Summary
Background
Cardiomyocytes produce a wide variety of bioactive molecules that regulate numerous physiological and pathophysiological processes. Recently, it has been recognized that changes in microribonucleic acid (miRNA) expression may lead to cardiac dysfunction.
Aims
To assess the expression of circulating miRNAs (miR-1, miR-21 and miR-208a) in patients with symptomatic heart failure (HF), and to investigate the relationship between expression of these miRNAs and secretion of N-terminal pro-B-type natriuretic peptide (NT-proBNP) and galectin-3.
Methods
Thirty-five patients in New York Heart Association (NYHA) class II/III (age: 68.8 ± 13.0 years) and 26 patients in NYHA class IV (age: 72.0 ± 10.4 years) hospitalized in the intensive coronary care unit participated in the study. Serum concentrations of miRNAs were measured by quantitative real-time polymerase chain reaction. Basic biochemical assays were carried out, and NT-proBNP and galectin-3 concentrations were measured in all serum samples.
Results
miR-1 was downregulated in patients with symptomatic HF and its expression decreased with severity of NYHA class ( P = 0.007). In contrast, overexpression of miR-21 was seen in all patients, independent of HF severity. Results suggest no miR-208a leakage into the circulation in patients with symptomatic HF. There was an inverse relationship between miR-1 expression and NT-proBNP concentration (Spearman’s rank correlation coefficient [ r ] = –0.389; P = 0.023) in patients in NYHA class II/III. Overexpression of miR-21 correlated significantly with galectin-3 concentration ( r = 0.422; P = 0.032).
Conclusion
Dysregulation of miR-1 and miR-21 expression may be essential for the development of HF; miR-1 might become a biomarker for predicting HF exacerbation.
Résumé
Justification
Les cardiomyocytes produisent une large variété de molécules bioactives régulant différents processus physiologiques et physiopathologiques. Récemment, il a été montré que l’expression des modifications d’ARN-mi pouvait conduire à une dysfonction cardiaque.
Objectifs
Évaluer l’expression des ARN-mi circulant (miR-1, miR-21 et miR-208a) chez des patients ayant une insuffisance cardiaque symptomatique. Nous avons également investigué la relation entre l’expression de ces micro-ARN et la sécrétion de NT-pro-BNP et de galectine-3.
Méthode
Trente-cinq patients en classe fonctionnelle NYHA II à III (âge moyen : 68,8 ± 13 ans) et 26 patients en classe fonctionnelle NYHA IV (âge moyen : 72,0 ± 10,4 ans) ont été hospitalisés dans une unité de soins intensifs cardiaque et ont participé à cette étude. Les concentrations sériques de micro-ARN ont été mesurées par méthode de PCR quantitative. L’évaluation biochimique a été effectuée dans les prélèvements sanguins initiaux avec détermination des taux de NT-pro-BNP et de galectine-3.
Résultats
miR-1 était régulé de façon négative chez les patients ayant une insuffisance cardiaque symptomatique et leur expression décroissait avec la sévérité de la classe fonctionnelle NYHA ( p = 0,007). En revanche, la surexpression de miR-21 était observée chez tous les patients, indépendamment de la sévérité de l’insuffisance cardiaque. Nos résultats suggèrent qu’il n’y a pas de libération de miR-208a dans la circulation sanguine chez les patients ayant une insuffisance cardiaque symptomatique. Nous avons noté une relation inverse entre l’expression de miR-1 et les concentrations de NT-pro-BNP (coefficient de corrélation de Spearman [ r ] = 0,389 ; p = 0,023) chez les patients en classe fonctionnelle NYHA II ou III. La surexpression miR-21 est corrélée avec les concentrations de galectine-3 ( r = 0,422 ; p = 0,032).
Conclusion
La dysrégulation de l’expression de miR-1 et miR-21 pourrait être essentielle pour l’apparition d’une insuffisance cardiaque ; miR-1 pourrait dans ces conditions devenir un biomarqueur prédisant les poussées d’insuffisance cardiaque.
Background
Microribonucleic acids (miRNAs) are recently discovered, small, endogenous, single-stranded, non-coding RNAs comprising 18–25 nucleotides. They are initially transcribed as primary-miRNA with a characteristic stem-loop structure. The primary-miRNA stem-loop structure is cleaved by the enzyme Drosha to ∼70 nucleotides in length within the nucleus, and is called precursor-miRNA. Precursor-miRNAs are then exported from the nucleus into the cytoplasm, where the mature miRNA strands regulate gene expression post transcriptionally .
More than 600 miRNAs have been discovered; they participate in various physiological processes, including heart development, but are also involved in the pathogenesis of heart failure (HF), myocardial hypertrophy and arrhythmia . Cardiac fibroblasts play a key role in the adverse myocardial remodelling that occurs with hypertension, myocardial infarction and HF. The involvement of miRNAs in these pathological processes has been recently recognized. Indeed, altered miRNA expression during cardiac remodelling has been reported in mice and humans . Aberrant expression of selected miRNAs has been linked with various pathological conditions such as cardiac fibrosis . Recent evidence suggests that miRNAs are differentially expressed in the failing myocardium and play an important role in the progression of HF . miRNAs are fundamentally involved in and have an effect on cardiac fibrosis . In addition, miRNA controls cardiac fibroblast differentiation .
Among the numerous miRNAs reported to influence the process of maladaptive cardiac remodelling, the strongest preclinical and clinical data appear to support the roles of miR-1, miR-21 and miR-208 in this process . miR-1 is one of the most abundant miRNA in the heart, and plays a protective role against cardiac hypertrophy or HF by regulating several hypertrophy-associated genes, which include transcription factors, receptor ligands, apoptosis regulators and ion channels, as well as exacerbating arrhythmogenesis when overexpressed . Predominantly expressed in cardiac fibroblasts, miR-21 is one of the most greatly upregulated miRNAs during cardiac hypertrophy; miR-21 induces cardiac fibrosis and protects cardiomyocytes against apoptosis. Interestingly, pharmacological antagonism of miR-21 suppresses cardiac remodelling after pressure overload in the heart . Furthermore, in experimental studies, miR-208a was sufficient to induce cardiac remodelling and modulate the expression of hypertrophy-associated genes, and the systemic delivery of miR-208a inhibitors prevented pathological myosin switching and cardiac remodelling while improving cardiac function and survival .
The aims of this study were to assess the level of expression of miRNA-1, miRNA-21 and miRNA-208a in the sera of patients with symptomatic HF, and to evaluate the relationship between serum miRNAs and the serum concentrations of galectin-3 and N-terminal prohormone of B-type natriuretic peptide (NT-proBNP) in relation to the severity of decompensated HF.
Methods
Study population
Sixty-one patients hospitalized for symptomatic HF in the 1st Chair and Department of Cardiology, Medical University of Warsaw, between October 2013 and August 2014 were enrolled in this prospective, single-centre registry. Symptomatic HF comprised acute decompensated heart failure – de novo or decompensation of chronic heart failure – as well as symptomatic chronic HF, with New York Heart Association (NYHA) class ≥ II.
Patients with symptomatic HF requiring hospitalization were included within 24 hours of admission. The inclusion criteria also involved clinical or radiological signs of pulmonary congestion and left ventricular ejection fraction (LVEF) <50%, computed according to Simpson’s method .
Patients with acute coronary syndromes, active infection, Cushing’s syndrome, primary hyperaldosteronism, Addison’s disease, liver cirrhosis, acute renal failure, paraneoplastic syndromes, subarachnoid haemorrhage, chronic lung diseases, acute and chronic pulmonary embolism, and myopathies were excluded from the study.
Data on time of chronic HF diagnosis, risk factors, cardiovascular conditions, family history and previously used medications were collected. All enrolled patients underwent a physical examination, with special emphasis on clinical features of HF, an electrocardiogram and routine laboratory tests.
The local ethics committee approved the protocol of the study, and all patients provided written informed consent.
Echocardiographic studies
Each patient underwent a complete echocardiographic examination using an iE33 ultrasound system (Philips, Amsterdam, Netherlands). LVEF was calculated according to Simpson’s method .
Sample collection
Blood samples were collected during the 24 hours after admission. Blood samples were collected after overnight fasting into plastic tubes with clot activator. Large gauge needles were used to avoid potential platelet activation and miRNA release, as suggested by Witwer et al. .
Circulating RNA extraction
Total RNA was extracted from 300 μL of serum using a commercial column-based system (NucleoSpin ® miRNA Plasma kit; Macherey-Nagel, Düren, Germany), according to the manufacturer’s instructions. Before RNA isolation, serum samples were thawed and vortexed. Total RNA was eluted by adding 30 μL of ribonuclease-free water to the membrane of the spin column. RNA was stored at −80 °C until further analysis. A fixed volume of 4 μL RNA solution from the 30 μL RNA isolation eluate was put into the reverse transcription reaction.
Circulating miRNA reverse transcription and amplification
Complementary deoxyribonucleic acid (cDNA) synthesis was performed with the Universal cDNA Synthesis kit (Exiqon, Vedbaek, Denmark) according to the manufacturer’s protocol. The reaction volume was 20 μL. Endogenous abundance of miRNA was measured using quantitative real-time polymerase chain reaction (qRT-PCR) on the ViiA™ 7 Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA). The triplicate reactions in 15 μL were carried out using ExiLENT SYBR ® Green Master Mix (Exiquon, Vedbaek, Denmark) with the locked nuclei acid (LNA™) primer sets for miRNA-1 (MIMAT0000416), miRNA-21-5p (MIMAT0000076) and miRNA-208a (MIMAT0000241), plus miRNA-103-3p (MIMAT0000101) as an internal control. In each reaction, 6 μL of 10 × diluted cDNA was used. Thermal cycling consisted of an initial denaturation followed by 50 cycles of amplification. A melt curve was performed after each cycle to indicate the specificity of the primers. The threshold cycle (Ct) for each reaction was determined, defined as a cycle number at which exponential phase of the amplification plot crosses the threshold, significantly above the background. If the Ct value was >40, the miRNA concentration was considered undetectable. Relative expression of investigated miRNAs compared with healthy volunteers was calculated using the ΔΔCt method, the most usual way to analyse gene expression experiments. For each sample, ΔCt was obtained, which indicates the difference in Ct values between the miRNA of interest and the endogenous control (miR-103-3p) (ΔCt = Ct miR-x − Ct miR-103 ). Then, the mean ΔCt value for the control group was calculated and subtracted from the ΔCt value obtained for the individuals from the experimental group (ΔΔCt = ΔCt ex − ΔCt co ). To find the relative expression, the fold value was calculated using the 2 -ΔΔCt formula. A fold value (2 -ΔΔCt ) lower or higher than 1 indicates down- or upregulation, respectively, compared with the healthy controls.
The results are presented as fold change in relation to the miRNA expression in the serum samples of the control subjects, who were 17 age- and sex-matched healthy volunteers, age: 59 ± 12.4 years; 13 (76%) were men. Normalization for variations in RNA input was conducted using miR-103-3p, which, in our samples, varied little between patients, with a coefficient of variation = 7.94%. Moreover, this miRNA was found to be stably expressed in plasma. In addition, we analysed the expression of miR-16, but it occurred to be less favourable, as the coefficient of variation occurred to be higher (11.6%).
Biochemical analysis
Blood samples were collected after overnight fasting into plastic tubes with clot activator. After centrifugation, part of the serum samples were used for determination of the concentrations of creatinine, high-sensitivity C-reactive protein, NT-proBNP, troponin I; the remaining serum was immediately frozen at −70 °C for later measurements of the concentration of galectin-3. Serum galectin-3 concentration was measured using the reagents and instrument from the VIDAS ® family (bioMérieux SA, Marcy-l’Étoile, France). The assay principle combines a one-step immunoassay sandwich method with final fluorescent detection (enzyme-linked fluorescence assay [ELFA]). The concentrations of creatinine, high-sensitivity C-reactive protein, troponin I and NT-proBNP were measured using the Flex ® Reagent Cartridge and the Dimension Xpand instrument (Siemens Health Care Diagnostics, Erlangen, Germany).
Statistical analysis
For statistical analyses, the data analysis software system STATISTICA, version 10 (StatSoft, Inc., Tulsa, OK, USA) was used. Continuous variables were tested for normal distribution by the Shapiro-Wilk test. Results for normally distributed continuous variables are expressed as means ± standard deviations, and we used the unpaired Student’s t test to compare mean values. Continuous variables with non-normal distribution are presented as median values and interquartile ranges (range from the 25th to the 75th percentile). Between-group comparisons of distributions were performed using the Mann-Whitney U test and Wilcoxon’s signed-rank sum test. Correlations among continuous variables were assessed using Spearman’s rank correlation coefficient [ r ]. Categorical variables are expressed as numbers (percentages) and were compared using Fisher’s exact test. To evaluate the expression levels of miRNAs circulating within the two groups, we decided to use Student’s t test for two independent groups. P -values < 0.05 were considered statistically significant.
Results
Patients’ characteristics
Sixty-one patients were divided into two subgroups according to NYHA functional class. The first group included 35 patients with cardiac functional classes II and III, with a mean age of 68.8 ± 13.0 years; the second group included 26 patients with cardiac functional class IV, with a mean age of 72.0 ± 10.4 years. There were no statistically significant differences in terms of age and sex between these two groups ( Table 1 ).
