MicroRNA-21 (miR-21) is one of the most abundant miRNAs in the cardiovascular system and strongly upregulated in failing myocardium. Despite multiple studies that indicate therapeutic efficacy of antimiRs directed against miR-21, its targetome in the myocardium is largely unknown. We have recently developed an approach to identify high affinity and highly regulated microRNA targets using Argonaute2-ribonucleoprotein immunoprecipitation in the presence of an antimiR directed against the microRNA of interest followed by next generation RNA sequencing (Werfel et al. Nucl Acids Res 2017). Using this approach, we aimed to identify the mRNA targetome regulated by miR-21 in defined cardiac cell fractions in vivo.
Mice were subjected to pressure overload of the left ventricle by transverse aortic constriction and antimiR-21, a synthetic inhibitor of miR-21, was administered intravenously two weeks after surgery for three consecutive days. Two days later, an Argonaute2-ribonucleoprotein immunoprecipitation from the heart was performed. RNA-sequencing of the input and AGO2-immunoprecipitated mRNA, followed by bioinformatic analysis identified the miR-21 targets related to heart failure by means of their de-enrichment in the Ago-bound transcriptome (RIP fold change) and their de-repression in the total transcriptome. We performed analogous experiments in defined isolated primary cells and cell lines (myocytes, fibroblasts, macrophages). The Ago2-RIP fold changes showed highly significant differences for targets versus non-targets in each cell fraction (p < 0.05) after antimiR-21 treatment. Finally, we used cell deconvolution methodology utilizing the identified miR-21 targets in the different cell types compared to the identified targets of the whole heart to determine the myocardial cell fractions where miR-21 exerts its pathological effect.

Taken together, this approach has allowed us to delineate the targetome of miR-21 in defined cardiac cell fractions in vivo. These data aid our understanding of miR-21 in the mammalian heart and the mechanisms underlying the therapeutic efficacy of antimiR-21 in cardiac failure. Our approach should be applicable to the analysis of the targetome of any microRNA in complex mammalian tissues.