Human cardiomyocyte derived extracellular vesicles regulate cardiac fibroblast activation through miR-24
Authors: Senesi G , Lodrini A , Mohammed S , Mosole S , Ceresa D , Bolis S , Malatesta P , Goumans M J , Paneni F , Barile L , Balbi C , Vassalli G 
Published: May 2024

Abstract

Funding Acknowledgements
Type of funding sources: Foundation. Main funding source(s): Fondazione Leonardo

Introduction
Cardiovascular diseases are associated with increase in interstitial fibrosis due to cardiac fibroblast (CF) activation into myofibroblast leading to the secretion and deposition of extracellular matrix (ECM). The intricate functional activation of these processes is not well understood, especially concerning the contribution of the microenvironment. One of the most important mechanisms of paracrine communication are due to Extracellular vesicles (EVs). EVs are cell-released nanoparticles carrying proteins, lipids, and nucleic acids, particularly miRs. To note, previous transcriptomic analysis of healthy human heart demonstrated that miR24-3p is one of the most abundant miRs [1], suggesting an important role associated with its expression.
Aim of the work
Here we aim to investigate the role of miR24-3p as regulator of cardiac fibroblast activation, focusing on healthy and pathological conditions.

Materials and methods
Bioinformatic analysis of open-data, single-cell RNA sequencing from heart tissue was conducted to assess the expression of miR24-3p and its specific targets in both healthy and infarcted myocardium. To explore the functional role of miR24-3p, loss- and gain-of-function experiments were performed by transfecting cardiac fibroblasts (CF) with specific antimiR or mimic oligonucleotides, respectively. In investigating the influence of cardiomyocytes (CM) on CF regulation, extracellular vesicles (EVs) from human-induced pluripotent stem cell-derived CM were isolated and utilized in in vitro experiments. The expression levels of miRNA24-3p were also evaluated in the plasma of healthy individuals and patients who had experienced myocardial infarction. Finally, cardiac slices from human tissue were employed as ex-vivo microtissues to functionally validate the in vitro findings.

Results
RealTime analysis of CF, CM and cardiac endothelial cells (EC) revealed a CM specific expression of miR24-3p, not only at cellular level but also in EVs. Upregulation of miR24-3p in CF was able to significantly reduce protein expression of FURIN, CYCLD1 and SMAD4, resulting in decrease of CF activation and ECM secretion. Interestingly, treatment of CF with CM-EVs recapitulate miR24-3p transfection, while EVs derived from hypoxia cultured CM (MI-like) does not. RNAScope and ddPCR analysis on human cardiac slice cultured under hypoxia conditions confirmed decrease in miR24-3p expression upon induction of stress. Ultimately, a significant decrease in miR24-3p in circulating serum EVs of MI patients’ was observed.

Conclusions
These data demonstrate the role of miR24-3p in CF regulation through the paracrine activity mediated by CM-EVs. Further analysis, including the use of a 3D human heart microtissue, will confirm the mechanism of cross-talk between CM and CF.