Time matters: non-coding RNA in the detection of a developing myocardial infarction
The aim of the project "Non-coding RNAs to diagnose developing myocardial infarction – a prospective study using next-generation sequencing" by Ewelina Błażejowska is to find molecules that can "reveal" a clot in the coronary artery that is just developing before irreversible damage to the heart muscle occurs.
The project received the PRELUDIUM 24 grant of the National Science Centre.
Troponin: the gold standard with a gap at the start
Myocardial infarction is one of the most severe forms of cardiovascular disease and remains one of the leading causes of death worldwide. Rapid diagnosis and treatment make a difference: the sooner we clear the vessel, the smaller part of the heart is damaged and the greater the chance of recovery.
Today, troponin testing constitutes the biochemical basis for the diagnosis of myocardial infarction. Troponin is a protein released into the blood when heart muscle cells are damaged. Troponin is a great marker of necrosis, but when it comes to a really early detection of a heart attack, there are key limitations. In myocardial infarction, troponin only grows when necrosis has already begun – that is, it shows the effect, not the very beginning of the thrombotic process. Moreover, if myocardial infarction is suspected, troponin has to be determined twice. This prolongs making the diagnosis – the patient has to wait for the next blood test and the result, and the decision on treatment or safe discharge is made later. Since troponin remains in the bloodstream for a long time, it can also make it difficult to assess whether another incident has occurred. In addition, troponin may also be elevated in other conditions which are not directly related to myocardial infarction, such as pulmonary embolism, sepsis, or renal failure, which sometimes hinders result interpretation.
In practice, this means many hours of observation and repeating tests in some patients. Meanwhile, in many patients, a clot in the coronary artery forms more than a day before the onset of chest pain, and an "older" clot is associated with a poorer prognosis. It is this stage – critical, because it is potentially still reversible – that we want to capture.
Non-coding RNA: small molecules, a lot of information
They used to be called "genetic junk". Today, we know that non-coding RNAs (ncRNAs) are one of the most important regulators of cell function. They do not produce proteins, but they can act as microswitches – activating or silencing entire sets of genes. Various classes of ncRNA circulate in the bloodstream: microRNA (miRNA), PIWI-interacting RNA (piRNA), small nuclear RNA (snoRNA) and fragments derived from tRNA (tRF).
From the diagnostic point of view, the most interesting thing is that ncRNAs may appear in the blood quickly in response to cellular stress and vascular processes. They are also exceptionally stable, largely because they often "travel" in protective media that protect them from disintegration. Therefore, they may be measured in a repetitive manner with laboratory methods, which may become quick in the future. Importantly, previous research showed that some miRNAs could increase in the blood before troponin, but results are not always consistent between studies and populations. Therefore, a broad and "unbiased" approach is needed. It should not be limited to a few selected molecules.
Extracellular vesicles: couriers from inside the vessels
Extracellular vesicles (EVs) are such non-coding RNA carriers. They are microscopic “packets” surrounded by a membrane, actively released by cells. EVs also carry a variety of other molecules, including RNA, and are involved in cell-to-cell communication. It is particularly interesting because EVs may selectively "package" RNAs released by cells under stress. Thus, they may carry more specific information about the thrombotic process than RNAs circulating freely in the plasma.
This approach stems from our previous experience from the LEMONADE project, where we examined vesicles in patients presenting to hospital with chest pain. We noted that selected platelet-derived vesicles might have a diagnostic value even when baseline troponin was still negative. The results were promising, but the measurements of the number of the vesicles and markers on their surface alone did not yet provide enough accuracy to develop a simple test for routine use. Hence the idea to go further – towards the analysis of their "charge", i.e., ncRNA.
How are we going to look for new biomarkers?
We work on plasma samples obtained from 98 patients participating in the LEMONADE study: 34 people were definitively diagnosed with myocardial infarction and 64 people had a different cause of chest pain (cardiac or extra-cardiac). The material has already been collected and biobanked. In such projects, the devil is in the details, which is why the samples were prepared according to a unified protocol so that the differences in the results were due to the biology of the disease, and not due to the technical nuances of blood collection or preparation.
In the first stage, we are going to isolate small ncRNAs from the plasma and EV-enriched fraction. We are planning to isolate the vesicles using a method that acts as a very precise sieve (so-called size exclusion chromatography), thanks to which we are going to get a fraction rich in EV, and poor in "random" proteins. Then, in the selected group of 20 samples, we are going to perform next-generation sequencing (NGS). It is a "discovery" method: instead of looking for one pre-selected marker, we can see a broad ncRNA profile and select molecules that best distinguish patients with ACS (acute coronary syndromes) from the others – already on admission to the hospital.
During the second stage, we are going to select up to ten most promising candidates and verify them in all 98 patients with qRT PCR. The technique is fast, sensitive and standardizable in diagnostic laboratories – i.e., one that stands a real chance of being translated into a future test. We are also planning to look at how levels of selected ncRNAs are associated with the patients' clinical features (e.g., pain characteristics), with troponin on admission, and with inflammatory markers.
The third stage focuses on the prognosis. We are going to examine whether the acute phase ncRNA profile is associated with the risk of recurrence of ischemic events within 3 years of follow-up (e.g., reinfarction, rehospitalization, or sudden cardiac death). We are planning to combine molecular data with clinical and laboratory information to develop a risk model useful to physicians and patients, and to better understand what biological processes underlie the observed RNA changes.
The novelty lies in the presence of two "sources" of information and a broader view of RNA
Numerous previous studies have focused mainly on microRNAs. We want to look more broadly – also taking account of piRNA and snoRNA, i.e., molecules less often analyzed in the context of ACS. At the same time, we are going to compare two "carriers" of information: ncRNA circulating freely in the plasma and ncRNA enclosed in the extracellular vesicles. This may answer the question of where the signal is more specific to the developing clot and whether there is "clearer" information about the mechanism of the disease in the vesicles.
What can the patient gain?
If it is possible to identify an ncRNA panel that increases earlier than troponin, the benefits may be very practical. In patients at a high risk of myocardial infarction, the diagnosis could be made faster, which would shorten the path to the treatment and a possible urgent intervention. On the other hand, in some patients with pain of a different cause, it would be possible to exclude myocardial infarction faster and safer without unnecessarily long observation and some invasive tests. For the healthcare system, this means a better use of resources: fewer delays in the diagnostic process in the ER and more targeted referral of patients to the right path.
The project is carried out at the Medical University of Warsaw in cooperation with centres experienced in the analysis of extracellular vesicles. Perhaps it is the "microscopic messages" stored in RNA that will make it possible to tell the patient in the future: a heart attack is just developing and we still have time to prevent it. We believe this is a step towards faster, more personalized diagnosis and a better prognosis.