A fibroblast-specific miRNA functional cluster in remote exosomes aggravates myocardial ischaemia/reperfusion injury via the Rap1b/ERK1/2 axis.

BACKGROUND AND PURPOSE: Communication between various cardiac cells by paracrine factors and exosomes has an important role in myocardial ischaemia/reperfusion (I/R) injury. It remains unclear whether exosomes derived from healthy cardiac fibroblasts affect I/R injury and, if so, what are the underlying mechanisms?

EXPERIMENTAL APPROACH: Cardiac fibroblasts were isolated from neonatal rats, adult rats and adult rats subjected to I/R. Their exosomes were designated as follows: neonatal cardiac fibroblasts (N-Exo), adult rat cardiac fibroblasts (A-Exo) and cardiac fibroblasts in a remote non-ischaemic area (R-Exo). Apoptosis of cardiomyocytes and the role of a cluster of microRNAs from exosomes in I/R injury were investigated.

KEY

RESULTS

N-Exo, A-Exo and R-Exo were taken up by ischaemic cardiomyocytes through clathrin heavy chain (Cltc)-mediated endocytosis, enhancing cardiomyocyte apoptosis and increasing myocardial infarct size in rats and mice. Inhibiting Cltc-mediated endocytosis with chlorpromazine reduced the pro-apoptotic effects of N-Exo in neonatal rat cardiomyocytes under anoxia/reoxygenation. A functional cluster of miRNAs (miR-9a-5p, miR-92b-3p, miR-181a-5p, miR-494-3p and miR-708-5p) from exosomes was identified and promoted cardiomyocyte apoptosis via a common gene, Rap1b. R-Exo downregulated cardiac Rap1b and Bcl2 in I/R rats. Mimics of these miRNAs reduced luciferase activity of the Rap1b gene and were blocked by site-directed mutagenesis of the Rap1b gene at miRNAs binding sites. Co-immunoprecipitation demonstrated that Rap1b protein bound to ERK1/2 and Cltc.

CONCLUSIONS AND IMPLICATIONS: Exosomes from non-ischaemic fibroblasts worsen I/R injury by promoting apoptosis of ischaemic cardiomyocytes through a cluster of miRNAs targeting the Rap1b/ERK1/2 pathway, highlighting Rap1b restoration as a potential therapeutic strategy.