Extracellular vesicles (EVs) mediate intercellular signaling by transferring their cargo to recipient cells, but the functional consequences of signaling are not fully appreciated. Red blood cell (RBC)-derived EVs are abundant in circulation and have been implicated in regulating immune responses. Here, we use a transgenic mouse model for fluorescence-based mapping of RBC-EV recipient cells to assess the role of this intercellular signaling mechanism in heart disease. Using fluorescent-based mapping, we detected an increase in RBC-EV-targeted cardiomyocytes in a murine model of ischemic heart failure. Single cell nuclear RNA sequencing of the heart revealed a complex landscape of cardiac cells targeted by RBC-EVs, with enrichment of genes implicated in cell proliferation and stress signaling pathways compared to non-targeted cells. Correspondingly, cardiomyocytes targeted by RBC-EVs more frequently express cellular markers of DNA synthesis, suggesting the functional significance of EV-mediated signaling. In conclusion, our mouse model for mapping of EV-recipient cells reveals a complex cellular network of RBC-EV mediated intercellular communication in ischemic heart failure and suggests a functional role for this mode of intercellular signaling.

Previous studies have demonstrated the importance of extracellular vesicle (EV) cargo RNAs, notably miRNAs in modulating recipient cell function. We therefore sought to determine the small RNA content of RBC-EVs and their possible regulation of pathways highlighted by the snRNA seq described above. Human red blood cell (RBC)-EVs were isolated using immunoaffinity-bead-based flow sorting with a validated antibody against Glycophorin-A (CD235a). We explored RBC-EV content in a clinically well-characterized biorepository of human plasma from patients with myocardial infarction (MI, n=5) and control patients referred for supraventricular tachycardia (SVT, n=5). After confirming the specificity of the Glycophorin-A positive EV isolation method using flow cytometry, microscopy, and digital PCR for RBC-enriched RNAs, we performed small RNA-seq to analyze RNA content in Glycophorin-A (CD235a)-immunopositive EVs from the plasma of human subjects. After analyzing pooled RNA-seq data for samples that passed minimal QC, we identified a total of 105 distinct miRNAs in the plasma derived RBC-EVs.