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Functional high-throughput analysis of the role of microRNAs in cardiac ischemia-reperfusion injury

Periodic Reporting for period 1 - microCardio (Functional high-throughput analysis of the role of microRNAs in cardiac ischemia-reperfusion injury)

Reporting period: 2016-03-01 to 2018-02-28

Ischemia-reperfusion injury is a major cause of morbidity and mortality transversal to different clinical settings and pathologies, including a wide range of cardiovascular diseases including myocardial infarction and cerebral ischemic stroke, the main cause of death worldwide. Ischemia is characterised by insufficient supply of blood to tissues causing tissue oxygen deprivation. Importantly, damage by prolonged ischemia is further aggravated by reperfusion, leading to irreversible tissue damage. As consequence, cardiac cells at the site of injury die and a fibrotic scar, composed mainly of extracellular matrix, is formed. Cardiac fibrosis greatly affects cardiac function due to the loss of myocardial contractility, which can progress to heart failure and eventually cause death. Identifying and understanding the factors contributing to cardiac fibrosis is essential to counteract the irreversible damage and to potentially develop novel therapeutic approaches to intervene in myocardial infarction. Recent studies have shown that microRNAs control various aspects of heart disease, including ischemia-reperfusion injury and cardiac fibrosis. Although a few microRNAs have already been implicated in this process, a comprehensive analysis of the functional role of microRNAs in cardiac fibrosis and ischemia-reperfusion injury is still missing.
The main goal of this project was to identify microRNAs which modulate cardiac fibrosis and characterise the molecular mechanisms underlying their function. Particular emphasis was put in studying how miRNAs modulate cardiac fibroblasts’ proliferation and differentiation, and extracellular matrix deposition. This was achieved through gain-of-function high-throughput screenings using genome-wide microRNA libraries in the presence or absence of a stimulus for extracellular matrix deposition. The identification and characterisation of the molecular targets of the selected microRNAs is underway and will entail a combination of computational and experimental approaches.
This project used innovative experimental approaches to unravel a previously unappreciated network of microRNAs and microRNA targets critical to cardiac fibrosis, which may reveal novel opportunities for therapeutic intervention, with important clinical implications.
To achieve the goals set out at the beginning of the project, high-content microscopy screenings were performed using a library of microRNA mimics corresponding to all the annotated microRNAs (2,588 mature microRNAs) to analyse fibroblast proliferation and differentiation, and collagen I deposition, as models of the fibroblast activation and extracellular matrix deposition that is seen in vivo. Using this approach, 145 microRNAs that increase and 45 microRNAs that decrease fibroblast differentiation by more than 4-fold relative to control were identified, as well as a number of microRNAs that strongly impact fibroblast proliferation. Furthermore, several miRNAs that decrease collagen I deposition by cardiac fibroblasts were found. Demonstrating the relevance and success of this experimental approach is the identification of master regulators of fibrosis, such as the miR-29 family. Interestingly, close to 200 microRNAs that promote cardiac fibroblast differentiation, but decrease collagen I deposition could be selected. By facilitating the differentiation of fibroblasts into contractile cells, but inhibiting the deposition of extracellular matrix in excess, these microRNAs could promote the regenerative process after a myocardial infarction, yet maintaining the necessary contractile function of the heart, thereby preventing heart failure to ensue.
These results were presented by the fellow at the Keystone’s symposium on RNA-Based Approaches in Cardiovascular Disease held in March 2017 at Keystone Resort, Colorado, USA. A manuscript is being prepared for publication. Furthermore, throughout the duration of the project, the fellow participated in several outreach activities, namely in the European Researcher’s Night and in the Science and Technology week where she visited secondary schools to present her work.
To date, the study of microRNA function in the context of cardiac fibrosis has been limited to studies examining microRNA expression in animal models, to functional studies with a small subset of microRNAs, or to analysis of whole tissues rather than pure cardiac fibroblast cultures. By contrast, this project resorted to state-of-the art genome-scale experimental approaches to explore the role of microRNAs in cardiac fibrosis. Namely, a systematic and unbiased approach based on the combination of gain- and loss-of-function high-throughput screenings using genome-wide libraries of miRNA mimics was employed. The systematic nature of the applied experimental approaches led to the identification of miRNAs not previously associated with cardiac fibrosis.
This project addressed one of the societal challenges objectives (SC1 – Health, demographic changes and wellbeing; HCO13-2015: ERA-NET) in the Horizon 2020 programme and one of the goals in the UN 2030 Agenda for Sustainable Development (Goal 3.4. By 2030, reduce by one third premature mortality from non communicable diseases through prevention and treatment and promote mental health and wellbeing), by studying how miRNAs modulate cardiac fibrosis, a process associated to many cardiovascular diseases, the leading cause of death worldwide. The results obtained during this project are highly innovative and may constitute the basis for the development of novel RNA-based therapeutic approaches for cardiac fibrosis.
Furthermore, this fellowship allowed the fellow to return to her home country and contribute to the development of research in molecular cardiology and RNA biology. It also enabled her to acquire the necessary tools and contacts to transition into a career in science management.
microCardio workflow