Cardiac macrophages have been suggested to exert important functions in myocardial homeostasis and disease. Yet, the mechanisms controlling the function of these macrophages have remained largely enigmatic and the role of noncoding RNAs, specifically microRNAs (miRs) in this immune cell population is entirely unkown. Here, we sought to determine the entire miRnome of cardiac macrophages and specifically investigated the function of the highest expressed microRNA, namely miR-21, in this cell population. 

We isolated cardiac macrophages using a FACS-based approach and performed small RNA sequencing that identified miR-21 as the single most abundant miR in this cell type. Although this miR is already known to be expressed in a wide variety of cardiac cell types including fibroblasts and myocytes, cardiac macrophages emerged as the cell type with the highest levels of miR-21 compared to all other cell types. Cardiac remodeling - induced by left ventricular pressure overload - led to a further upregulation of miR-21 in cardiac macrophages. 

In view of these findings, we hypothesized that miR-21 may function as an important regulator of cardiac macrophage function and possibly influences the processes that govern cardiac remodeling. To further investigate the role of miR-21 in cardiac macrophages in vivo, we generated macrophage-specific miR-21^-/--mice by crossing miR-21^fl/fl-mice with a mouse line expressing Cre-recombinase driven by the Cx3cr1-promotor. Cx3cr1^Cre/0-miR-21^fl/fl-mice (KO) exhibited normal cardiac function at baseline with preserved ejection fraction as determined by echocardiography. These animals were subjected to pressure overload of the left ventricle induced by aortic constriction (TAC), which induced cardiac hypertrophy, myocardial fibrosis and left ventricular dysfunction. Mice deficient in miR-21 displayed significantly reduced cardiac hypertrophy, myocardial fibrosis (interstitial fibrosis (%): WT Sham 1.5 ±0.2, KO Sham 0.7±0.1, WT TAC 2.8±0.4, KO TAC 1.3±0.2) and preserved LV function (Ejection fraction (%): WT Sham 49 ±0.7, KO Sham 51±0.7, WT TAC 26±2.0, KO TAC 37±0.6). 

This vast improvement in cardiac function suggested that miR-21 in cardiac macrophages affects the extracellular matrix composition and the adaption of other cardiac cell types. To investigate this interaction we performed single cell RNA Sequencing (scRNA-Seq, 10xGenomics platform) on the cardiac non-myocyte population 6 days after TAC. In our analysis we could identify two separate macrophage populations with substantially different expression profiles, one leaning towards a pro-inflammatory phenotype and the other expressing M2-like macrophage markers. In miR-21 deficient mice, the polarization changed in favour of the less inflammatory phenotype. These clusters also showed a distinct interaction pattern of ligands and receptors with other cardiac cell types, most importantly fibroblasts.

In our study, we could show that miR-21 is the single highest expressed microRNA in cardiac macrophages and is further upregulated upon induction of cardiac dysfunction. Genetic deletion of miR-21 specifically in cardiac resident macrophages prevented pressure-overload induced cardiac remodeling and heart failure. Mechanistically, deletion of miR-21 determined macrophage polarization thereby preventing cardiac fibroblast activation.