyngeal infection.^[3] Autoimmunity is the failure of an individual to recognize its own constituent parts as self, thus leading to an immune response against its own cells and tissues. The repeated or prolonged exposure to Group A streptococcus (GAS) infections increases the risk of developing RHD. GAS is a Gram-positive bacterium with human as a unique reservoir and an array of virulence factors allowing for a very broad spectrum of clinical expression. ARF occur in children aged 5–15 years, whereas the prevalence of RHD peaks in early adulthood with approximately 60% of ARF cases progressing to RHD.^[4],[5] At present, ARF/RHD is an uncommon disease in developed countries, but 350,000 deaths occur in developing world annually.^[6],[7]

Rheumatic mitral stenosis is associated with thickening of the mitral valve leaflets, fusion of commissures, and chordae tendinae together with fibrosis and calcification.^[8] Patients with mitral stenosis are prone to develop atrial fibrillation (AF), because of left atrial dilation in response to valve obstruction, inflammatory and fibrotic changes caused by the rheumatic process. AF is a common arrhythmic condition that is highly prevalent in 0.4% of the overall population.^[9] Echocardiography, cardiovascular magnetic resonance, and cardiac computed tomography are the imaging techniques used to assess patients with mitral stenosis and regurgitation. Moreover, being a high workforce-dependent technique, there is a chance of over or under diagnosis of rheumatic mitral stenosis with these imaging techniques. Therefore, an identification of a biomarker combined with the echocardiography-based approach is essential for the prediction, diagnosis, and management of RHD in patients with irreversible valvular dysfunction and recommended for valvular surgery. The detection of an ideal biomarker help in studying the early pathophysiological process of RHD.

Proteomics is the large-scale study of proteins, especially their structures and functions which enables detection and identification of low abundance proteins. Serum a complex body fluid comprises 10,000 with a majority of them secreted or shed by cells during different physiological or pathological processes can potentially be used to assess both normal and disease conditions. Till date, only a few reports have mentioned the relationship between valvular disease and serum proteomic profiling in RHD patients.^{[10],[11],[12],[13],[14],[15]} Thus, the present work is focused on the investigation of proteomic alterations in patients with RHD in comparison to normal controls, which could pave the way to identify the pathological changes.

Materials and Methods

Chemicals

Urea, Thiourea, CHAPS, 100% Glycerol, Sodium dodecyl sulphate, Acrylamide and Bisacrylamide, Glycine, Tris Cl, TEMED, DTT, IAA, Ammonium per sulphate, Trypsin were purchased from Sigma (Sigma-Aldrich, St Louis, MO). Acetonitrile, methanol and formic acid were purchased from Biosolve (Biosolve, Valkenswaard, The Netherlands). Coomassie Brilliant Blue R-250 dye was acquired from Thermofisher scientific (Landsmeer, The Netherlands).

Study design and population

The study population consisted of 81 participants that include 49 cases and 32 healthy controls aged between 17 and 60 years with informed consent. This study complies with the approval of the Institutional Ethical Committee of Madurai Kamaraj University. RHD patients admitted in Saravana Hospital, Madurai, from March 2014 to October 2016 were selected into the study as cases. All patients had morphologic cardiac valve lesions and most of them presented severe mitral stenosis. Healthy individuals chosen within the vicinity of Madurai Kamaraj University with no evidence or family history of cardiac abnormality or any medication were treated as controls. Participants having clinically documented rheumatoid arthritis, diabetes, hypertension, severe systemic illness, renal failure, liver failure, or any heart disease except RHD were summarily excluded from the study.

Biochemical parameter analysis

All participants were examined by physicians, and their clinical data were entered in a structured pro forma. Systolic blood pressure and diastolic blood pressure were determined. Pulse rate was observed, and occurrence of any irregularity was noted. The murmur of the heart confirmed by echocardiography during mitral stenosis was considered as clinical evidence of cardiac inflammation.

Blood samples were collected from all participants in a 5 ml sterile tube without anticoagulant and centrifuged at 3000 rpm for 10 min at 4°C. The collected serum was aliquoted and stored at − 80°C until further use. A part of serum samples was used for the analysis of biochemical parameters such as serum glucose urea, potassium, chloride, and sodium using electrolyte analyzer (Sensacore medical instrumentation Pvt. Ltd., India). Another aliquot of serum sample was subjected to two-dimensional electrophoresis (2DE).

Sample preparation for two-dimensional electrophoresis

Serum protein (450 μg) was mixed with 250 μl of rehydration buffer (7 M Urea, 2 M Thiourea, 4% CHAPS, 50 mM DTT, 0.5% ampholytes, and 0.004% bromophenol blue as tracking dye). The sample was loaded onto a 13-cm immobilized pH gradient (IPG) strip (PH 4–7, GE Healthcare, Sweden) for passive rehydration overnight and focused in an IPG Phor 3 (GE Healthcare, Sweden) according to the modified manufacturer's protocol (GE Healthcare, Sweden). During the first dimension electrophoresis, the Isoelectric focusing (IEF) of proteins was performed in four steps (500 v for 1 h, 1000 v for 1 h, 8000 v for 2 h 30 min and 8000 for 55 min). After IEF, consecutive reduction and alkylation of the IPG strips were carried out using 20 mg/ml DTT and 25 mg/ml Iodoacetamide (IAA), respectively, in an equilibration buffer (50 mM Tris-HCL PH 8.8, 6 M Urea, 30% Glycerol, 2% SDS). Then, the second dimension was performed in 12.5% polyacrylamide gel in SE 600 Ruby (GE Healthcare, Sweden) at 50 V followed by coomassie staining.^[16] The stained gels were scanned using 2D platinum image scanner (Amersham, Germany), and gel images were analyzed using Image Master Platinum Software version 7.0 (IMP7, GE Healthcare, Sweden).

Image analysis

After 2-DE separation, the 15 gels were stained with Coomassie blue R-250 (Sigma, St Louis, MO, USA). Spot detection and quantification were carried out using Image Master Platinum Software version 7.0 (IMP7). Spot intensity was quantified automatically by the calculation of spot volume after normalization of the image by taking the ratio of intensity of one spot to the total spots and expressed as the intensity. Only those spots with significant change (un-paired t-test, P = 0.01) in expression intensity were selected for mass spectrometry (MS) analysis.

In-gel tryptic digestion

Proteins were subjected to in-gel trypsin digestion according to Jiménez et al.^[17] Excised gel spots were destained with 200 μl of wash solution containing 50% methanol and 5% acetic acid for 4 h. 200 μl of 25 mM ammonium bicarbonate in 50% acetonitrile was added, and the gel was dehydrated. The gel was dried in a speed vacuum for 30 min. 40 μl of 10 mM DTT in 25 mM NH₄HCO₃ was added and incubated at 56°C for 1 h. The same volume of 55 mM IAA in 25 mM NH₄HCO₃ was added and incubated at the room temperature for 45 min in the dark with vortexing. 25 μl of 12.5 ng/μl in 25 mM NH₄HCO₃ to the dry gel pieces and incubated for overnight. 30 μl of 5% formic acid in 50% acetonitrile was added and agitated for 30–60 min to extract the peptides from the gel. 15 μl of 5% formic acid was added and incubated at the room temperature for 10 min and then 15 μl of 100% acetonitrile was added and agitated for 30–60 min. The two extract solutions were combined and dried in a speed vac. The dry pellet was dissolved in 10–20 μl of 0.1% formic acid and stored at − 20°C till further analysis.

Electrospray ionization-mass spectrometry analysis

The in-gel tryptic digestion was performed,^[18],[19] and the resulting peptides were identified by Electrospray ionization (ESI)-MS (LC2 fleet, Thermo Fisher Instruments limited, US) in positive ion mode. The scan range of mass spectrum was 300–2000 m/z. After data acquisition, the generated XML files were used to perform database searches using the MASCOT software v-2.2 (Matrix Science, London, UK). Search was carried out in NCBInr, MSDB and the Swiss-Prot databases with the following parameters such as species homo sapiens, Peptide tolerance 0.2 Da; MS/MS ion mass tolerance 0.1 Da; allowed up to one missed cleavage: Fixed modification, cysteine carboxyamido methylation, variable modification, oxidation of methionine, and propionamide (for cysteine modification by acrylamide). The mascot score was considered significant if P ≤ 0.05.

Western blotting

The serum samples were subjected for determining the upregulated expression of RHAMM by Western blotting.^[20] 12% sodium dodecyl polyacrylamide gel electrophoresis and immunoblotting were performed according to the standard protocol with equal amounts of proteins (100 μg). The antibodies used for this study are as follows: Mouse anti-β actin (1:100 dilutions), rabbit anti-rhamm antibody (1:1000 dilutions) purchased from Santa Cruz Biotechnology. β actin was used as a normalization control. Detection was realized by enhanced chemiluminescence with an ECL prime Western blotting detection reagent (Amersham Pharmacia Biotech, Buckinghamshire, UK) and horseradish peroxidase conjugated secondary antibody corresponding to each primary antibody. The luminolabelled membranes were analyzed by the Biorad chemiluminescent analyzer.

Histopathological analysis

The mitral valve tissue samples from RHD patients who exhibited significant differential expression of proteins (P ≤ 0.01) were collected during their valve replacement surgery, fixed in 10% formalin, embedded in paraffin. Mitral valve tissue sections (5 μm) are subjected to H and E staining (Sigma Chemical Co., St Louis, MO, USA) as described by Rashed et al.^[21] Sections were examined under an Olympus BX51 (Olympus Corporation, Tokyo, Japan) microscope, and images were captured with a digital camera attached to it.

Statistical analysis

All the data are expressed as the mean ± standard deviation. Repeated measure one-way analysis of variance (ANOVA) was used to compare the values of measurements between the groups. The experimental group gels were matched with reference gel (control) for analyzing the level of expression. Values were considered statistically significant if P ≤ 0.01. For analyzing other physiological and biochemical parameters, one-way ANOVA followed by Post hoc Tukey test was carried out using the Statistical Package for the Social Sciences (SPSS software version 16.0, Chicago, IL, USA).

Results

Baseline characteristics

A total of 49 patients with chronic RHD were enrolled in this study. Baseline characteristics of the patients are shown in Annexure 1. RHD patients were grouped based on their ECG data; AF (5), sinus tachycardia (25), sinus bradycardia (10), and other 9 patients were found to have normal rhythm. No significant changes in serum glucose, triglycerides, high-density lipoprotein, low-density lipoprotein, potassium, sodium, and chloride level were observed in RHD patients. Blood urea level (28.23 ± 7.8 mg/dl) was significantly higher in RHD patients.

Serum proteomics analysis in rheumatic heart disease patients and control samples

In the present study, serum from 49 RHD patients was subjected to differential protein analysis using 2DE, and the results were compared with 32 normal individuals. The proteins were resolved by 2DE gel using narrow range (pI 4–7, linear) IPG strips. [Figure 1] shows the representative image of 2DE gels of RHD patient samples with AF. A total of 1082 protein spots were detected in the serum of five patient samples, 36 protein spots were showing differential expression when compared to healthy controls. All the 36 protein spots were trypsin–digested and 30 spots could not be recognized with the information available in the peptide mass fingerprinting (PMF). Six protein spots were identified based on PMF after in-gel tryptic digestion and of which 4 protein spots was found to be significantly up-regulated (P ≤ 0.01 versus control, unpaired t-test) and 2 protein spots were significantly downregulated (P ≤ 0.01 versus control, unpaired t-test) [Figure 2] in RHD patients with AF. There is no significant difference in the proteomic alterations from the serum in RHD patients with sinus bradycardia and tachycardia.

Figure 1: Representative two-dimensional gel electrophoresis of serum in rheumatic heart disease patients and control individuals. (a) Aliquots containing 450μg of proteins from control samples were subjected to isoelectric focusing and separated by mass via Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) (Coomassie stained). Protein spots were circled and labelled were identified using mass spectrometry. (b-d) Representative image of two-dimensional electrophoresis gels of patient samples with Atrial fibrillation. The samples were separated using linear immobilized pH gradient gel (pH 4–7, 13 cm) in the first phase and 12.5% SDS-PAGE. Black circle represents up-regulation and red circles represents down-regulation

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Figure 2: Representative two-dimensional electrophoresis gel of serum in valvular heart disease and normal controls. (a) The intensity of serum RHAMM was up-regulated in Rheumatic heart disease when compared to normal controls. (b) The intensity of Troponin 1 in Rheumatic heart disease was up-regulated in Rheumatic heart disease compared with the normal control groups. (c) The intensity of Jakmip 1 in Rheumatic heart disease was up-regulated in Rheumatic heart disease individuals in comparison with normal control groups. (d) The intensity of nuclear ubiquitous casein and cyclin-dependent kinase substrate in Rheumatic heart disease was up-regulated compared with normal control groups. (e and f) The intensity of muscle related coiled coil protein and Basal body orientation factor 1 in Rheumatic heart disease was down-regulated in Rheumatic heart disease compared with the normal controls groups. Data are shown as mean ± standard error of the mean. (n = 5, **P ≤ 0.01 vs. control group, un-paired t-test)

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Identification of peptide fragments using electrospray ionization-mass spectrometry analysis

Six of the protein spots and their corresponding polypeptides were successfully identified by ESI-MS as Hyaluronan-mediated motility receptor (RHAMM) (26256.6 ± 500.6), troponin 1 (14162.63 ± 80.17), Janus kinase and microtubule interacting protein 1 (Jakmip1) (16263.67 ± 13.6), nuclear ubiquitous casein and cyclin-dependent kinase substrate1 (NUCKS1) (13272.33 ± 278.28), basal body-orientation factor 1 (BBOF1) (16542.5 ± 46.9), and muscle-related coiled-coil protein (14267.3 ± 54.2). Annexure 2 shows the MS data from the differentially expressed protein that depicts the sequence for human Hyaluronan-mediated motility receptor. Among six proteins, two were found to be down-regulated, in which one protein from patients with sinus bradycardia and other in AF condition. There were four up-regulated proteins found in AF. From the ESI-MS analysis, peptides belonging to Hyaluronan-mediated motility receptor (RHAMM), troponin 1, Janus kinase and microtubule interacting protein 1, NUCKS1 were found to be up-regulated as compared to the control. Basal BBOF1, muscle-related coiled-coil protein containing two coiled-coil regions were found to be down-regulated when compared to control. The differentially expressed proteins are represented in [Table 1].

Table 1: Differentially expressed protein spots identified using electrospray ionization mass spectrometers analysis in rheumatic heart disease patients and its function

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Overexpression of RHAMM in serum samples by Western blots

To validate the protein expression of RHAMM, Western blots were performed for RHD patients with AF and control samples. The peptide RHAMM significantly over expressed in the patient samples when compared to control. Beta actin was validated as an internal control in all the patient samples with the comparison to controls.

Histopathology analysis

The microscopic examination of mitral valve sections from three rheumatic mitral stenosis patients showed an enhanced degree of fibrosis. Aschoff bodies were observed as an important pathological condition in RHD patients. Neovascularization and valvular endothelial surface lining ulceration were observed. Microscopically, mitral valve calcification was also present in relation to valvular dysfunction. [Figure 3] shows the histopathological changes observed in the mitral valves of RHD patients.

Figure 3: Histopathological analysis of hematoxylin- eosin stained sections of 3 stenotic mitral valve samples from Rheumatic heart disease patients (×100 magnification). denotes aschoff bodies; denotes fibrosis condition; denotes Neovascularizationdenotes calcification

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Discussion

In the results of the present study, four proteins were overexpressed in the experimental group (Hyaluronan-mediated motility receptor (RHAMM), troponin 1, Janus kinase and microtubule interacting protein 1, NUCKS1) and two proteins less expressed in the experimental group (basal BBOF1, muscle-related coiled-coil protein). To the best of our knowledge, most of the proteins identified in this study have not been previously studied in the context of RHD. The differential expression of proteins in the patients reflects the pathophysiological changes, whereas proteins that are differentially abundant between the two groups are potential serological biomarkers for mitral stenosis arising from abnormal mitral valve structure and function.

In order to gain insight into the biological significance of the altered proteins in the disease, the differentially expressed proteins are categorized according to their reported biological functions. The results revealed that most of the identified proteins are related to inflammatory and immune response. The significant changes observed in the abundance of cardiovascular proteins such as RHAMM, troponin 1, and Janus kinase microtubule interacting protein 1 strongly suggested that they can be involved in the pathogenesis of RHD, but in different ways and represent a molecular signature in valve lesions. Histopathological analysis on tissue sections of stenotic mitral valves has confirmed the presence of valvular lesions. The pathological changes in the valvular specimens from patients with RHD have been defined in earlier literature, but these have been mainly in postmortem studies.^[22] The present study emphasizing the surgical specimen of the excised valves indicates that the main pathological process in chronic RHD is a progressive fibrosis, particularly affecting the heart valves. In RHD patients, the persistent inflammatory process in the heart tissue and the altered hemodynamic stress on the valve is responsible to extend the damage.^[23] Chronic inflammation in the valves of the heart results in narrowing of the valves which decreases the blood flow through the heart or leakage of the valves causing abnormal blood flow. This may lead to arrhythmias such as AF or the heart failure, where the heart is unable to pump enough blood to other parts of the body.^[24],[25] Studies reported that Aschoff nodules are characteristic lesions and can be taken as an indicator of a recent episode of RF that were absent in cases with AF.^[21],[24] However, in the present study, AF was clinically diagnosed in five cases and the histology report of two patient was identified with Aschoff bodies. Rheumatic valve calcification is an inflammatory process, associated with the expression of osteoblastic markers and neoangiogenesis that was highly associated with AF.^[9]

The increased intensity of RHAMM (26256.6 ± 500.6) in RHD patients with AF made the study to further focus on the concept of RHAMM as a potential marker in the diagnosis of RHD. RHAMM is located intracellularly in the cytoplasm, nucleus, and on the cell surface which has been implicated in regulating cellular responses to growth factors and plays a role in cell migration during wound repair.^[26],[27] Previous studies have reported the elevation of RHAMM level in knee synovial tissue of patients with advanced osteoarthritis.^[28] In accordance with the previous study,^[29],[30] the serum HA concentration was significantly higher in rheumatic arthritis patient which was similar to our results where the concentration of Hyaluronan binding receptor protein is significantly increased in RHD patients. The increased levels of Hyaluronan binding receptor protein in plasma from patients with rheumatic arthritis might cause cardiovascular diseases which is a common cause of death in patients with rheumatic arthritis.^[31] Hence, the expression of RHAMM may be increased due to the inflammation reaction caused in the mitral valve of the heart. Cardiac troponins are the components of the cardiomyocytes contractile apparatus and circulating concentrations are elevated during the myocardial injury. Studies reported that even low level of cardiac troponin elevation is associated with increased risk of heart failure and cardiovascular death in patients with coronary artery disease.^[32] Braunwald 2008^[33] reported that the underlying mechanism of troponin 1 release in the myocardium is due to subendocardial ischemia leading to myocytes necrosis, cardiomyocytes injury from inflammatory cytokines, hibernating myocardium, and apoptosis. In concordance with the previous studies done by Gupta et al., 2002,^[34] Williams et al., 2002,^[35] Alehan et al., 2003,^[36] troponin 1 is a significant marker for identifying myocardial injury due to hypoxic condition and viral myocarditis in the present study also the up-regulation of troponin 1 was observed in patients because of the myocardial injury due to RHD. The physiological mechanisms for the release of troponin T in the population were hypothesized as multifactorial and include the following: Transient, clinically silent ischemic episodes, small vessel occlusions, inflammatory processes, cardiomyocyte apoptosis, and increased myocardial strain due to pressure or volume overload. Due to increased afterload secondary to AS, left ventricular hypertrophy can result in ischemia due to supply/demand mismatch as well as myocardial fibrosis that can serve as an arrhythmogenic substrate in this patient population.

The present study shows the increased expression of Janus kinase and microtubule interacting protein (Jakmip) which plays a pivotal role in the signaling of numerous cytokines that have been implicated in the pathogenesis of inflammatory diseases including rheumatoid arthritis. The JAK proteins are key proteins that transduce the signals from the variety of cytokine and growth factor receptors. Four JAK's were identified (JAK1, JAK2, JAK3, and TYK1) which can form heterodimers and homodimers. The benefit of upregulated expression of JAK1 during host defense requires further study. The identification of proteins such as Troponin 1 and Jakmip has been shown to involve in multiple cellular functions during pathological processes of inflammatory disease such as RA. Jakmip was identified for its role in binding to the N-terminal FERM homology domain of Tyk2, a member of the JAK family of nonreceptor tyrosine kinases, central elements of cytokine signaling cascade.^[37] The results showed a down-regulation of muscle-related coiled-coil protein and basal body orientation factor 1. Muscle related coiled-coil protein enhances Rho/ROCK (Rho-kinase) signaling in cardiac muscles cells that facilitate myofibrillar organization. The mechanistic explanation of the differential expression of NUCKS1, muscle related coiled-coil protein and basal body orientation factor 1 is not known. Basal BBOF1 was found to be associated with heart septal defects. Thus, the results of the present study indicated that the altered expression of these proteins might be related to the valvular pathological changes which may also due to the infection by GAS in chronic RHD patients. The present findings provided the description of the changes in the proteomic profile in RHD patients based on the limited number of representative patient samples. Hence, recruiting larger multicentric population would be more effective for the verification studies. The alteration of proteins in mitral valve damage is influenced by the factors such as age, left atrial diameter, mitral valve area, and pulmonary artery systolic pressure. We found alterations in quite a few abundant serum proteins, but proteins in the ng/ml range such as cytokines, small peptides, and growth factors were not detected. This is the most comprehensive study performed till date on the serum proteome in mitral valve damaged severe RHD patients with AF and sinus bradycardia. This study highlights the importance of the differential protein expression profile in the progression to RHD which reflects the dysfunction of interstitial cells that are responsible for the tissue maintenance. Our results promote a deeper understanding of mitral valve lesion formation mediated by the autoimmune responses. Therefore, the specific proteins RHAMM, Janus kinase 1, and muscle-related coiled-coil protein may represent as potential biomarkers for RHD.

The molecular events leading to disease development are complex, diverse, and remain incompletely characterized so far, and to the best of our knowledge, none of the work related to stenotic mitral valve. The serum protein profile data serve as a central point for future mechanistic studies and a document for the proteins differentially abundant in disease which considered as candidate biomarkers for the diagnosis and prognosis. Therefore, the results of the present study help to provide information related to the molecular mechanisms of mitral valve damages due to RHD and improve the existing diagnostic strategies.

Acknowledgment

The authors express their gratitude to volunteers for providing the blood samples. The authors also thank ICMR for Junior Research Fellowship, UGC-CEGS, UPE, UGC-CAS and UGC-NRCBS program for the central instrumentation facility at SBS, MKU.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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