Cardiac resident macrophages were recently shown to participate in cardiac conduction and hypertrophy, but underlying molecular mechanism are unclear. To understand whether Long non-coding RNAs (lncRNAs) play important roles in cardiac resident macrophages, we employed deep RNASeq (>100 million reads/sample) of purified murine cardiac macrophages and single cell sequencing of over 30,000 cells (total myocardial cells and purified leukocyte fraction). First, re-clustering analyses of the single cell data uncovered a general importance of lncRNAs for cell type identity. Using different gene sets as input, we could show that lncRNA genes improve clustering sensitivity not only as a whole group, but already by using only a few lncRNAs (p<0.05). Secondly, based on our sequencing data, we could define the entire lncRNome of cardiac macrophages from healthy and diseased murine myocardium (pressure-overload to the left ventricle). We identified 250 novel lncRNA genes of which 162 lncRNAs were enriched in cardiac macrophages with log2 fold change >1. In total, we uncovered a set of 56 lncRNAs highly enriched (fold change >10) in cardiac resident macrophages and 36 lncRNAs also deregulated during disease (p<0.05). Integrating scSeq and RNASeq expression data as well as the de-regulation during disease yielded the lncRNA Schlafenlnc as the top candidate for further study. 

Schlafenlnc is encoded within the Schlafen protein family locus, its proteins play important roles in immune cells such as differentiation, invasion and migration. To analyze the function of Schlafenlnc, we deleted the whole locus based on promoter and gene body chromatin marks in a Cas9-transgenic macrophage cell line. Employing RNASeq of unstimulated, as well as M1-like (LPS-treated) and M2-like (IL4-treated) Schlafenlnc-deficient macrophages, we could show that – similarly to the function of some of the Schlafen proteins – Schlafenlnc impacted on genes with gene ontology terms involved in differentiation and particularly migration. Most prominently, one gene of the matrix metalloproteinase family (degradation of extracellular matrix) was strongly down-regulated in Schlafenlnc-deficient cells. To validate this phenotype, we conducted different migration assays (random migration on Fibronectin as well as directed migration (chemotaxis) against a chemokine gradient), which showed both a strongly decreased migration of Schlafenlnc-deficient cells in comparison to controls.

Collectively, our data establish the entire lncRNome in cardiac resident macrophages, highlighting especially their importance for cell identity. The lncRNA Schlafenlnc was identified as a critical regulator of macrophage migratory functions and we could describe for the first time a function for a lncRNA in this crucial cardiac cell type.