Single-cell analysis of the long noncoding RNA transcriptome identifies novel therapeutic targets for cardiac fibrosis

Abstract

Coronary artery disease is a leading cause of death worldwide and typically leads to acute myocardial infarction (MI). Long noncoding RNAs (lncRNAs) are importance in cardiac hypertrophy and fibrosis. The aim of this project is therefore to characterize cardiac non-myocyte cells before and after MI via a single-cell analysis of the lncRNA transcriptome. Non-myocyte fractions of mouse hearts were isolated using a Langendorff approach, 3 days after MI. FACS was used to remove leukocytes (CD45 + ) and endothelial cells (CD31 + ) from metabolically active (Calcein + ) and viable (DAPI − ) cells. Then, sorted cells were subjected to a single-cell analysis using a 10X Genomics platform. An average number of reads of 200,000 per cell allows capturing a total of 73,828 transcripts (coding and noncoding RNAs) in 8413 cells. We were able to identify 15 different cell clusters. Two clusters appeared to be infarction-specific. According to the expression of key marker genes, cells belonging to these two clusters were identified as myofibroblasts. We selected a few intergenic transcripts for further validation. Then, we further filtered lncRNAs for those that were association with enhancer elements and multi-exonic with significant basal expression. At the end, the novel enhancer-associated lncRNA CF31245 was selected for functional analysis. This lncRNA is highly enriched in cardiac tissues as compared to 10 other tissues. Interestingly, CF31245 knockdown using specific GapmeRs in adult cardiac fibroblasts led to a significant decrease in expression of genes encoding extracellular matrix proteins, and inhibit thereby myofibroblast differentiation. Altogether, these results validate the single-cell approach for identifying novel subpopulation-specific lncRNAs. They further suggest that the fibroblast-specific CF31245 lncRNA represents an attractive therapeutic target to blunt the fibrotic response after MI.