Dissection of the role of microRNAs in cardiac fibrosis through functional genomics screenings

Abstract

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): FCT, Portuguese Foundation for Science and Technology Cardiac fibrosis is a maladaptive remodelling process of the myocardium, which results from an over-activation of cardiac fibroblasts into myofibroblasts, excessive deposition of ECM proteins (particularly collagen) and scar formation, ultimately leading to stiffening of the heart wall and greatly compromising heart function. A deeper knowledge of the molecular and cellular fibrogenic processes is crucial to identify novel targets for therapeutic interventions aimed at diminishing cardiac fibrosis and preventing/reversing cardiac remodelling. Recent studies have shown that microRNAs control several features of cardiovascular diseases, including cardiac fibrosis. To systematically identify microRNAs that modulate cardiac fibrosis, we performed a series of high-content microscopy functional screenings using a genome-wide library of microRNA mimics (2,588 mature microRNAs). We focused on key phenotypes relevant to cardiac fibrosis, specifically human primary cardiac fibroblast proliferation, myofibroblast differentiation, and deposition of fibrillary collagen under unstimulated and stimulated conditions. Using this approach, we identified 134 microRNAs that strongly modulate myofibroblast differentiation (4-fold change), as well as a high number of microRNAs that completely block cardiac fibroblast proliferation (166 microRNAs) or that strongly modulate collagen deposition (4-fold change; 227 and 96 microRNAs in unstimulated and stimulated conditions, respectively). Of note, we observed that myofibroblast differentiation is not a pre-requisite for collagen deposition, and that specific microRNAs can modulate these phenotypes independently. Clustering performed on the global analysis of the phenotypes elicited by each microRNA led to the selection of 92 microRNAs. We confirmed that the phenotypes could be reproduced in human cardiac fibroblasts from different donors and that some of the phenotypes were conserved in fibroblasts isolated from different tissues, including aorta, skin, and lung. Additionally, we performed high-throughput qPCR analysis to identify molecular signatures associated with the effects of these microRNAs on 92 genes implicated in fibrosis-related processes. Based on these data, we selected 8 microRNAs for detailed mechanistic characterization. Seven of these microRNAs strongly block collagen deposition in stimulated conditions, while exhibiting differential effects on cardiac fibroblast differentiation and/or tissue-specific activities, and 1 microRNA increases collagen deposition. Mechanistic studies showed that 3 of these microRNAs target the CDS or 3’UTR of collagen (Col1a1), while the others act through alternative mechanisms. Overall, our work establishes microRNAs as powerful modulators of multiple processes critical to cardiac fibrosis and offers a unique opportunity for testing different anti-fibrotic approaches based on the differential modulation of these processes by microRNAs.