PhD Scientific Days 2022

Molecular Sciences V.

Investigation of extracellular vesicles derived from cardiac cell lines under normoxia and hypoxia

Text of the abstract

Introduction: Myocardial dysfunctions are one of the leading cause of death globally. Extracellular vesicles (EVs), released by cells may help in early diagnostics and therapy. Cardiac cell lines (such as H9c2, AC16 and HL1) are widely used in cardiovascular EV research as in vitro models. Increasing number of studies are using these cell lines, although they have significant limitations. We have decided to perform comparative characterization of EVs released by differentiated H9c2, AC16 and HL1 cells. The aim of the recent project was to compare cardiomyocyte derived EVs under normal and hypoxic conditions.
Methods: Cell lines including the AC16 (human), HL1 (mouse) and H9c2 (rat) cardiomyoblast cells were used in this study. Following differentiation protocols, small- medium- and large-sized EVs (sEV, mEV, lEVs) were collected from conditioned medium of normal and hypoxia treated cells. EVs were separated by a combination of gravity filtration, differential centrifugation and tangential flow filtration (TFF). EVs were characterized according to MISEV 2018 by MicroBCA, SPV-Lipid assay, Nanoparticle Tracking Analysis (NTA) and transmission electron microscopy (TEM). The protein profile of sEV and mEV were determined by mass spectrometry. Some of the proteomic hits were validated by Western blotting, flow cytometry and immunogold TEM. EV release was examined by fluorescent live-cell imaging using GFP-fusion protein transfected cells.
Results: One of our most relevant proteomic hits was endoplasmin (ENPL, grp94, gp96). It was specifically present in EVs released by hypoxia-treated cells. Even though ENPL is an endoplasmic reticulum resident chaperon, its presence in EVs were confirmed by Western blotting, flow cytometry and Immunogold TEM. Secretion of ENPL containing EVs was analyzed by confocal microscopy using fixed and living HL1 cells.
Conclusions: We provide evidence that ENPL-containing EVs are released by cardiac cell lines. We suggest that ENPL may take part in cardio-protection during hypoxia through reducing ER stress.
Funding: This work was funded by the EFOP-3.6.3-VEKOP-16-2017-00009 grant, NVKP_16-1-2016-0004 grant of the Hungarian National Research, Development and Innovation Office (NKFIH), Semmelweis Innovation Fund (STIA 2020 KFI), Hungarian Scientific Research Fund (OTKA K120237), VEKOP-2.3.2-16-2016-00002, VEKOP-2.3.3-15-2017-00016,