Physiol. Res. 65: 751-762, 2016 https://doi.org/10.33549/physiolres.933253 Identification of Novel Biomarker Candidates for Hypertrophic Cardiomyopathy and Other Cardiovascular Diseases Leading to Heart Failure H. REHULKOVA1, P. REHULKA1, A. MYSLIVCOVA FUCIKOVA1, J. STULIK1, R. PUDIL2 1Department of Molecular Pathology and Biology, Faculty of Military Health Sciences, University of Defence, Hradec Kralove, Czech Republic, 21st Department of Internal Medicine – Cardioangiology, Faculty Hospital, Hradec Kralove, Czech Republic Received November 9, 2015 Accepted April 8, 2016 On-line July 15, 2016 Summary Introduction In-depth proteome discovery analysis represents new strategy in an effort to identify novel reliable specific protein markers for Heart failure (HF) is not a uniform syndrome. It hypertrophic cardiomyopathy and other life threatening encompasses various causes as cardiomyopathy, cardiovascular diseases. To systematically identify novel protein congenital heart diseases, hypertension and ischemic biomarkers of cardiovascular diseases with high mortality we heart disease. The molecular basis of this entity is not employed an isobaric tag for relative and absolute quantitation completely understood (Yang et al. 2015). Hypertrophic (iTRAQ) proteome technology to make comparative analysis of cardiomyopathy (HCM), one of the pathologies plasma samples obtained from patients suffering from non- contributing to HF, has been defined morphologically by obstructive hypertrophic cardiomyopathy, stable dilated unexplained hypertrophy in the absence of cardiomyopathy, aortic valve stenosis, chronic stable coronary haemodynamic stress, and at the histological level by artery disease and stable arterial hypertension. We found myocyte disarray, fibrosis, and abnormalities of the 128 plasma proteins whose abundances were uniquely regulated intramyocardial small vessels. HCM is a monogenic among the analyzed cardiovascular pathologies. 49 of them have cardiac disease with an autosomal dominant pattern of not been described yet. Additionally, application of statistical heritability and with prevalence in the general population exploratory analyses of the measured protein profiles indicated of 1/500. More than 1,400 different mutations have been the relationship in pathophysiology of the examined discovered so far, involving mutations in 11 genes of cardiovascular pathologies. contractile sarcomeric proteins that have been shown to produce the disease (Hensley et al. 2015). Asymmetrical Key words septal hypertrophy leads to a significant pressure gradient Cardiovascular diseases • Biomarkers • Proteome analysis • between the apical left ventricular (LV) chamber and the Statistical evaluation left ventricular outflow tract (LVOT), resulting in the so- called ‘hypertrophic obstructive cardiomyopathy’ Corresponding author (obstructive HCM) presentation (Hensley et al. 2015). J. Stulik, Department of Molecular Pathology and Biology, Faculty The prevalence of LVOT obstruction in HCM is 20-30 % of Military Health Sciences, University of Defence, at rest and up to 70 % with provocation (Cooper et al. Trebesska 1575, 500 01 Hradec Kralove, Czech Republic. E-mail: 2015). Majority of patients with obstructive HCM are [email protected] asymptomatic but some can present with heart failure or arrhythmias. Just arrhythmias, premature sudden cardiac PHYSIOLOGICAL RESEARCH • ISSN 0862-8408 (print) • ISSN 1802-9973 (online) 2016 Institute of Physiology of the Czech Academy of Sciences, Prague, Czech Republic Fax +420 241 062 164, e-mail: [email protected], www.biomed.cas.cz/physiolres 752 Rehulkova et al. Vol. 65 deaths, heart failure and less frequently thromboembolic recommendations (Lang et al. 2015). The term “stable” events are primary causes of mortality amongst HCM means that patient is clinically stable at least for one patients (Kumar et al. 2015). Ultimately, about 10 % of month before the enrollment to the study. obstructive HCM patients progress to an end-stage dilated Patients were enrolled into the study phase with LV wall thinning, cavity enlargement, and consecutively. There was no selection bias according to systolic dysfunction that resembles a dilated age, gender and medication. However, the patients with cardiomyopathy (DCM) (Xiao et al. 2015). significant concomitant disease, such as pulmonary The classification of heart failure is traditionally disease, malignancy, autoimmune disorders, neurodege- based on the pathological cause of failure of the cardiac nerative disorders, thyroid disease, or concurrent viral pump, the pathophysiological characteristics, and the disease were excluded. Baseline characteristics of the acuity and severity of the heart failure. There is general population of this study are shown in Table 1. assumption that identification of biomarker profiles of Results were compared to control group of pathologies threatening by heart failure can provide healthy 46 blood donors aged 55 to 67 years (median age additional criteria for risk stratification among HF 60 years) with no evidence of cardiovascular disease or patients (Braunwald 2008). From this point of view other severe disorders. Statistical analysis of the age plasma is very useful clinical specimen for the differences showed no significant differences between all identification of novel markers of cardiovascular studied groups with one exception of aortic stenosis. diseases. Some plasma proteins like proteins of There the median of age was significantly higher than in coagulation cascade, proteins of lipid transfer, proteins the other groups, because of the higher prevalence of interacting with vessel walls or platelets and, finally, degenerative etiology aortic stenosis. inflammatory proteins were found to be directly involved The study protocol conformed to the ethical pathogenesis of the cardiovascular system (Anderson guidelines of the 1975 Declaration of Helsinki, and was 2005). Plasma also represents the largest proteome of the approved by the ethical committee of our institution. human body exhibiting extremely dynamic ranges in Informed consent was obtained from each patient. protein concentration. This makes its quantitative analysis highly challenging. Nevertheless, current advanced mass Plasma sample collection spectrometry approaches preceded with different pre- Blood samples (before echocardiography and fractionation techniques enable to quantitate several cardiac catheterization) were obtained from left forearm thousand plasma proteins in one run (Keshishian et al. after an overnight fasting at the First Department of 2015). Internal Medicine of the University Hospital in Hradec In our study we employed isobaric tag for Kralove. The control samples were obtained from healthy relative and absolute quantitation (iTRAQ) technology donors at the Transfusion Department of the same labeling of peptides (Wiese et al. 2007) for comparison of hospital. The BD P100v1.1 vacuum tubes containing plasma protein profiles of patients with HCM and other protease inhibitors and EDTA anticoagulant were used diseases of the cardiovascular system, which are (Becton Dickinson, Franklin Lakes, NJ). Within one accompanied with structural changes of the myocardium hour, the blood samples were centrifuged for 20 min at and can lead to its failure. 2500 g at 22 °C and the supernatant was removed, aliquoted and kept at -80 °C until a further analysis was Methods performed. Study population Plasma immunoaffinity enrichment Study population consisted of five groups: An equal volume was taken from each patient patients with non-obstructive HCM, patients with aortic sample to create pooled aliquots of each group, all in valve stenosis (AS), chronic stable coronary artery duplicate. These pooled aliquots were depleted from disease (CAD), stable arterial hypertension (AH), and fourteen most abundant plasma proteins (albumin, IgG, stable DCM. The diagnosis of HCM was based on antitrypsin, IgA, transferrin, haptoglobin, fibrinogen, a history of illness, physical examination, alpha-2-macroglobulin, alpha-1-acid glycoprotein, IgM, echocardiography, and cardiac catheterization in apolipoprotein A1, apolipoprotein A2, complement C3, accordance with European Society of Cardiology and transthyretin) using HPLC immunoaffinity column 2016 Identification of Novel Biomarkers in Cardiovascular Diseases 753 MARS Hu-14 (Multiple Affinity Removal System of plasma proteins were then concentrated and desalted Agilent Technologies, Santa Clara, CA) according to using 3K Amicon Ultra centrifugal filter units (Millipore manufacturer´s instructions. Obtained depleted fractions Corporation, Bedford, MA). Table 1. Baseline characteristics of patients groups in the study. HCM DCM CAD AS AH (n=47) (n=21) (n=20) (n=24) (n=20) Age (years) 58.4±12.4 57.6±12.0 62.8±9.2 70.6±11.6 54.7±15.8 Female gender, n (%) 12 (26) 4 (19) 3 (15) 6 (25) 7 (35) NYHA functional class, n (%) I 21 (45) 4 (19) 13 (65) 3 (13) 20 (100) II 18 (38) 14 (67) 5 (25) 15 (63) 0 III-IV 8 (17) 3 (14) 2 (10) 6 (25) 0 Atrial fibrillation, n (%) 14 (30) 6 (29) 3 (15) 6 (25) 2 (10) Creatinine level (μmol.l-1) 94.0±29.8 88.3±18.0 89.7±29.0 109.9±44.7 91.7±18.9 Diabetes mellitus, n (%) 13 (28) 4 (19) 4 (20) 9 (38) 4 (20) Hyperlipidemia, n (%) 23 (49) 9 (43) 19 (95) 15 (63) 13 (65) Smoking, n (%) 20 (43) 12 (57) 15 (75) 9 (38) 9 (45) Echocardiography LA (mm) 48.2±7.2 42.1±12.3 43.1±7.4 44.0±6.0 42.7±2.9 RV (mm) 26.1±3.8 26.5±7.7 26.8±2.8 26.7±3.4 27.2±2.4 IVST (mm) 19.4±4.4 10.4±2.6 11.3±1.9 13.9±2.1 12.1±1.3 LV ESD (mm) 31.9±7.1