digital-pcr-for-quantifying-circulating-micrornas-acute-myocardial

Digital PCR for Quantifying Circulating MicroRNAs in Acute Myocardial Infarction and Cardiovascular Disease

Circulating serum microRNAs (miRNAs) have shown promise as biomarkers for the cardiovascular disease and acute myocardial infarction (AMI), being released ...

University of Freiburg

Residual platelet reactivity is a predictor of poor prognosis in patients with acute coronary syndromes (ACSs) undergoing percutaneous coronary intervention. Thrombin is a major platelet activator and upon initiation of the coagulation cascade, it is subsequently produced downstream of factor IXa, which itself is known to be increased in ACS. Pegnivacogin is a novel RNA-aptamer based factor IXa inhibitor featuring a reversal agent, anivamersen. We hypothesized that pegnivacogin could reduce platelet reactivity.

Direct factor IXa inhibition with the RNA-aptamer pegnivacogin reduces platelet reactivity in vitro and residual platelet aggregation in patients with acute coronary syndromes.

The extent of myocardial damage in patients with ST-segment elevation myocardial infarction (STEMI) depends on both the time to reperfusion as well as injury induced by ischaemia-reperfusion resulting in a cascade of cellular and humoral reactions. As a consequence of ischaemia-reperfusion in the heart, the high-temperature requirement serine peptidase 2 (HtrA2) is translocated from the mitochondria to the cytosol, whereupon it induces protease activity-dependent apoptosis mediated via caspases. Myocardial damage induced by reperfusion cannot be monitored due to a current lack in specific biomarkers. We examined the serum level of HtrA2 as a potentially novel biomarker for mitochondrial-induced cardiomyocyte apoptosis.

The mitochondria-targeting peptide elamipretide diminishes circulating HtrA2 in ST-segment elevation myocardial infarction.

Ischemia-reperfusion (I/R) injury in ST-segment elevation myocardial infarction (STEMI) significantly contributes to overall myocardial damage. As a consequence of I/R injury in the heart, the high-temperature requirement protein A2 (HtrA2) is released from the mitochondrial intermembrane space of cardiomyocytes to the cytoplasm, whereupon it induces apoptosis.

Circulating HtrA2 as a novel biomarker for mitochondrial induced cardiomyocyte apoptosis and ischemia-reperfusion injury in ST-segment elevation myocardial infarction.

Circulating HtrA2 after primary percutaneous coronary intervention in ST-segment elevation myocardial infarction: A potential novel biomarker for mitochondrial induced cardiomyocyte apoptosis and ischemia-reperfusion injury.

miRNAs have shown promise as potential biomarkers for acute myocardial infarction (AMI). However, the current used quantitative real-time PCR (qRT-PCR) allows solely for relative expression of nucleic acids and it is susceptible to day-to-day variability, which has limited the validity of using the miRNAs as biomarkers. In this study we explored the technical qualities and diagnostic potential of a new technique, chip-based digital PCR, in quantifying the miRNAs in patients with AMI and ischaemia-reperfusion injury (I/R). In a dilution series of synthetic C.elegans-miR-39, chip-based digital PCR displayed a lower coefficient of variation (8.9% vs 46.3%) and a lower limit of detection (0.2 copies/μL vs 1.1 copies/μL) compared with qRT-PCR. In the serum collected from 24 patients with ST-elevation myocardial infarction (STEMI) and 20 patients with stable coronary artery disease (CAD) patients after percutaneous coronary intervention (PCI), we used qRT-PCR and multiplexed chip-based digital PCR to quantify the serum levels of miRNA-21 and miRNA-499 as they have been validated in AMI in prior studies. In STEMI, I/R injury was assessed via measurement of ST-segment resolution (ST-R). Chip-based digital PCR revealed a statistical significance in the difference of miR-21 levels between stable CAD and STEMI groups (118.8 copies/μL vs 59 copies/μL; P=0.0300), whereas qRT-PCR was unable to reach significance (136.4 copies/μL vs 122.8 copies/μL; P=0.2273). For miR-499 levels, both chip-based digital PCR and qRT-PCR revealed statistically significant differences between stable CAD and STEMI groups (2 copies/μL vs 8.5 copies/μL, P=0.0011; 0 copies/μL vs 19.4 copies/μL; P<0.0001). There was no association between miR-21/499 levels and ST-R post-PCI. Our results show that the chip-based digital PCR exhibits superior technical qualities and promises to be a superior method for quantifying miRNA levels in the circulation, which may become a more accurate and reproducible method for directly quantifying miRNAs, particularly for use in large multi-centre clinical trials.

Chip-based digital PCR as a novel detection method for quantifying microRNAs in acute myocardial infarction patients.

micro-RNAs have shown promise as potential biomarkers for acute myocardial infarction and ischemia-reperfusion injury (I/R). Most recently droplet digital polymerase chain reaction (ddPCR) has been introduced as a more reliable and reproducible method for detecting micro-RNAs.

Article	Year
Direct factor IXa inhibition with the RNA-aptamer pegnivacogin reduces platelet reactivity in vitro and residual platelet aggregation in patients with acute coronary syndromes. European heart journal. Acute cardiovascular care\| PubMed ID: 28403626	2017
The mitochondria-targeting peptide elamipretide diminishes circulating HtrA2 in ST-segment elevation myocardial infarction. European heart journal. Acute cardiovascular care\| PubMed ID: 28534645	2017
Circulating HtrA2 as a novel biomarker for mitochondrial induced cardiomyocyte apoptosis and ischemia-reperfusion injury in ST-segment elevation myocardial infarction. International journal of cardiology\| PubMed ID: 28602551	2017
Circulating HtrA2 after primary percutaneous coronary intervention in ST-segment elevation myocardial infarction: A potential novel biomarker for mitochondrial induced cardiomyocyte apoptosi... International journal of cardiology\| PubMed ID: 29121749	2017
Chip-based digital PCR as a novel detection method for quantifying microRNAs in acute myocardial infarction patients. Acta pharmacologica Sinica\| PubMed ID: 29188800	2017
Droplet digital PCR as a novel detection method for quantifying microRNAs in acute myocardial infarction. International journal of cardiology\| PubMed ID: 29506703	2018

Marcus Hortmann

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