MicroRNA Profiling of Pericardial Fluid Samples from Patients with Heart Failure

<div><p>Aims</p><p>Multicellular organisms maintain vital functions through intercellular communication. Release of extracellular vesicles that carry signals to even distant target organs is one way of accomplishing this communication. MicroRNAs can also be secreted from the cells in exosomes and act as paracrine signalling molecules. In addition, microRNAs have been implicated in the pathogenesis of a large number of diseases, including cardiovascular diseases, and are considered as promising candidate biomarkers due to their relative stability and easy quantification from clinical samples. Pericardial fluid contains hormones secreted by the heart and is known to reflect the cardiac function. In this study, we sought to investigate whether pericardial fluid contains microRNAs and if so, whether they could be used to distinguish between different cardiovascular pathologies and disease stages.</p><p>Methods and Results</p><p>Pericardial fluid was collected from heart failure patients during open-heart surgery. MicroRNA profiles of altogether 51 patients were measured by quantitative real-time PCR (qPCR) using Exiqon human panels I and II. On the average, 256 microRNAs were detected per sample, and 70 microRNAs out of 742 profiled microRNAs were detected in every sample. The five most abundant microRNAs in pericardial fluid were miR-21-5p, miR-451a, miR-125b-5p, let-7b-5p and miR-16-5p. No specific signatures for cardiovascular pathologies or clinically assessed heart failure stages could be detected from the profiles and, overall, microRNA profiles of the samples were found to be very similar despite the heterogeneity in the study population.</p><p>Conclusion</p><p>Measured microRNA profiles did not separate the samples according to the clinical features of the patients. However, several previously identified heart failure marker microRNAs were detected. The pericardial fluid microRNA profile appeared to be a result of an active and selective secretory process indicating that microRNAs may act as paracrine signalling factors by mediating the local crosstalk between cardiac cells.</p></div

Cardiovascular disease groups.

<p>Cardiovascular disease groups.</p

Differentially detected microRNAs by ANOVA.

<p>The presence of miRNAs with highest standard deviation between <b>a)</b> disease groups (Group 1: Coronary artery disease, Group 2: Mitral valve insufficiency, Group 3: Aortic stenosis, Group 5: Other cardiovascular disease, and Group 7: Coronary artery disease and aortic stenosis) and <b>b)</b> NYHA classes were measured using qPCR. Results for <b>c)</b> miR-106b-3p and <b>d)</b> miR-215 are shown by disease groups and NYHA classes, respectively. Results are depicted as individual points for each measured sample (<i>n</i> = 45 for disease, and <i>n</i> = 51 for NYHA classes) lines indicating the overall mean for each miRNA. All miRNAs were not detected in every sample. *<i>p</i><0.05, **<i>p</i><0.01.</p

Clinical characteristics.

<p>NYHA class = New York Heart Association class</p><p>* Mean ± SD.</p><p>Clinical characteristics.</p

MicroRNAs found in all pericardial fluid samples.

<p>MicroRNAs found in all pericardial fluid samples.</p

MicroRNA gene family miR-30.

<p>The presence of all mir-30 family members in pericardial fluid was investigated using qPCR. Results are depicted as individual points for each measured sample (<i>n</i> = 51) lines indicating the mean expression for each miRNA.</p

Cardiac microRNAs in pericardial fluid.

<p>*Average on all samples</p><p>Cardiac microRNAs in pericardial fluid.</p

Heart failure marker miR-423–5p in pericardial fluid samples.

<p>The presence of miRNAs by <b>a)</b> disease groups with three or more members (Group 1: Coronary artery disease, Group 2: Mitral valve insufficiency, Group 3: Aortic stenosis, Group 5: Other cardiovascular disease, and Group 7: Coronary artery disease and aortic stenosis), and <b>b)</b> NYHA grading for miR-423–5p and by <b>c)</b> disease group and <b>d)</b> NYHA grading for miR-423–3p were measured using qPCR. Results are depicted as individual points for each measured sample (<i>n</i> = 45 for disease, and <i>n</i> = 51 for NYHA classes) lines indicating the overall mean for each group.</p

Heat map and unsupervised hierarchical clustering for disease aetiology.

<p>The clustering is performed on top 50 miRNAs with highest standard deviation between groups and groups with three or more members. Samples were grouped according to the disease aetiologies of the patients: Group 1: Coronary artery disease (red), Group 2: Mitral valve insufficiency (blue), Group 3: Aortic stenosis (darkgreen), Group 5: Other cardiovascular disease (orange), and Group 7: Coronary artery disease and aortic stenosis (brown). The normalized (dCp) values were used for the analysis. The colour scale illustrates the relative expression level of miRNAs across all samples: red colour represents an expression level above mean, blue colour lower than the mean. Missing values are shown in grey.</p

Blood cell microRNAs in pericardial fluid.

<p>*Average on all samples</p><p>Blood cell microRNAs in pericardial fluid.</p