Cardiospecific microRNA Plasma Levels Correlate with Troponin and Cardiac Function in Patients with ST Elevation Myocardial Infarction, Are Selectively Dependent on Renal Elimination, and Can Be Detected in Urine Samples

Objectives: Circulating microRNAs (miRNAs) are promising as biomarkers for various diseases. We examined the release patterns of cardiospecific miRNAs in a closed-chest, large animal ischemia-reperfusion model and in patients with ST elevation myocardial infarction (STEMI). Methods: Six anesthetized pigs were subjected to coronary occlusion-reperfusion. Plasma, urine, and clinical parameters were collected from 25 STEMI patients undergoing primary percutaneous coronary intervention. miRNA was extracted and measured with qPCR. Results: In the pig reperfusion model miR-1, miR133a, and miR-208b increased rapidly in plasma with a peak at 120 min, while miR-499-5p remained elevated longer. In patients with STEMI all 4 miRNAs increased abruptly from 70-fold to 3,000-fold in plasma, with a peak within 12 h (p ! 0.01). miR-1 and miR-133a both correlated strongly with the glomerular filtration rate (GFR), indicating renal elimination. This was confirmed by detection of miR-1 and miR-133a, but not miR-208b or miR-499-5p, in urine. Peak values of miR208b correlated with peak troponin and the ejection fraction. Conclusion: We demonstrate a distinct and rapid inReceived: November 30, 2010 Accepted after revision: April 27, 2011 Published online: June 24, 2011 Prof. David Erlinge, MD, PhD Department of Cardiology Lund University Hospital SE–221 85 Lund (Sweden) Tel. +46 46 17 25 97, E-Mail david.erlinge @ med.lu.se © 2011 S. Karger AG, Basel 0008–6312/11/1184–0217$38.00/0 Accessible online at: www.karger.com/crd D ow nl oa de d by : 54 .7 0. 40 .1 1 11 /1 9/ 20 17 3 :0 0: 15 P M Gidlöf/Andersson/van der Pals/Götberg/ Erlinge Cardiology 2011;118:217–226 218 pression. These noncoding RNA molecules act by pairing with complimentary regions in the 3 -untranslated region of target mRNA, thereby suppressing gene expression [5] . At present, more than 900 human miRNA species have been reported in miRBase [6] release 15 (http:// mirbase.org), and many of them appear to be expressed in a tissue-specific manner [7]. Differential expression of certain miRNA species has been linked with pathological processes such as cancer [8] , inflammation [9] , and cardiovascular disease [10–12]. miRNA has been shown to be important for cardiac development, and knockout of miR-1 results in cardiomyopathy [13]. The remarkable stability of miRNAs in blood [14] and urine [15] have also made them interesting candidates as biomarkers for various pathological conditions. Certain miRNA species that have been shown to be highly enriched either in cardiac and skeletal muscle in general (miR-1, miR-133a, and miR-499-5p) or specifically in cardiomyocytes (miR-208a and miR-208b) are released into the circulation following myocardial infarction. Several recent studies have aimed to demonstrate the usefulness of some of these miRNAs as cardiac biomarkers with promising results [16–20]. The aim of our study was to investigate the levels of cardiospecific miRNAs in plasma following myocardial infarction and to evaluate their usefulness as markers of cardiac cell death and cardiac function. Materials and Methods Porcine Model of Myocardial Infarction The procedure has been described in detail previously [21]. Briefly, 6 healthy 40to 50-kg domestic pigs were anesthetized and a 3.0–3.5 ! 20 mm Maverick Monorail angioplasty balloon (Boston Scientific Scimed, Maple Grove, Minn., USA) was positioned in the proximal left anterior descending artery (LAD). Ischemia was induced by inflation of the angioplasty balloon for 40 min. Total occlusion of the LAD during inflation of the balloon and restoration of blood flow after deflation was verified with angiograms. Blood samples were drawn before occlusion and every 30 min for a total of 150 min after inflation of the balloon. All pigs were sacrificed after 4 h. Patients Patients eligible for inclusion were those undergoing primary percutaneous coronary intervention at Skåne University Hospital Lund due to an STEMI. All patients gave their written approval for participating in the study. The study group included 25 patients, 20 (80%) of whom were men. The mean age of the patients was 64.56 years (SEM = 2.70). Patient characteristics are summarized in table 1 . Healthy volunteers recruited at the Biomedical Centre in Lund, Sweden, were used as controls. The control group included 7 men (64%) and 4 women (36%) with a mean age of 65.09 (SEM = 3.51). This study was carried out according to the principles of the Declaration of Helsinki and was approved by the local ethics committee of Skåne University Hospital. Sample Collection and Handling The first blood sample was obtained by venipuncture within 24 h of the onset of symptoms, the second within 48 h, and the third within 72 h. Elevation of cardiospecific miRNAs in the setting of myocardial infarction has previously been shown to increase rapidly, peaking within hours of the presentation of symptoms [16]. The initial samples were therefore divided into 2 groups depending on whether they were collected before or after a cutoff value of 12 h (n = 9, ! 12 h; n = 16, 1 12 h). Additional blood samples were collected 3–6 months after discharge. The samples were centrifuged at 1,600 g for 15 min within 2 h of collection, followed by aspiration of the plasma, which was stored at –80 ° C. A urine samTable 1. Patient characteristics Patients 25 Women 5 (20) Hypertension 9 (36) Diabetes mellitus 3 (12) Hyperlipidemia at admission 0 Current smoker 10 (40) Infarct-related artery LAD 14 (56) Cx 5 (20) RCA 6 (24) Symptoms-to-reperfusion time (min) <100 4 (16) 100–180 10 (40) 181–360 5 (20) 361–600 2 (8) >600 1 (4) Unknown 3 (12) TIMI 3 flow post-PCI 24 (96) Ejection fraction >55 11 (44) 45–55 2 (8) 35–45 10 (40) 25–35 2 (8) <25 0 Cardiovascular death within 3 months of AMI 1 (4) Rehospitalization for ACS within 3 months of AMI 0 GFR (ml/min) >90 10 (40) 60–90 11 (44) 30–59 3 (12) <30 1 (4) A ll values are presented as numbers (%). All patients received hyperlipidemia treatment at admission. Cx = Circumflex; RCA = right coronary artery; TIMI = thrombolysis in myocardial infarction; PCI = percutaneous coronary intervention; AMI = acute myocardial infarction; ACS = acute coronary syndrome. D ow nl oa de d by : 54 .7 0. 40 .1 1 11 /1 9/ 20 17 3 :0 0: 15 P M miRNA in Acute Myocardial Infarction Cardiology 2011;118:217–226 219 ple was collected in a subset of STEMI patients (n = 8) within 24 h of the onset of symptoms and stored at –80 ° C. To test the stability of miRNA in urine during freezing/thawing, urine samples from 3 patients were subjected to 4 cycles of freezing/thawing and aliquots were taken in each cycle for RNA preparation. RNA Isolation and Real-Time Quantitative RT-PCR Plasma or urine was mixed with TRIzol LS (Invitrogen, Carlsbad, Calif., USA) in a 1: 3 ratio and the samples were homogenized by vortexing 1 30 s. RNA was then isolated using an miRNeasy mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. RNA quantity and quality were assessed by NanoDrop (NanoDrop Products, Wilmington, Del., USA) and a Bioanalyzer (Agilent, Santa Clara, Calif., USA) using the 2100 Small RNA assay. cDNA was synthesized from 20 ng of total RNA using an miRCURY LNA Universal cDNA synthesis kit (Exiqon, Vedbaek, Denmark) according to the manufacturer’s instructions. Quantitative RT-PCR (qRT-PCR) was carried out in 20 l triplicate reactions with Fast SYBR Green Master Mix (Applied Biosystems, Carlsbad, Calif., USA) and LNA primer sets (Exiqon) specific for miR-1, miR-133a, miR-208a, miR-208b, miR-4995p, and miR-16 according to the manufacturer’s protocol on a StepOnePlus Real-Time PCR System (Applied Biosystems). Analysis of qRT-PCR Data The threshold cycle (Ct) is defined as the fractional PCR cycle number at which the fluorescence reaches a given threshold. For the plasma samples, Ct values for the miRNAs of interest were normalized against the arbitrary value of 40 and expressed relative to the mean of the control samples according to the 2 – Ct method. The levels of miRNA in urine samples were normalized to an exogenous miRNA spike-in (Exiqon) and expressed as relative quantities (2 – Ct ). The porcine miRNA data was normalized against baseline values and the maximum value in each pig during the time course. Samples where no amplification could be detected were defined as having a Ct value of 40. An interplate calibrator sample was used to adjust for run-to-run variation. Statistical Analysis All statistical analyses were carried out using GraphPad Prism 4.0 software (GraphPad Software, Inc., La Jolla, Calif., USA). Differences in miRNA levels were analyzed using 1-way ANOVA with Bonferroni’s multiple comparisons post hoc test. Normality tests of all parameters were performed using the D’Agostino-Pearson omnibus method. Correlations between circulating miRNA levels and clinical parameters were performed using Spearman’s rank correlation coefficient. p ! 0.05 was considered statistically significant. All error bars represent the standard error of the mean (SEM).