MicroRNA-21–Dependent Macrophage-to-Fibroblast Signaling Determines the Cardiac Response to Pressure Overload

Abstract

Background: Cardiac macrophages (cMP) are increasingly recognized as important regulators of myocardial homeostasis and disease, yet the role of noncoding RNA in these cells is largely unknown. Small RNA sequencing of the entire miRNomes of the major cardiac cell fractions revealed microRNA-21 (miR-21) as the single highest expressed microRNA in cMPs, both in health and disease (25% and 43% of all microRNA reads respectively). MiR-21 has been previously reported as a key microRNA driving tissue fibrosis. Here, we aimed to determine the function of macrophage miR-21 on myocardial homeostasis and disease-associated remodeling. Methods: Macrophage-specific ablation of miR-21 in mice driven by Cx3cr1-Cre was used to determine the function of miR-21 in this cell type. As a disease model, mice were subjected to pressure overload for 6 and 28 days. Cardiac function was assessed in vivo by echocardiography, followed by histological analyses and single cell sequencing. Co-cultures of macrophages and cardiac fibroblasts were employed to study macrophage-to-fibroblast signaling. Results: Mice with macrophage-specific genetic deletion of miR-21 were protected from interstitial fibrosis and cardiac dysfunction when subjected to pressure overload of the left ventricle. Single cell sequencing of pressure-overloaded hearts from these mice revealed that miR-21 in macrophages is essential for their polarization towards a M1-like phenotype. Systematic quantification of intercellular communication mediated by ligand-receptor interactions across all cell types revealed that miR-21 primarily determined macrophage-fibroblast communication, promoting the transition from quiescent fibroblasts to myofibroblasts. Polarization of isolated macrophages in vitro towards a pro-inflammatory (M1) phenotype activated myofibroblast transdifferentiation of cardiac fibroblasts in a paracrine manner and was dependent on the rapid induction of miR-21 in cMPs. Conclusions: Our data indicate a critical role of cMPs in pressure overload-induced cardiac fibrosis and dysfunction and reveal macrophage miR-21 as a key molecule for the pro-fibrotic role of cMPs.

Modulation of Cardiac Fibrosis in and Beyond Cells

“…The activation of cardiac fibroblasts could also be suppressed by macrophage-specific deletion of microRNA-21 which decreased interstitial fibrosis in mice following pressure overload ( Ramanujam et al, 2021 ). Deletion of microRNA-21 in macrophages increased M2-polarized macrophages, while decreased M1-like proinflammatory macrophages which might disrupted the macrophage-to-fibroblast signaling and activation of cardiac fibroblasts ( Table 1 ) ( Ramanujam et al, 2021 ).…”

“…The activation of cardiac fibroblasts could also be suppressed by macrophage-specific deletion of microRNA-21 which decreased interstitial fibrosis in mice following pressure overload ( Ramanujam et al, 2021 ). Deletion of microRNA-21 in macrophages increased M2-polarized macrophages, while decreased M1-like proinflammatory macrophages which might disrupted the macrophage-to-fibroblast signaling and activation of cardiac fibroblasts ( Table 1 ) ( Ramanujam et al, 2021 ). However, during the proliferative phase of MI, CD226 deletion also increased M2-macrophages and decreased M1-macrophages, but it resulted in accumulation of myofibroblasts and collagen deposition in the infarct area, with reduced interstitial fibrosis in the infarct border area though ( Li et al, 2020 ).…”

Organized Chaos: Deciphering Immune Cell Heterogeneity’s Role in Inflammation in the Heart

“…This may be because multiple pathways lead to activation of fibroblasts and different macrophage populations might use distinct pathways especially in the context of the method used to induce PO, i.e., Ang II versus transverse aortic constriction (TAC). For example, using TAC, a murine model of PO, expression of microRNA-21 (mir-21) in macrophages was shown to induce a pro-inflammatory macrophage phenotype [ 45 ]. Furthermore, mice with deleted macrophage expression of miR-21 demonstrated reduced fibrosis and fewer myofibroblasts suggesting that pro-inflammatory macrophages were primary drivers of fibrosis [ 45 ].…”

“…For example, using TAC, a murine model of PO, expression of microRNA-21 (mir-21) in macrophages was shown to induce a pro-inflammatory macrophage phenotype [ 45 ]. Furthermore, mice with deleted macrophage expression of miR-21 demonstrated reduced fibrosis and fewer myofibroblasts suggesting that pro-inflammatory macrophages were primary drivers of fibrosis [ 45 ]. In another model of PO using Ang II infusion, Falkenham et al demonstrated that depletion of macrophages using clodronate liposomes reduced levels of αSMA+ myofibroblasts, decreased collagen content, and decreased levels of mRNA encoding Cxcl3 , a nonclassical macrophage marker supporting an essential role of non-classical macrophages in fibrosis.…”

Recent Progress in Cardiovascular Research Involving Single-Cell Omics Approaches

“…Ramanujam et al applied ligand–receptor pair analysis to scRNA-seq data from non-CMs in pressure-overloaded murine hearts by analyzing the transcripts of secreted protein ligands in one cell type and their receptors in another cell type, and predicted that M1 like macrophages interacted with activated fibroblasts via secreted proteins such as TGFβ and fibronectin. These interactions were demonstrated to be mediated by macrophage miR-21, depletion of which rescued hypertrophic phenotype after TAC ( 76 ). Martini et al, by sequencing cardiac CD45 + cells, showed increases in Osm + pro-inflammatory macrophages, as well as Pdcd 1 + Tregs, in pressure-overloaded murine hearts when compared with healthy hearts ( 77 ).…”