Characterization of cardiac extracellular vesicles in heart failure

Abstract

Heart failure (HF) is a primary cause of death worldwide. Three miRNAs, miRNA 21-5p, 23a-3p, and 222-3p, were significantly upregulated in the heart and plasma of HF-rats compared to sham-rats. Extracellular vesicles (EVs) are prominent carriers of diagnostically relevant molecules, such as regulatory proteins and miRNAs. EVs can be categorized as large or small based on their biogenesis. We investigated small EVs in the context of HF due to their prominent role in transporting extracellular miRNAs. Our aim was to develop an efficient and reproducible EV isolation technique and to identify specific markers of cardiac EVs in order to investigate the role of these EVs in transporting miRNAs involved in HF. Four EV isolation techniques were compared to find the most optimal approach to isolate vesicles from rat cardiac tissue. EVTRAP (EV total recovery and purification) followed by LC-MS were performed on rat heart and plasma samples to identify protein markers that are enriched in cardiac EVs. RT-qPCR was used to detect the presence of the three miRNAs in cardiac-derived small EVs. Differential ultracentrifugation (UC) was the most efficient technique for cardiac EV isolation. Surprisingly, classical small EV markers were not detected in cardiac small EVs. LC-MS of cardiac and plasma total EVs isolated by EVTRAP revealed two potential markers enriched in cardiac EVs: Alpha B-Crystallin (CRYAB) and LIM domain biding 3 (LDB3). These proteins were confirmed to be cardiac-specific by comparing their expression in different tissue types by western blot, and their presence was confirmed in cardiac small EVs isolated using UC. Finally, RT-qPCR revealed that the three miRNAs involved in HF are indeed loaded in cardiac-derived small EVs. We found that UC is the most efficient technique for isolating EVs from cardiac tissue, and identified CRYAB and LDB3 as cardiac EV markers. Moreover, we showed that cardiac-derived small EVs transport the three miRNAs known to be increased in the heart and plasma of HF-rats. Going forward, we will compare the quantity of the three miRNAs in cardiac small EVs from HF- and sham-rats to confirm their transport into the circulation via EVs during HF.