MicroRNAs in Peripheral Mononuclear Cells as Potential Biomarkers in Hypertensive Patients With Heart Failure With Preserved Ejection Fraction

MicroRNAs in Peripheral Mononuclear Cells as Potential Biomarkers in Hypertensive Patients With... Abstract BACKGROUND MicroRNAs (miRs) regulate gene expression and play an important role in ventricular and vascular remodeling. However, there are limited data regarding their role in heart failure with preserved ejection fraction (HFpEF). The aim of this study was to assess gene expression of miR-1, miR-133a, miR-21, miR-208b, miR-499, and miR-26b in peripheral blood mononuclear cells (PBMCs) in hypertensive patients with HFpEF and to evaluate their association with their exercise capacity. METHODS We included 56 hypertensive patients with HFpEF (age 67.29 ± 7.75 years). Forty-two hypertensive patients without HFpEF (age 66.83 ± 7.17 years) served as controls. All subjects underwent a cardiopulmonary exercise test (CPXT). PBMCs were isolated and levels of miRs were determined by quantitative real-time reverse transcription polymerase chain reaction. RESULTS For hypertensive patients with HFpEF, higher expression levels in PBMCs were found only for miR-26b (7.6 ± 7.3 vs. 4.0 ± 3.6, P = 0.002), miR-208b (28.8 ± 35.3 vs. 7.5 ± 13.3, P < 0.001), and miR-499 (14.2 ± 22.4 versus 3.5 ± 2.9, P = 0.001). The strongest correlations with CPXT parameters were found for miR-208b levels, which had a positive correlation with maximal oxygen uptake (peakVO2) (r = 0.671, P < 0.001), exercise duration (r = 0.445, P = 0.001), and minute ventilation–carbon dioxide production relationship (VE/VCO2) (r = 0.437, P = 0.001) in the HFpEF group. CONCLUSIONS miR-26b, miR-208b, and miR-499 show a distinct in profile in hypertensive patients with HFpEF that is related with functional capacity. Further studies are needed to assess the role of miRs as prognostic tools or as therapeutic targets in those patients. blood pressure, cardiopulmonary exercise, heart failure, hypertension, microRNA Heart failure (HF) with preserved ejection fraction (HFpEF) is a common clinical entity and accounts for a significant proportion of patients with HF. Previous studies found that the prevalence of HFpEF is increasing and might reach 55% among patients with HF diagnosis.1 Patients with HFpEF are more likely to be female and have hypertension.1 HFpEF has reached epidemic levels and is soon likely to predominate, because the incidence of HFpEF is increasing faster than that of HF with reduced EF.1 This will have serious consequences, since it is associated with markedly increased morbidity, mortality, and health-care expenditure. Despite the high prevalence and poor prognosis of HFpEF, there is still no effective treatment in clinical practice, apart from management of its comorbidities. This is due in part to our insufficient understanding of the pathophysiology underlying HFpEF. Exercise intolerance, dyspnea, and fatigue during exertion are the predominant symptoms in HFpEF and have a decisive effect on the patients’ quality of life. Cardiopulmonary exercise response in these patients is markedly abnormal. The cardiopulmonary exercise test (CPXT) is a very valuable examination that reflects the disease severity of HF with both reduced and preserved EF. Recent advances in the research field suggest that the pathophysiology of exercise intolerance in HFpEF patients is complex and that noncardiac “peripheral” factors contribute to the reduced maximal oxygen uptake (peak VO2) they exhibit.2 Recent data in the literature have shown that worsening of cardiopulmonary exercise test parameters, mainly peakVO2 and minute ventilation-carbon dioxide production relationship (VE/VCO2), corresponds to a worsening in diastolic dysfunction, which is the main feature of HFpEF.3 microRNAs (miRs) are powerful regulators of biological processes and can modulate the expression of a large number of genes, while multiple miRs can target the same gene, as well as interact with each other, and can be actively exported or imported by cells.4 It has been estimated that more than 60% of all protein-coding genes are controlled by miRs.5 miRs have been shown to play an important role in the manifestations of cardiovascular diseases. It has been reported that heart-associated miRs (miR-1, miR-208b, miR-499, miR-133, etc.) are released into plasma during acute cardiac damage, serving as a stable biomarker.6,7 The study of miRs has the potential to help us better understand the biological mechanisms responsible for the development of cardiovascular diseases, including HF.8 On the other hand, the importance of transcriptomic and proteomic profile changes of peripheral blood mononuclear cells (PBMCs) has been noted in several cardiovascular conditions, including HF.9,10 In addition, in a recent study, Gupta MK et al. showed that there is dysregulation of miRs in PBMCs of HF patients and reported a specific miRNA signature in PBMCs of HF patients.11 They also showed that miRs alterations in PBMCs from HF patients do not mirror or overlap with cardiac miRs changes, suggesting that PBMCs may have their unique miRs signature in response to cardiac function. In addition, it has been reported that exercise changes miRs profiles in PBMCs of healthy individuals.12 Although a potential role of muscle and heart specific miRs in cardiovascular adaptation processes after aerobic exercise has been noted in marathon athlets,13 the role of miRs in exercise intolerance of HFpEF patients has not been investigated. In the present study, we sought to identify miRs in HFpEF patients that could be of prognostic validity and might be associated with those patients’ exercise functional parameters. We measured miR-1, miR-133a, miR-21, miR-208b, miR-499, and miR-26b gene expression levels in PBMCs, cells that contain subpopulations that are known to play a significant part in the pathophysiology of hypertension and HF.14,15 These miRs were chosen because they play a central role in cardiogenesis, heart function, and pathology,16 they have been implicated in several different cardiovascular pathologies including vascular and heart remodeling and have a distinct expression profile in cardiovascular diseases.17,18 For the evaluation of exercise capacity, we used the most important parameters of CPXT, which is the widely accepted method for this purpose. METHODS We enrolled 56 patients with HFpEF and essential hypertension and 42 patients with essential hypertension without symptoms or signs of HF. We applied the diagnostic criteria of the European Working Group for the diagnosis of HFpEF.19 Briefly, we defined HFpEF as follows: patients with HF syndrome and (i) left ventricular ejection fraction >50% and (ii) ratio between early mitral inflow velocity and mitral annular early diastolic velocity (E/e′) ≥15 or 8 < E/e′ < 15 and N-terminal pro-brain natriuretic peptide (NT-proBNP) levels >220 pg/ml. The diagnosis of hypertension was based on the participants’ medical history, in accordance with the recommendations of the European Society of Hypertension/European Society of Cardiology.20 The HFpEF patients were well-compensated, ambulatory outpatients who had been stable with no medication changes for at least 6 weeks. Patients with evidence of significant coronary, valvular, or pulmonary disease documented intracardiac shunting or any other medical condition that could mimic HF symptoms (anemia, chronotropic incompetence, thyroid dysfunction, etc.) were excluded. We also excluded patients with an implantable pacemaker, cardioverter–defibrillator, primary hypertrophic or restrictive cardiomyopathy, or any systemic illness associated with infiltrative heart disease (e.g., cardiac amyloidosis), inability to exercise due to other comorbidities that might affect the performance of an exercise test (e.g., osteoarthritis, peripheral vessel disease), submaximal exercise as evidenced by a peak respiratory exchange ratio <1.0, or participation in another trial. Patients with atrial fibrillation or flutter; ischemic heart disease, body mass index >36 kg/m2, or other significant comorbidities were also excluded. All participants underwent a physical examination, an electrocardiogram and a comprehensive noninvasive diagnostic workup at baseline. Blood samples were taken at baseline for assessment of miR expression levels in PBMCs and for estimation of NT-proBNP serum levels. More specifically, on the first visit in all patients, after a rest of 20 minutes, blood was drawn from a superficial brachial vein via a 21-gauge needle, with care to avoid stasis, hemolysis, and contamination by tissue fluids or exposure to glass, and was transferred to ethylenediaminetetraacetic acid collection tubes for PBMC isolation and to serum collection tubes. A full echocardiographic study was performed and left ventricular mass index was calculated by the Devereux formula.21 Our study complies with the Declaration of Helsinki. The Institutional Ethics Committee approved the study and written informed consent was obtained from all participants. RNA isolation and miR quantification PBMCs were isolated from 3 ml blood samples by density gradient centrifugation using Lymphoprep (Stem Cell Technologies, Vancouver, British Columbia, Canada). Total RNA was isolated using the TRI-Reagent (Ambion, Life Technologies, Carlsbad, CA) and reverse-transcribed using the Mir-X miRNA First-Strand Synthesis kit (Clontech, Takara Bio, Kisatsu, Shiga, Japan). Measurements of miRs levels were performed by quantitative real-time polymerase chain reaction using the Corbett Research 6000 detection system. Quantitative real-time polymerase chain reaction assays were performed using the KAPA SYBR FAST qPCR Kit (Kapa Biosystems, Woburn, MA). Primers used were 5′-TTC AAG TAA TTC AGG ATA GGT-3′ for miR-26b-5p, 5′-ATA AGA CGA ACA AAA GGT TTG T-3′ for miR-208b3p, 5′-TTA AGA CTT GCA GTG ATG TTT-3′ for miR-499a-5p, 5′-TGG AAT GTA AAG AAG TAT GTA T-3′ for miR-1, 5′-TTT GGT CCC CTT CAA CCA GCT G-3′ for miR-133a, and 5′-TAG CTT ATC AGA CTG ATG TTG A-3′ for miR-21. U6 expression was used as a normalization standard and relative quantification of the amplification products was performed using the comparative Ct (2−ddCt) method.22 All samples were run in duplicate and Ct values were averaged for the replicates. The standard curve method was used for absolute quantification of the amplification products and specificity was determined by performing a melting curve analysis. NT-proBNP measurement Serum samples were collected at baseline and kept at −80 °C until analysis. Serum levels of NT-proBNP were determined using an Enzyme-Linked Immunosorbent Assay kit (Cloud-Clone, Houston, TX) according to the manufacturer’s instructions. Cardiopulmonary exercise test A symptom-limited CPXT with simultaneous expired ventilatory gas analysis was performed using a treadmill.23 The system was carefully calibrated before each study. Breath-by-breath online gas measurements were obtained at resting baseline and throughout the exercise protocol to measure minute ventilation (VE), tidal volume, respiratory rate, oxygen uptake (VO2), and carbon dioxide production (VCO2). Peak VO2 was defined as the highest VO2 in the last minute of symptom-limited exercise. The ventilatory anaerobic threshold was determined by the V-slope method. The VE/VCO2 slope for the entire duration of exercise was calculated based on 10-second averaged VE (l/min) and VCO2 (l/min) data. Patients were encouraged to reach an respiratory exchange ratio (respiratory exchange ratio = VCO2/VO2) >1.0, which serves as a measure of effort. Gas exchange collection continued into recovery with 1 minute of active cool-down at 1.5 mph, 0% elevation, followed by seated rest until a return to baseline VO2 was observed or up to 6 minutes. Statistical analysis Summary descriptive statistics are given as mean ± SD or frequencies (%), as appropriate. Comparisons of continuous variables between the hypertensive patients with HFpEF and those without were performed using 2-sided independent samples t-tests. Categorical variables were compared using chi-square tests. The associations between continuous variables were assessed with Pearson’s correlation coefficient. All statistical tests were carried out at the 5% level of significance using IBM-SPSS 22 software. RESULTS A total of 98 patients (mean age 67.5 ± 7.4 years) were recruited from the outpatients’ hypertension clinic of our department. The main demographic, echocardiographic, and clinical characteristics of the overall sample are reported in Table 1. HFpEF patients were in New York Heart Association (NYHA) class II (87.5%) or III (12.5%). CPTX parameters of our population are shown in Table 2. Table 1. Participants’ demographic and clinical data   Hypertensives with HFpEF (n = 56)  Hypertensives without HFpEF (n = 42)  P  Sex (male/female)  24/32  13/29  0.293  Age (years)  67.2 ± 7.75  66.83 ± 7.17  0.769  Creatinine (mg/dl)  0.98 ± 0.12  0.99 ± 0.08  0.512  Hemoglobin (mg/dl)  13.80 ± 3.50  14.21 ± 4.91  0.64  Total cholesterol (mg/dl)  204.61 ± 32.70  215.43 ± 35.02  0.119  Uric acid (mg/dl)  6.18 ± 1.25  6.29 ± 1.21  0.646  Diabetes mellitus (+/−)  23/33  18/24  0.1  Systolic blood pressure (mm Hg)  143.45 ± 7.62  145.79 ± 9.47  0.179  Diastolic blood pressure (mm Hg)  86.27 ± 9.21  89.14 ± 11.73  0.177  Heart rate (bpm)  74.16 ± 8.55  72.12 ± 7.72  0.226  Body mass index (kg/m2)  29.63 ± 2.84  28.95 ± 2.19  0.198  Left ventricular mass index (g/m2)  108.08 ± 24.46  98.14 ± 20.72  0.036  E/e′  13.87 ± 1.03  8.04 ± 0.70  <0.001  Ejection fraction (%)  58.6 ± 7.7  60.63 ± 5.91  0.76  NT-proBNP (pg/ml)  421.01 ± 232.68  161.46 ± 29.66  <0.001  Patients receiving renin–angiotensin–aldosterone system inhibitor  52  39  0.54  Patients receiving calcium channel blockers  26  25  0.13  Patients receiving beta-blockers  10  7  0.88  Patients receiving diuretics  42  36  0.68    Hypertensives with HFpEF (n = 56)  Hypertensives without HFpEF (n = 42)  P  Sex (male/female)  24/32  13/29  0.293  Age (years)  67.2 ± 7.75  66.83 ± 7.17  0.769  Creatinine (mg/dl)  0.98 ± 0.12  0.99 ± 0.08  0.512  Hemoglobin (mg/dl)  13.80 ± 3.50  14.21 ± 4.91  0.64  Total cholesterol (mg/dl)  204.61 ± 32.70  215.43 ± 35.02  0.119  Uric acid (mg/dl)  6.18 ± 1.25  6.29 ± 1.21  0.646  Diabetes mellitus (+/−)  23/33  18/24  0.1  Systolic blood pressure (mm Hg)  143.45 ± 7.62  145.79 ± 9.47  0.179  Diastolic blood pressure (mm Hg)  86.27 ± 9.21  89.14 ± 11.73  0.177  Heart rate (bpm)  74.16 ± 8.55  72.12 ± 7.72  0.226  Body mass index (kg/m2)  29.63 ± 2.84  28.95 ± 2.19  0.198  Left ventricular mass index (g/m2)  108.08 ± 24.46  98.14 ± 20.72  0.036  E/e′  13.87 ± 1.03  8.04 ± 0.70  <0.001  Ejection fraction (%)  58.6 ± 7.7  60.63 ± 5.91  0.76  NT-proBNP (pg/ml)  421.01 ± 232.68  161.46 ± 29.66  <0.001  Patients receiving renin–angiotensin–aldosterone system inhibitor  52  39  0.54  Patients receiving calcium channel blockers  26  25  0.13  Patients receiving beta-blockers  10  7  0.88  Patients receiving diuretics  42  36  0.68  Values are given as mean ± SD. Abbreviations: E/e′, ratio between early mitral inflow velocity and mitral annular early diastolic velocity; HFpEF, heart failure with preserved ejection fraction; NT-proBNP, N-terminal pro-brain natriuretic peptide View Large Table 1. Participants’ demographic and clinical data   Hypertensives with HFpEF (n = 56)  Hypertensives without HFpEF (n = 42)  P  Sex (male/female)  24/32  13/29  0.293  Age (years)  67.2 ± 7.75  66.83 ± 7.17  0.769  Creatinine (mg/dl)  0.98 ± 0.12  0.99 ± 0.08  0.512  Hemoglobin (mg/dl)  13.80 ± 3.50  14.21 ± 4.91  0.64  Total cholesterol (mg/dl)  204.61 ± 32.70  215.43 ± 35.02  0.119  Uric acid (mg/dl)  6.18 ± 1.25  6.29 ± 1.21  0.646  Diabetes mellitus (+/−)  23/33  18/24  0.1  Systolic blood pressure (mm Hg)  143.45 ± 7.62  145.79 ± 9.47  0.179  Diastolic blood pressure (mm Hg)  86.27 ± 9.21  89.14 ± 11.73  0.177  Heart rate (bpm)  74.16 ± 8.55  72.12 ± 7.72  0.226  Body mass index (kg/m2)  29.63 ± 2.84  28.95 ± 2.19  0.198  Left ventricular mass index (g/m2)  108.08 ± 24.46  98.14 ± 20.72  0.036  E/e′  13.87 ± 1.03  8.04 ± 0.70  <0.001  Ejection fraction (%)  58.6 ± 7.7  60.63 ± 5.91  0.76  NT-proBNP (pg/ml)  421.01 ± 232.68  161.46 ± 29.66  <0.001  Patients receiving renin–angiotensin–aldosterone system inhibitor  52  39  0.54  Patients receiving calcium channel blockers  26  25  0.13  Patients receiving beta-blockers  10  7  0.88  Patients receiving diuretics  42  36  0.68    Hypertensives with HFpEF (n = 56)  Hypertensives without HFpEF (n = 42)  P  Sex (male/female)  24/32  13/29  0.293  Age (years)  67.2 ± 7.75  66.83 ± 7.17  0.769  Creatinine (mg/dl)  0.98 ± 0.12  0.99 ± 0.08  0.512  Hemoglobin (mg/dl)  13.80 ± 3.50  14.21 ± 4.91  0.64  Total cholesterol (mg/dl)  204.61 ± 32.70  215.43 ± 35.02  0.119  Uric acid (mg/dl)  6.18 ± 1.25  6.29 ± 1.21  0.646  Diabetes mellitus (+/−)  23/33  18/24  0.1  Systolic blood pressure (mm Hg)  143.45 ± 7.62  145.79 ± 9.47  0.179  Diastolic blood pressure (mm Hg)  86.27 ± 9.21  89.14 ± 11.73  0.177  Heart rate (bpm)  74.16 ± 8.55  72.12 ± 7.72  0.226  Body mass index (kg/m2)  29.63 ± 2.84  28.95 ± 2.19  0.198  Left ventricular mass index (g/m2)  108.08 ± 24.46  98.14 ± 20.72  0.036  E/e′  13.87 ± 1.03  8.04 ± 0.70  <0.001  Ejection fraction (%)  58.6 ± 7.7  60.63 ± 5.91  0.76  NT-proBNP (pg/ml)  421.01 ± 232.68  161.46 ± 29.66  <0.001  Patients receiving renin–angiotensin–aldosterone system inhibitor  52  39  0.54  Patients receiving calcium channel blockers  26  25  0.13  Patients receiving beta-blockers  10  7  0.88  Patients receiving diuretics  42  36  0.68  Values are given as mean ± SD. Abbreviations: E/e′, ratio between early mitral inflow velocity and mitral annular early diastolic velocity; HFpEF, heart failure with preserved ejection fraction; NT-proBNP, N-terminal pro-brain natriuretic peptide View Large Table 2. Cardiopulmonary exercise parameters   Hypertensives with HFpEF (n = 56)  Hypertensives without HFpEF (n = 42)  P  Exercise duration (minute)  5.9 ± 1.11  7.13 ± 1.41  <0.001  Peak exercise respiratory exchange ratio  1.06 ± 0.72  1.11 ± 0.84  0.2  Peak VO2 (ml kg−1 min−1)  20.62 ± 6.59  27.53 ± 8.23  <0.001  VE/VCO2  31.99 ± 6.99  30.81 ± 7.61  0.45    Hypertensives with HFpEF (n = 56)  Hypertensives without HFpEF (n = 42)  P  Exercise duration (minute)  5.9 ± 1.11  7.13 ± 1.41  <0.001  Peak exercise respiratory exchange ratio  1.06 ± 0.72  1.11 ± 0.84  0.2  Peak VO2 (ml kg−1 min−1)  20.62 ± 6.59  27.53 ± 8.23  <0.001  VE/VCO2  31.99 ± 6.99  30.81 ± 7.61  0.45  Abbreviations: HFpEF, heart failure with preserved ejection fraction; peakVO2, maximal oxygen uptake; VE/VCO2, minute ventilation-carbon dioxide production relationship. View Large Table 2. Cardiopulmonary exercise parameters   Hypertensives with HFpEF (n = 56)  Hypertensives without HFpEF (n = 42)  P  Exercise duration (minute)  5.9 ± 1.11  7.13 ± 1.41  <0.001  Peak exercise respiratory exchange ratio  1.06 ± 0.72  1.11 ± 0.84  0.2  Peak VO2 (ml kg−1 min−1)  20.62 ± 6.59  27.53 ± 8.23  <0.001  VE/VCO2  31.99 ± 6.99  30.81 ± 7.61  0.45    Hypertensives with HFpEF (n = 56)  Hypertensives without HFpEF (n = 42)  P  Exercise duration (minute)  5.9 ± 1.11  7.13 ± 1.41  <0.001  Peak exercise respiratory exchange ratio  1.06 ± 0.72  1.11 ± 0.84  0.2  Peak VO2 (ml kg−1 min−1)  20.62 ± 6.59  27.53 ± 8.23  <0.001  VE/VCO2  31.99 ± 6.99  30.81 ± 7.61  0.45  Abbreviations: HFpEF, heart failure with preserved ejection fraction; peakVO2, maximal oxygen uptake; VE/VCO2, minute ventilation-carbon dioxide production relationship. View Large We observed higher expression levels in PBCM of miR-26b (7.6 ± 7.3 versus 4.0 ± 3.6, P = 0.002), miR-208b (28.8 ± 35.3 versus 7.5 ± 13.3, P < 0.001), and miR-499 (14.2 ± 22.4 versus 3.5 ± 2.9, P = 0.001) (Figure 1) in hypertensive patients with HFpEF compared with the group without HFpEF. No statistically significant difference was found between hypertensive patients with HFpEF compared with the group without HFpEF in expression levels of miR-1 (21.7 ± 27.4 versus 29.0 ± 26.7, P = 0.186), miR-133a (5.2 ± 6.8 versus 6.1 ± 6.2, P = 0.491), and miR-21 (10.2 ± 35.7 versus 2.3 ± 2.9, P = 0.195). Figure 1. View largeDownload slide (a) MicroRNA-208b, (b) microRNA-26b and (c) microRNA-499 gene expression levels in peripheral blood mononuclear cells in hypertensive patients with and without heart failure with preserved ejection fraction (HFpEF). Figure 1. View largeDownload slide (a) MicroRNA-208b, (b) microRNA-26b and (c) microRNA-499 gene expression levels in peripheral blood mononuclear cells in hypertensive patients with and without heart failure with preserved ejection fraction (HFpEF). All miRs whose level of expression was significantly elevated in patients with HFpEF (Figure 1) showed significant correlation with peakVO2 in patients with HFpEF. More specifically, a strong positive correlation was observed between peakVO2 and miR-208b expression levels in PBMC (r = 0.671, P < 0.001) (Figure 2), and there were weaker but statistically significant correlations with miR-26b (r = 0.345, P = 0.009) and miR-499a (r = 0.364, P = 0.006). In addition, miR-208b expression levels in PBMCs revealed a significant positive correlation with exercise duration (r = 0.445, P = 0.001) and VE/VCO2 (r = 0.437, P = 0.001) in hypertensives with HFpEF (Figure 2). Finally, miR-499a showed a weak but significant correlation with exercise duration in those patients (r = 0.307, P = 0.021). Notably, no other miR revealed a significant correlation with CPXT parameters or with levels of NT-proBNP in our group of patients (data not shown). Interestingly, miR-208b expression levels in PBMCs also showed a significant positive correlation with VE/VCO2 (r = 0.445, P = 0.004) in the group of hypertensives without HFpEF. Figure 2. View largeDownload slide Correlation of microRNA-208b gene expression levels in peripheral blood mononuclear cells with (a) peakVO2, (r = 0.671, P < 0.001) (b) exercise duration (r = 0.445, P = 0.001) (c) VE/VCO2 (r = 0.437, P = 0.001) in hypertensive patients with heart failure with preserved ejection fraction. Abbreviations: peakVO2, maximal oxygen uptake; VE/VCO2, minute ventilation-carbon dioxide production relationship. Figure 2. View largeDownload slide Correlation of microRNA-208b gene expression levels in peripheral blood mononuclear cells with (a) peakVO2, (r = 0.671, P < 0.001) (b) exercise duration (r = 0.445, P = 0.001) (c) VE/VCO2 (r = 0.437, P = 0.001) in hypertensive patients with heart failure with preserved ejection fraction. Abbreviations: peakVO2, maximal oxygen uptake; VE/VCO2, minute ventilation-carbon dioxide production relationship. DISCUSSION The aim of our study was to investigate levels of various miRs as biomarkers for HFpEF in hypertensive patients and to evaluate their association with those patients’ functional exercise parameters. We found that hypertensive patients with HFpEF showed significantly elevated levels of miR-26b, miR-208b, and miR-499 in PBMCs compared to hypertensive subjects without HFpEF. Of those miRs, miR-208b had the strongest correlation with the HFpEF hypertensive patients’ functional capacity, while miR-26b and miR-499 showed a weaker correlation in those patients. HFpEF remains a challenging clinical presentation to diagnose and manage. A striking majority of these patients suffer from hypertension,24 which is known to be one of the most significant risk factors for HF. Natriuretic peptides are moderately elevated in HFpEF patients25 but have so far proved to be of only limited use. HFpEF is an emerging clinical problem that requires a much better understanding of its pathophysiology and the discovery of new biomarkers of severity that can help improve the treatment and management of patients. Exercise tolerance and functional capacity have been used as primary endpoints in many clinical studies of HF. Peak VO2 and VE/VCO2 are 2 of the most investigated indices in relation to the functional stage of HF and are therefore enjoying great clinical recognition and acceptance of their prognostic value.26 Although the usefulness of peakVO2 has been established in HF, there are studies indicating that VE/VCO2 slope is more powerful. Particularly in patients with HFpEF, VE/VCO2 slope provides independent and additive prognostic information for defining their cardiovascular risk.27 It is characteristic that the primary chronic symptom in HFpEF patients, even when well compensated, is severe exercise intolerance. The CPXΤ response in patients with HFpEF is markedly abnormal3 dyspnea with fatigue during exertion is the major symptomatology in HFpEF patients and is a strong determinant of their reduced quality of life. Previous studies have shown that the deterioration of diastolic dysfunction in these patients is associated with a worsening of their functional stage, as expressed by CPXT.3 The pathophysiology of exercise intolerance in HFpEF is not well understood. Recent studies in HFpEF patients suggest that extracardiac factors contribute to the reduced exercise capacity. HFpEF is associated with significant maladaptive changes in peripheral arterial28 and muscular functions.29 However, it seems that cardiac fibrosis might play a significant role. Myocardial collagen type I and type III are elevated in HFpEF and increased tissue inhibitor of matrix metalloproteinase-1 expression was also reported which may further enhance fibrosis.30 Patients with HFpEF who exhibit a significant reduction in cardiopulmonary reserve also show elevation of proinflammatory indices and pro-oxidative status.31 miR-208b and miR-499 are involved in myogenesis and cardiogenesis, they are abundantly expressed in the heart and skeletal muscle and are specific for myocardial and muscular tissue. During cardiogenesis, they are related to the commitment procedures of cardiomyocytes from myoblasts.16 They are involved in the skeletal muscle development.32 Also, miR-208b, miR-499, and miR-26b are major regulators of cardiac hypertrophy and left ventricular remodeling.16,33,34 They regulate several mRNA targets involved in developmental and metabolic pathways, such as myocyte enhancer factor 2C (MEF2C), SRY box 6 (Sox6), thyroid hormone receptor-associated protein 1 (THRAP1), insulin-like growth factor-1 (IGF-1), pyruvate dehydrogenase subunit X (PDHX), and MED13.34,35 MED13 is a component of the Mediator Complex, which plays a central role in cardiac and muscle metabolic processes.36 In addition, during exercise, miR-499 and miR-208b may also modify several mRNA targets, such as VEGF and Sox6 expression, that in turn regulate several metabolic pathways.34,37,38 Since they are major regulators of myogenic and cardiomyogenic commitment decisions and they are involved in stem/progenitor cell differentiation procedures, they possibly originate from circulating mesenchymal stem cells with myogenic and cardiomyogenic potential (myocyte and cardiomyocyte progenitors), which are mobilized in circulation under cardiovascular pathological conditions.39 In addition, the possibility of their presence in PBMCs’ subpopulations as a result of intercellular communication cannot be excluded.40 Previous studies have noted the importance of transcriptomic and proteomic profile changes of PBMCs in several cardiovascular conditions, including HF.9,10 In addition, there is evidence indicating dysregulation of miRs specific for PBMCs in HF patients, suggesting that miRs in PBMCs may be implicated in cardiovascular pathology.11 We chose to study miRs expression levels in PBMCs, as changes in PBMC trancriptome are linked to the pathogenesis and the complications of both hypertension and HF. In recent years, studies of miRs in systolic HF have been performed in PBMCs indicating several miRs in PBMCs that correlate with disease severity and pathophysiology.41,42 In addition, there are published data suggesting that miRs evaluation in PBMCs from HF patients, may vary in response to specific pathology and could be used to detect early HF.11 We hypothesized that miR expression in these cell populations are likely to have a distinct profile in HFpEF patients compared to control group, which might be related with their exercise capacity. Although there are studies available that have investigated the impact of exercise stimulus on miR expression by studying their levels in serum13 as well as in PBMC12 of healthy individuals, the role of miRs in exercise intolerance of HFpEF patients has not been investigated. It has been shown that while exercise increase the serum levels of miR-499 and miR-208b, their serum levels return to normal after exercise, they do not correlate with aerobic exercise capacity and thus they cannot be used as biomarkers of aerobic exercise.13 Because of the above-mentioned reasons for the role of PBMCs, we selected to study miR expression in those cells and not in serum as previous studies did. In addition, we measured the levels of NT-proBNP in all our patients and correlated them with our findings, as NT-proBNP is an established diagnostic and prognostic factor that interacts with other clinical parameters addressing of functional capacity in those patients.43 We do not know whether our findings correlate pathophysiologically with the deterioration in exercise capacity, or whether they represent a compensatory mechanism that modifies gene expression related with HF. However, it appears that miR-208b is implicated in HF clinical presentation, since it is involved in ventricular remodelling.33 Our findings with respect to miR-26b, miR-208b, and miR-499 show a distinct difference in profile in hypertensive patients with HFpEF compared with hypertensives without HFpEF. These findings open new perspectives for our knowledge and understanding of HFpEF. However, further research is needed in order to clarify whether these miRs are a marker or modulator of functional capacity in those patients. This study has several limitations. It did not include a group of normotensive individuals. We did not consider this necessary, since our group has thoroughly investigated the behavior of the above miRs in hypertensives who were followed in comparison with controls and we have reported these patients’ distinct expression profile in previous publications.15 In addition, the study was started before the publication of the latest HF guidelines.44 In consequence, we used higher NT-pro BNP levels than recommended as a criterion for patients’ inclusion in the study. It is thus likely that we excluded some symptomatic hypertensive patients who might have been enrolled in the group of patients with HF. No member of the group without HFpEF showed any symptoms or signs of HF. We cannot rule out the possibility that medication may had some influence on our findings. However, we consider their effect to be small since in any case both groups were taking those medications in similar proportions. DISCLOSURE The authors declared no conflict of interest. REFERENCES 1. Owan TE, Hodge DO, Herges RM, Jacobsen SJ, Roger VL, Redfield MM. Trends in prevalence and outcome of heart failure with preserved ejection fraction. N Engl J Med  2006; 355: 251– 259. Google Scholar CrossRef Search ADS PubMed  2. Dhakal BP, Malhotra R, Murphy RM, Pappagianopoulos PP, Baggish AL, Weiner RB, Houstis NE, Eisman AS, Hough SS, Lewis GD. Mechanisms of exercise intolerance in heart failure with preserved ejection fraction: the role of abnormal peripheral oxygen extraction. Circ Heart Fail  2015; 8: 286– 294. Google Scholar CrossRef Search ADS PubMed  3. Guazzi M, Myers J, Peberdy MA, Bensimhon D, Chase P, Arena R. Cardiopulmonary exercise testing variables reflect the degree of diastolic dysfunction in patients with heart failure-normal ejection fraction. J Cardiopulm Rehabil Prev  2010; 30: 165– 172. Google Scholar CrossRef Search ADS PubMed  4. Zampetaki A, Willeit P, Drozdov I, Kiechl S, Mayr M. 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Google Scholar CrossRef Search ADS PubMed  26. Arena R, Myers J, Guazzi M. The clinical and research applications of aerobic capacity and ventilatory efficiency in heart failure: an evidence-based review. Heart Fail Rev  2008; 13: 245– 269. Google Scholar CrossRef Search ADS PubMed  27. Yan J, Gong SJ, Li L, Yu HY, Dai HW, Chen J, Tan CW, Xv QH, Cai GL. Combination of B-type natriuretic peptide and minute ventilation/carbon dioxide production slope improves risk stratification in patients with diastolic heart failure. Int J Cardiol  2013; 162: 193– 198. Google Scholar CrossRef Search ADS PubMed  28. Puntawangkoon C, Kitzman DW, Kritchevsky SB, Hamilton CA, Nicklas B, Leng X, Brubaker PH, Hundley WG. Reduced peripheral arterial blood flow with preserved cardiac output during submaximal bicycle exercise in elderly heart failure. J Cardiovasc Magn Reson  2009; 11: 48. Google Scholar CrossRef Search ADS PubMed  29. 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All rights reserved. For Permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png American Journal of Hypertension Oxford University Press

MicroRNAs in Peripheral Mononuclear Cells as Potential Biomarkers in Hypertensive Patients With Heart Failure With Preserved Ejection Fraction

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Oxford University Press
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© American Journal of Hypertension, Ltd 2018. All rights reserved. For Permissions, please email: journals.permissions@oup.com
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0895-7061
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1941-7225
D.O.I.
10.1093/ajh/hpy035
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Abstract

Abstract BACKGROUND MicroRNAs (miRs) regulate gene expression and play an important role in ventricular and vascular remodeling. However, there are limited data regarding their role in heart failure with preserved ejection fraction (HFpEF). The aim of this study was to assess gene expression of miR-1, miR-133a, miR-21, miR-208b, miR-499, and miR-26b in peripheral blood mononuclear cells (PBMCs) in hypertensive patients with HFpEF and to evaluate their association with their exercise capacity. METHODS We included 56 hypertensive patients with HFpEF (age 67.29 ± 7.75 years). Forty-two hypertensive patients without HFpEF (age 66.83 ± 7.17 years) served as controls. All subjects underwent a cardiopulmonary exercise test (CPXT). PBMCs were isolated and levels of miRs were determined by quantitative real-time reverse transcription polymerase chain reaction. RESULTS For hypertensive patients with HFpEF, higher expression levels in PBMCs were found only for miR-26b (7.6 ± 7.3 vs. 4.0 ± 3.6, P = 0.002), miR-208b (28.8 ± 35.3 vs. 7.5 ± 13.3, P < 0.001), and miR-499 (14.2 ± 22.4 versus 3.5 ± 2.9, P = 0.001). The strongest correlations with CPXT parameters were found for miR-208b levels, which had a positive correlation with maximal oxygen uptake (peakVO2) (r = 0.671, P < 0.001), exercise duration (r = 0.445, P = 0.001), and minute ventilation–carbon dioxide production relationship (VE/VCO2) (r = 0.437, P = 0.001) in the HFpEF group. CONCLUSIONS miR-26b, miR-208b, and miR-499 show a distinct in profile in hypertensive patients with HFpEF that is related with functional capacity. Further studies are needed to assess the role of miRs as prognostic tools or as therapeutic targets in those patients. blood pressure, cardiopulmonary exercise, heart failure, hypertension, microRNA Heart failure (HF) with preserved ejection fraction (HFpEF) is a common clinical entity and accounts for a significant proportion of patients with HF. Previous studies found that the prevalence of HFpEF is increasing and might reach 55% among patients with HF diagnosis.1 Patients with HFpEF are more likely to be female and have hypertension.1 HFpEF has reached epidemic levels and is soon likely to predominate, because the incidence of HFpEF is increasing faster than that of HF with reduced EF.1 This will have serious consequences, since it is associated with markedly increased morbidity, mortality, and health-care expenditure. Despite the high prevalence and poor prognosis of HFpEF, there is still no effective treatment in clinical practice, apart from management of its comorbidities. This is due in part to our insufficient understanding of the pathophysiology underlying HFpEF. Exercise intolerance, dyspnea, and fatigue during exertion are the predominant symptoms in HFpEF and have a decisive effect on the patients’ quality of life. Cardiopulmonary exercise response in these patients is markedly abnormal. The cardiopulmonary exercise test (CPXT) is a very valuable examination that reflects the disease severity of HF with both reduced and preserved EF. Recent advances in the research field suggest that the pathophysiology of exercise intolerance in HFpEF patients is complex and that noncardiac “peripheral” factors contribute to the reduced maximal oxygen uptake (peak VO2) they exhibit.2 Recent data in the literature have shown that worsening of cardiopulmonary exercise test parameters, mainly peakVO2 and minute ventilation-carbon dioxide production relationship (VE/VCO2), corresponds to a worsening in diastolic dysfunction, which is the main feature of HFpEF.3 microRNAs (miRs) are powerful regulators of biological processes and can modulate the expression of a large number of genes, while multiple miRs can target the same gene, as well as interact with each other, and can be actively exported or imported by cells.4 It has been estimated that more than 60% of all protein-coding genes are controlled by miRs.5 miRs have been shown to play an important role in the manifestations of cardiovascular diseases. It has been reported that heart-associated miRs (miR-1, miR-208b, miR-499, miR-133, etc.) are released into plasma during acute cardiac damage, serving as a stable biomarker.6,7 The study of miRs has the potential to help us better understand the biological mechanisms responsible for the development of cardiovascular diseases, including HF.8 On the other hand, the importance of transcriptomic and proteomic profile changes of peripheral blood mononuclear cells (PBMCs) has been noted in several cardiovascular conditions, including HF.9,10 In addition, in a recent study, Gupta MK et al. showed that there is dysregulation of miRs in PBMCs of HF patients and reported a specific miRNA signature in PBMCs of HF patients.11 They also showed that miRs alterations in PBMCs from HF patients do not mirror or overlap with cardiac miRs changes, suggesting that PBMCs may have their unique miRs signature in response to cardiac function. In addition, it has been reported that exercise changes miRs profiles in PBMCs of healthy individuals.12 Although a potential role of muscle and heart specific miRs in cardiovascular adaptation processes after aerobic exercise has been noted in marathon athlets,13 the role of miRs in exercise intolerance of HFpEF patients has not been investigated. In the present study, we sought to identify miRs in HFpEF patients that could be of prognostic validity and might be associated with those patients’ exercise functional parameters. We measured miR-1, miR-133a, miR-21, miR-208b, miR-499, and miR-26b gene expression levels in PBMCs, cells that contain subpopulations that are known to play a significant part in the pathophysiology of hypertension and HF.14,15 These miRs were chosen because they play a central role in cardiogenesis, heart function, and pathology,16 they have been implicated in several different cardiovascular pathologies including vascular and heart remodeling and have a distinct expression profile in cardiovascular diseases.17,18 For the evaluation of exercise capacity, we used the most important parameters of CPXT, which is the widely accepted method for this purpose. METHODS We enrolled 56 patients with HFpEF and essential hypertension and 42 patients with essential hypertension without symptoms or signs of HF. We applied the diagnostic criteria of the European Working Group for the diagnosis of HFpEF.19 Briefly, we defined HFpEF as follows: patients with HF syndrome and (i) left ventricular ejection fraction >50% and (ii) ratio between early mitral inflow velocity and mitral annular early diastolic velocity (E/e′) ≥15 or 8 < E/e′ < 15 and N-terminal pro-brain natriuretic peptide (NT-proBNP) levels >220 pg/ml. The diagnosis of hypertension was based on the participants’ medical history, in accordance with the recommendations of the European Society of Hypertension/European Society of Cardiology.20 The HFpEF patients were well-compensated, ambulatory outpatients who had been stable with no medication changes for at least 6 weeks. Patients with evidence of significant coronary, valvular, or pulmonary disease documented intracardiac shunting or any other medical condition that could mimic HF symptoms (anemia, chronotropic incompetence, thyroid dysfunction, etc.) were excluded. We also excluded patients with an implantable pacemaker, cardioverter–defibrillator, primary hypertrophic or restrictive cardiomyopathy, or any systemic illness associated with infiltrative heart disease (e.g., cardiac amyloidosis), inability to exercise due to other comorbidities that might affect the performance of an exercise test (e.g., osteoarthritis, peripheral vessel disease), submaximal exercise as evidenced by a peak respiratory exchange ratio <1.0, or participation in another trial. Patients with atrial fibrillation or flutter; ischemic heart disease, body mass index >36 kg/m2, or other significant comorbidities were also excluded. All participants underwent a physical examination, an electrocardiogram and a comprehensive noninvasive diagnostic workup at baseline. Blood samples were taken at baseline for assessment of miR expression levels in PBMCs and for estimation of NT-proBNP serum levels. More specifically, on the first visit in all patients, after a rest of 20 minutes, blood was drawn from a superficial brachial vein via a 21-gauge needle, with care to avoid stasis, hemolysis, and contamination by tissue fluids or exposure to glass, and was transferred to ethylenediaminetetraacetic acid collection tubes for PBMC isolation and to serum collection tubes. A full echocardiographic study was performed and left ventricular mass index was calculated by the Devereux formula.21 Our study complies with the Declaration of Helsinki. The Institutional Ethics Committee approved the study and written informed consent was obtained from all participants. RNA isolation and miR quantification PBMCs were isolated from 3 ml blood samples by density gradient centrifugation using Lymphoprep (Stem Cell Technologies, Vancouver, British Columbia, Canada). Total RNA was isolated using the TRI-Reagent (Ambion, Life Technologies, Carlsbad, CA) and reverse-transcribed using the Mir-X miRNA First-Strand Synthesis kit (Clontech, Takara Bio, Kisatsu, Shiga, Japan). Measurements of miRs levels were performed by quantitative real-time polymerase chain reaction using the Corbett Research 6000 detection system. Quantitative real-time polymerase chain reaction assays were performed using the KAPA SYBR FAST qPCR Kit (Kapa Biosystems, Woburn, MA). Primers used were 5′-TTC AAG TAA TTC AGG ATA GGT-3′ for miR-26b-5p, 5′-ATA AGA CGA ACA AAA GGT TTG T-3′ for miR-208b3p, 5′-TTA AGA CTT GCA GTG ATG TTT-3′ for miR-499a-5p, 5′-TGG AAT GTA AAG AAG TAT GTA T-3′ for miR-1, 5′-TTT GGT CCC CTT CAA CCA GCT G-3′ for miR-133a, and 5′-TAG CTT ATC AGA CTG ATG TTG A-3′ for miR-21. U6 expression was used as a normalization standard and relative quantification of the amplification products was performed using the comparative Ct (2−ddCt) method.22 All samples were run in duplicate and Ct values were averaged for the replicates. The standard curve method was used for absolute quantification of the amplification products and specificity was determined by performing a melting curve analysis. NT-proBNP measurement Serum samples were collected at baseline and kept at −80 °C until analysis. Serum levels of NT-proBNP were determined using an Enzyme-Linked Immunosorbent Assay kit (Cloud-Clone, Houston, TX) according to the manufacturer’s instructions. Cardiopulmonary exercise test A symptom-limited CPXT with simultaneous expired ventilatory gas analysis was performed using a treadmill.23 The system was carefully calibrated before each study. Breath-by-breath online gas measurements were obtained at resting baseline and throughout the exercise protocol to measure minute ventilation (VE), tidal volume, respiratory rate, oxygen uptake (VO2), and carbon dioxide production (VCO2). Peak VO2 was defined as the highest VO2 in the last minute of symptom-limited exercise. The ventilatory anaerobic threshold was determined by the V-slope method. The VE/VCO2 slope for the entire duration of exercise was calculated based on 10-second averaged VE (l/min) and VCO2 (l/min) data. Patients were encouraged to reach an respiratory exchange ratio (respiratory exchange ratio = VCO2/VO2) >1.0, which serves as a measure of effort. Gas exchange collection continued into recovery with 1 minute of active cool-down at 1.5 mph, 0% elevation, followed by seated rest until a return to baseline VO2 was observed or up to 6 minutes. Statistical analysis Summary descriptive statistics are given as mean ± SD or frequencies (%), as appropriate. Comparisons of continuous variables between the hypertensive patients with HFpEF and those without were performed using 2-sided independent samples t-tests. Categorical variables were compared using chi-square tests. The associations between continuous variables were assessed with Pearson’s correlation coefficient. All statistical tests were carried out at the 5% level of significance using IBM-SPSS 22 software. RESULTS A total of 98 patients (mean age 67.5 ± 7.4 years) were recruited from the outpatients’ hypertension clinic of our department. The main demographic, echocardiographic, and clinical characteristics of the overall sample are reported in Table 1. HFpEF patients were in New York Heart Association (NYHA) class II (87.5%) or III (12.5%). CPTX parameters of our population are shown in Table 2. Table 1. Participants’ demographic and clinical data   Hypertensives with HFpEF (n = 56)  Hypertensives without HFpEF (n = 42)  P  Sex (male/female)  24/32  13/29  0.293  Age (years)  67.2 ± 7.75  66.83 ± 7.17  0.769  Creatinine (mg/dl)  0.98 ± 0.12  0.99 ± 0.08  0.512  Hemoglobin (mg/dl)  13.80 ± 3.50  14.21 ± 4.91  0.64  Total cholesterol (mg/dl)  204.61 ± 32.70  215.43 ± 35.02  0.119  Uric acid (mg/dl)  6.18 ± 1.25  6.29 ± 1.21  0.646  Diabetes mellitus (+/−)  23/33  18/24  0.1  Systolic blood pressure (mm Hg)  143.45 ± 7.62  145.79 ± 9.47  0.179  Diastolic blood pressure (mm Hg)  86.27 ± 9.21  89.14 ± 11.73  0.177  Heart rate (bpm)  74.16 ± 8.55  72.12 ± 7.72  0.226  Body mass index (kg/m2)  29.63 ± 2.84  28.95 ± 2.19  0.198  Left ventricular mass index (g/m2)  108.08 ± 24.46  98.14 ± 20.72  0.036  E/e′  13.87 ± 1.03  8.04 ± 0.70  <0.001  Ejection fraction (%)  58.6 ± 7.7  60.63 ± 5.91  0.76  NT-proBNP (pg/ml)  421.01 ± 232.68  161.46 ± 29.66  <0.001  Patients receiving renin–angiotensin–aldosterone system inhibitor  52  39  0.54  Patients receiving calcium channel blockers  26  25  0.13  Patients receiving beta-blockers  10  7  0.88  Patients receiving diuretics  42  36  0.68    Hypertensives with HFpEF (n = 56)  Hypertensives without HFpEF (n = 42)  P  Sex (male/female)  24/32  13/29  0.293  Age (years)  67.2 ± 7.75  66.83 ± 7.17  0.769  Creatinine (mg/dl)  0.98 ± 0.12  0.99 ± 0.08  0.512  Hemoglobin (mg/dl)  13.80 ± 3.50  14.21 ± 4.91  0.64  Total cholesterol (mg/dl)  204.61 ± 32.70  215.43 ± 35.02  0.119  Uric acid (mg/dl)  6.18 ± 1.25  6.29 ± 1.21  0.646  Diabetes mellitus (+/−)  23/33  18/24  0.1  Systolic blood pressure (mm Hg)  143.45 ± 7.62  145.79 ± 9.47  0.179  Diastolic blood pressure (mm Hg)  86.27 ± 9.21  89.14 ± 11.73  0.177  Heart rate (bpm)  74.16 ± 8.55  72.12 ± 7.72  0.226  Body mass index (kg/m2)  29.63 ± 2.84  28.95 ± 2.19  0.198  Left ventricular mass index (g/m2)  108.08 ± 24.46  98.14 ± 20.72  0.036  E/e′  13.87 ± 1.03  8.04 ± 0.70  <0.001  Ejection fraction (%)  58.6 ± 7.7  60.63 ± 5.91  0.76  NT-proBNP (pg/ml)  421.01 ± 232.68  161.46 ± 29.66  <0.001  Patients receiving renin–angiotensin–aldosterone system inhibitor  52  39  0.54  Patients receiving calcium channel blockers  26  25  0.13  Patients receiving beta-blockers  10  7  0.88  Patients receiving diuretics  42  36  0.68  Values are given as mean ± SD. Abbreviations: E/e′, ratio between early mitral inflow velocity and mitral annular early diastolic velocity; HFpEF, heart failure with preserved ejection fraction; NT-proBNP, N-terminal pro-brain natriuretic peptide View Large Table 1. Participants’ demographic and clinical data   Hypertensives with HFpEF (n = 56)  Hypertensives without HFpEF (n = 42)  P  Sex (male/female)  24/32  13/29  0.293  Age (years)  67.2 ± 7.75  66.83 ± 7.17  0.769  Creatinine (mg/dl)  0.98 ± 0.12  0.99 ± 0.08  0.512  Hemoglobin (mg/dl)  13.80 ± 3.50  14.21 ± 4.91  0.64  Total cholesterol (mg/dl)  204.61 ± 32.70  215.43 ± 35.02  0.119  Uric acid (mg/dl)  6.18 ± 1.25  6.29 ± 1.21  0.646  Diabetes mellitus (+/−)  23/33  18/24  0.1  Systolic blood pressure (mm Hg)  143.45 ± 7.62  145.79 ± 9.47  0.179  Diastolic blood pressure (mm Hg)  86.27 ± 9.21  89.14 ± 11.73  0.177  Heart rate (bpm)  74.16 ± 8.55  72.12 ± 7.72  0.226  Body mass index (kg/m2)  29.63 ± 2.84  28.95 ± 2.19  0.198  Left ventricular mass index (g/m2)  108.08 ± 24.46  98.14 ± 20.72  0.036  E/e′  13.87 ± 1.03  8.04 ± 0.70  <0.001  Ejection fraction (%)  58.6 ± 7.7  60.63 ± 5.91  0.76  NT-proBNP (pg/ml)  421.01 ± 232.68  161.46 ± 29.66  <0.001  Patients receiving renin–angiotensin–aldosterone system inhibitor  52  39  0.54  Patients receiving calcium channel blockers  26  25  0.13  Patients receiving beta-blockers  10  7  0.88  Patients receiving diuretics  42  36  0.68    Hypertensives with HFpEF (n = 56)  Hypertensives without HFpEF (n = 42)  P  Sex (male/female)  24/32  13/29  0.293  Age (years)  67.2 ± 7.75  66.83 ± 7.17  0.769  Creatinine (mg/dl)  0.98 ± 0.12  0.99 ± 0.08  0.512  Hemoglobin (mg/dl)  13.80 ± 3.50  14.21 ± 4.91  0.64  Total cholesterol (mg/dl)  204.61 ± 32.70  215.43 ± 35.02  0.119  Uric acid (mg/dl)  6.18 ± 1.25  6.29 ± 1.21  0.646  Diabetes mellitus (+/−)  23/33  18/24  0.1  Systolic blood pressure (mm Hg)  143.45 ± 7.62  145.79 ± 9.47  0.179  Diastolic blood pressure (mm Hg)  86.27 ± 9.21  89.14 ± 11.73  0.177  Heart rate (bpm)  74.16 ± 8.55  72.12 ± 7.72  0.226  Body mass index (kg/m2)  29.63 ± 2.84  28.95 ± 2.19  0.198  Left ventricular mass index (g/m2)  108.08 ± 24.46  98.14 ± 20.72  0.036  E/e′  13.87 ± 1.03  8.04 ± 0.70  <0.001  Ejection fraction (%)  58.6 ± 7.7  60.63 ± 5.91  0.76  NT-proBNP (pg/ml)  421.01 ± 232.68  161.46 ± 29.66  <0.001  Patients receiving renin–angiotensin–aldosterone system inhibitor  52  39  0.54  Patients receiving calcium channel blockers  26  25  0.13  Patients receiving beta-blockers  10  7  0.88  Patients receiving diuretics  42  36  0.68  Values are given as mean ± SD. Abbreviations: E/e′, ratio between early mitral inflow velocity and mitral annular early diastolic velocity; HFpEF, heart failure with preserved ejection fraction; NT-proBNP, N-terminal pro-brain natriuretic peptide View Large Table 2. Cardiopulmonary exercise parameters   Hypertensives with HFpEF (n = 56)  Hypertensives without HFpEF (n = 42)  P  Exercise duration (minute)  5.9 ± 1.11  7.13 ± 1.41  <0.001  Peak exercise respiratory exchange ratio  1.06 ± 0.72  1.11 ± 0.84  0.2  Peak VO2 (ml kg−1 min−1)  20.62 ± 6.59  27.53 ± 8.23  <0.001  VE/VCO2  31.99 ± 6.99  30.81 ± 7.61  0.45    Hypertensives with HFpEF (n = 56)  Hypertensives without HFpEF (n = 42)  P  Exercise duration (minute)  5.9 ± 1.11  7.13 ± 1.41  <0.001  Peak exercise respiratory exchange ratio  1.06 ± 0.72  1.11 ± 0.84  0.2  Peak VO2 (ml kg−1 min−1)  20.62 ± 6.59  27.53 ± 8.23  <0.001  VE/VCO2  31.99 ± 6.99  30.81 ± 7.61  0.45  Abbreviations: HFpEF, heart failure with preserved ejection fraction; peakVO2, maximal oxygen uptake; VE/VCO2, minute ventilation-carbon dioxide production relationship. View Large Table 2. Cardiopulmonary exercise parameters   Hypertensives with HFpEF (n = 56)  Hypertensives without HFpEF (n = 42)  P  Exercise duration (minute)  5.9 ± 1.11  7.13 ± 1.41  <0.001  Peak exercise respiratory exchange ratio  1.06 ± 0.72  1.11 ± 0.84  0.2  Peak VO2 (ml kg−1 min−1)  20.62 ± 6.59  27.53 ± 8.23  <0.001  VE/VCO2  31.99 ± 6.99  30.81 ± 7.61  0.45    Hypertensives with HFpEF (n = 56)  Hypertensives without HFpEF (n = 42)  P  Exercise duration (minute)  5.9 ± 1.11  7.13 ± 1.41  <0.001  Peak exercise respiratory exchange ratio  1.06 ± 0.72  1.11 ± 0.84  0.2  Peak VO2 (ml kg−1 min−1)  20.62 ± 6.59  27.53 ± 8.23  <0.001  VE/VCO2  31.99 ± 6.99  30.81 ± 7.61  0.45  Abbreviations: HFpEF, heart failure with preserved ejection fraction; peakVO2, maximal oxygen uptake; VE/VCO2, minute ventilation-carbon dioxide production relationship. View Large We observed higher expression levels in PBCM of miR-26b (7.6 ± 7.3 versus 4.0 ± 3.6, P = 0.002), miR-208b (28.8 ± 35.3 versus 7.5 ± 13.3, P < 0.001), and miR-499 (14.2 ± 22.4 versus 3.5 ± 2.9, P = 0.001) (Figure 1) in hypertensive patients with HFpEF compared with the group without HFpEF. No statistically significant difference was found between hypertensive patients with HFpEF compared with the group without HFpEF in expression levels of miR-1 (21.7 ± 27.4 versus 29.0 ± 26.7, P = 0.186), miR-133a (5.2 ± 6.8 versus 6.1 ± 6.2, P = 0.491), and miR-21 (10.2 ± 35.7 versus 2.3 ± 2.9, P = 0.195). Figure 1. View largeDownload slide (a) MicroRNA-208b, (b) microRNA-26b and (c) microRNA-499 gene expression levels in peripheral blood mononuclear cells in hypertensive patients with and without heart failure with preserved ejection fraction (HFpEF). Figure 1. View largeDownload slide (a) MicroRNA-208b, (b) microRNA-26b and (c) microRNA-499 gene expression levels in peripheral blood mononuclear cells in hypertensive patients with and without heart failure with preserved ejection fraction (HFpEF). All miRs whose level of expression was significantly elevated in patients with HFpEF (Figure 1) showed significant correlation with peakVO2 in patients with HFpEF. More specifically, a strong positive correlation was observed between peakVO2 and miR-208b expression levels in PBMC (r = 0.671, P < 0.001) (Figure 2), and there were weaker but statistically significant correlations with miR-26b (r = 0.345, P = 0.009) and miR-499a (r = 0.364, P = 0.006). In addition, miR-208b expression levels in PBMCs revealed a significant positive correlation with exercise duration (r = 0.445, P = 0.001) and VE/VCO2 (r = 0.437, P = 0.001) in hypertensives with HFpEF (Figure 2). Finally, miR-499a showed a weak but significant correlation with exercise duration in those patients (r = 0.307, P = 0.021). Notably, no other miR revealed a significant correlation with CPXT parameters or with levels of NT-proBNP in our group of patients (data not shown). Interestingly, miR-208b expression levels in PBMCs also showed a significant positive correlation with VE/VCO2 (r = 0.445, P = 0.004) in the group of hypertensives without HFpEF. Figure 2. View largeDownload slide Correlation of microRNA-208b gene expression levels in peripheral blood mononuclear cells with (a) peakVO2, (r = 0.671, P < 0.001) (b) exercise duration (r = 0.445, P = 0.001) (c) VE/VCO2 (r = 0.437, P = 0.001) in hypertensive patients with heart failure with preserved ejection fraction. Abbreviations: peakVO2, maximal oxygen uptake; VE/VCO2, minute ventilation-carbon dioxide production relationship. Figure 2. View largeDownload slide Correlation of microRNA-208b gene expression levels in peripheral blood mononuclear cells with (a) peakVO2, (r = 0.671, P < 0.001) (b) exercise duration (r = 0.445, P = 0.001) (c) VE/VCO2 (r = 0.437, P = 0.001) in hypertensive patients with heart failure with preserved ejection fraction. Abbreviations: peakVO2, maximal oxygen uptake; VE/VCO2, minute ventilation-carbon dioxide production relationship. DISCUSSION The aim of our study was to investigate levels of various miRs as biomarkers for HFpEF in hypertensive patients and to evaluate their association with those patients’ functional exercise parameters. We found that hypertensive patients with HFpEF showed significantly elevated levels of miR-26b, miR-208b, and miR-499 in PBMCs compared to hypertensive subjects without HFpEF. Of those miRs, miR-208b had the strongest correlation with the HFpEF hypertensive patients’ functional capacity, while miR-26b and miR-499 showed a weaker correlation in those patients. HFpEF remains a challenging clinical presentation to diagnose and manage. A striking majority of these patients suffer from hypertension,24 which is known to be one of the most significant risk factors for HF. Natriuretic peptides are moderately elevated in HFpEF patients25 but have so far proved to be of only limited use. HFpEF is an emerging clinical problem that requires a much better understanding of its pathophysiology and the discovery of new biomarkers of severity that can help improve the treatment and management of patients. Exercise tolerance and functional capacity have been used as primary endpoints in many clinical studies of HF. Peak VO2 and VE/VCO2 are 2 of the most investigated indices in relation to the functional stage of HF and are therefore enjoying great clinical recognition and acceptance of their prognostic value.26 Although the usefulness of peakVO2 has been established in HF, there are studies indicating that VE/VCO2 slope is more powerful. Particularly in patients with HFpEF, VE/VCO2 slope provides independent and additive prognostic information for defining their cardiovascular risk.27 It is characteristic that the primary chronic symptom in HFpEF patients, even when well compensated, is severe exercise intolerance. The CPXΤ response in patients with HFpEF is markedly abnormal3 dyspnea with fatigue during exertion is the major symptomatology in HFpEF patients and is a strong determinant of their reduced quality of life. Previous studies have shown that the deterioration of diastolic dysfunction in these patients is associated with a worsening of their functional stage, as expressed by CPXT.3 The pathophysiology of exercise intolerance in HFpEF is not well understood. Recent studies in HFpEF patients suggest that extracardiac factors contribute to the reduced exercise capacity. HFpEF is associated with significant maladaptive changes in peripheral arterial28 and muscular functions.29 However, it seems that cardiac fibrosis might play a significant role. Myocardial collagen type I and type III are elevated in HFpEF and increased tissue inhibitor of matrix metalloproteinase-1 expression was also reported which may further enhance fibrosis.30 Patients with HFpEF who exhibit a significant reduction in cardiopulmonary reserve also show elevation of proinflammatory indices and pro-oxidative status.31 miR-208b and miR-499 are involved in myogenesis and cardiogenesis, they are abundantly expressed in the heart and skeletal muscle and are specific for myocardial and muscular tissue. During cardiogenesis, they are related to the commitment procedures of cardiomyocytes from myoblasts.16 They are involved in the skeletal muscle development.32 Also, miR-208b, miR-499, and miR-26b are major regulators of cardiac hypertrophy and left ventricular remodeling.16,33,34 They regulate several mRNA targets involved in developmental and metabolic pathways, such as myocyte enhancer factor 2C (MEF2C), SRY box 6 (Sox6), thyroid hormone receptor-associated protein 1 (THRAP1), insulin-like growth factor-1 (IGF-1), pyruvate dehydrogenase subunit X (PDHX), and MED13.34,35 MED13 is a component of the Mediator Complex, which plays a central role in cardiac and muscle metabolic processes.36 In addition, during exercise, miR-499 and miR-208b may also modify several mRNA targets, such as VEGF and Sox6 expression, that in turn regulate several metabolic pathways.34,37,38 Since they are major regulators of myogenic and cardiomyogenic commitment decisions and they are involved in stem/progenitor cell differentiation procedures, they possibly originate from circulating mesenchymal stem cells with myogenic and cardiomyogenic potential (myocyte and cardiomyocyte progenitors), which are mobilized in circulation under cardiovascular pathological conditions.39 In addition, the possibility of their presence in PBMCs’ subpopulations as a result of intercellular communication cannot be excluded.40 Previous studies have noted the importance of transcriptomic and proteomic profile changes of PBMCs in several cardiovascular conditions, including HF.9,10 In addition, there is evidence indicating dysregulation of miRs specific for PBMCs in HF patients, suggesting that miRs in PBMCs may be implicated in cardiovascular pathology.11 We chose to study miRs expression levels in PBMCs, as changes in PBMC trancriptome are linked to the pathogenesis and the complications of both hypertension and HF. In recent years, studies of miRs in systolic HF have been performed in PBMCs indicating several miRs in PBMCs that correlate with disease severity and pathophysiology.41,42 In addition, there are published data suggesting that miRs evaluation in PBMCs from HF patients, may vary in response to specific pathology and could be used to detect early HF.11 We hypothesized that miR expression in these cell populations are likely to have a distinct profile in HFpEF patients compared to control group, which might be related with their exercise capacity. Although there are studies available that have investigated the impact of exercise stimulus on miR expression by studying their levels in serum13 as well as in PBMC12 of healthy individuals, the role of miRs in exercise intolerance of HFpEF patients has not been investigated. It has been shown that while exercise increase the serum levels of miR-499 and miR-208b, their serum levels return to normal after exercise, they do not correlate with aerobic exercise capacity and thus they cannot be used as biomarkers of aerobic exercise.13 Because of the above-mentioned reasons for the role of PBMCs, we selected to study miR expression in those cells and not in serum as previous studies did. In addition, we measured the levels of NT-proBNP in all our patients and correlated them with our findings, as NT-proBNP is an established diagnostic and prognostic factor that interacts with other clinical parameters addressing of functional capacity in those patients.43 We do not know whether our findings correlate pathophysiologically with the deterioration in exercise capacity, or whether they represent a compensatory mechanism that modifies gene expression related with HF. However, it appears that miR-208b is implicated in HF clinical presentation, since it is involved in ventricular remodelling.33 Our findings with respect to miR-26b, miR-208b, and miR-499 show a distinct difference in profile in hypertensive patients with HFpEF compared with hypertensives without HFpEF. These findings open new perspectives for our knowledge and understanding of HFpEF. However, further research is needed in order to clarify whether these miRs are a marker or modulator of functional capacity in those patients. This study has several limitations. It did not include a group of normotensive individuals. We did not consider this necessary, since our group has thoroughly investigated the behavior of the above miRs in hypertensives who were followed in comparison with controls and we have reported these patients’ distinct expression profile in previous publications.15 In addition, the study was started before the publication of the latest HF guidelines.44 In consequence, we used higher NT-pro BNP levels than recommended as a criterion for patients’ inclusion in the study. It is thus likely that we excluded some symptomatic hypertensive patients who might have been enrolled in the group of patients with HF. No member of the group without HFpEF showed any symptoms or signs of HF. We cannot rule out the possibility that medication may had some influence on our findings. However, we consider their effect to be small since in any case both groups were taking those medications in similar proportions. DISCLOSURE The authors declared no conflict of interest. REFERENCES 1. 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American Journal of HypertensionOxford University Press

Published: Mar 1, 2018

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