SARS-CoV-2 infects Human Cardiomyocytes via Vesicular Transport mediated by Inflammation and Oxidative stress

M. Tangos (Bochum)¹, H. Budde (Bochum)², M. Sieme (Bochum)¹, V. Nageswaran (Bochum)³, P. Ramezani Rad (Berlin)⁴, D. Kolijn (Bochum)⁵, S. Zhazykbayeva (Bochum)⁶, M. Lodi (Bochum)⁷, S.-R. Sadat-Ebrahimi (Bochum)³, M. Herwig (Bochum)¹, O. Overheu (Bochum)⁸, A. Reinacher-Schick (Bochum)⁸, C. Torres-Reyes (Bochum)⁹, M. Varatnitskaya (Bochum)¹⁰, M. Kacmaz (Bochum)¹, H. Osman (Bochum)¹¹, E. L. Robinson (Aurora)¹², K. Jaquet (Bochum)², I. Akin (Mannheim)¹³, J. Mustroph (Regensburg)¹⁴, K. Evert (Regensburg)¹⁵, N. Babel (Herne)¹⁶, D. Lindner (Freiburg im Breisgau)¹⁷, D. Westermann (Freiburg im Breisgau)¹⁸, H. G. Mannherz (Bochum)¹⁹, L. Leichert (Bochum)²⁰, A. Haghikia (Bochum)³, I. El-Battrawy (Bochum)³, S. T. Sossalla (Gießen)²¹, N. Hamdani (Bochum)^22
1Institut für Physiologie, Institut für Forschung und Lehre (IFL), Ruhr-Universität Bochum Abteilung für Zelluläre und Translationale Physiologie, Molekulare und Experimentelle Kardiologie Bochum, Deutschland; ²Abteilung für Zelluläre und Translationale Physiologie, Molekulare und Experimentelle Kardiologie,, Institut für Physiologie, Institut für Forschung und Lehre (IFL), Ruhr-Universität Bochum Bochum, Deutschland; ³Klinikum der Ruhr-Universität Bochum Medizinische Klinik II, Kardiologie Bochum, Deutschland; ⁴Deutsches Herzzentrum der Charite (DHZC) Klinik für Kardiologie, Angiologie und Intensivmedizin Berlin, Deutschland; ⁵Ruhr-Universität Bochum Institut für Physiologie, Abt. für Systemphysiologie - MA2/148 Bochum, Deutschland; ⁶Ruhr University Bochum Department of Cellular and Translational Physiology Bochum, Deutschland; ⁷Ruhr-Universität Bochum Abteilung für Systemphysiologie Bochum, Deutschland; ⁸Katholisches Klinikum Bochum, St. Josef-Hospital Klinik für Hämatologie und Onkologie mit Palliativmedizin Bochum, Deutschland; ⁹Katholisches Klinikum Bochum, St. Josef-Hospital Innere Medizin Bochum, Deutschland; ¹⁰Ruhr-Universität Bochum Zelluläre und Translationale Physiologie Bochum, Deutschland; ¹¹Kath. Klinikum Bochum Institut für Forschung und Lehre (IFL), St. Josef Hospital Bochum, Deutschland; ¹²University of Colorado Anschutz Medical Campus School of Medicine, Division of Cardiology Aurora, Deutschland; ¹³Universitätsklinikum Mannheim GmbH I. Medizinische Klinik Mannheim, Deutschland; ¹⁴Universitätsklinikum Regensburg Klinik und Poliklinik für Innere Med. II, Kardiologie Regensburg, Deutschland; ¹⁵Universität Regensburg Institut für Pathologie Regensburg, Deutschland; ¹⁶Marienhospital Herne Zentrum für Translationale Medizin mit S. Immunologie und Transplantation Herne, Deutschland; ¹⁷Universitäts-Herzzentrum Freiburg - Bad Krozingen Klinik für Kardiologie und Angiologie Freiburg im Breisgau, Deutschland; ¹⁸Universitäts-Herzzentrum Freiburg - Bad Krozingen Innere Medizin III, Kardiologie und Angiologie Freiburg im Breisgau, Deutschland; ¹⁹Ruhr-Universität Bochum Anatomie und Molekulare Embryologie Bochum, Deutschland; ²⁰Ruhr-Universität Bochum Mikrobielle Biochemie Bochum, Deutschland; ²¹Universitätsklinikum Gießen und Marburg GmbH Medizinische Klinik I - Kardiologie und Angiologie Gießen, Deutschland; ²²Kath. Klinikum Bochum Cellular Physiology Bochum, Deutschland

Background and aim: SARS-CoV-2 infection is associated with increased cardiovascular morbidity and mortality, including myocarditis. However, the mechanisms how SARS-CoV-2 enters and infects cardiomyocytes and endothelial cells remains unclear. Here, we explore the role of extracellular vesicles (EV) as mediators of communication between cardiomyocytes and endothelial cells during infection. EVs are lipid-bound vesicles released from a variety of cell types and can be found in all biological fluids. They are known for their pivotal role in transporting proteins, RNA, DNA, lipids, and metabolites between cells and organs.

Methods: We performed imaging of cardiac tissue, EVs, and serum derived from SARS-CoV-2 patients via confocal microscopy and transmission electron microscopy (TEM). Furthermore, cardiac tissue was used for a mass spectrometry protocol, specifically developed to detect oxidized cysteines in proteins (OxICAT labeling). The Gene Ontology (GO) analysis was performed to assess oxidative stress-affected pathways and cellular components. To elucidate the effects of SARS-CoV-2 patient-derived EVs on mitochondrial function, inflammatory, apoptotic, and glycolytic events, we conducted endothelial cell culture experiments. For this purpose, serum-derived EVs were isolated via polyethylene glycol precipitation and subsequent size exclusion chromatography (SEC).

Results: Our results verify the close proximity/interaction of EVs and SARS-CoV-2 components in cardiac tissue, as well as serum of infected patients. Mass spectrometry revealed a high burden of oxidative stress inside cardiac tissue, reflected by highly oxidized cysteines in various proteins involved in exosomal traffic and mitochondrial function. In addition, patient-derived EVs were able to trigger inflammatory events and reduce energy metabolism in endothelial cells.

Conclusion: Taken together, our data suggest that SARS-CoV-2 infects cardiomyocytes and endothelial cells. Extracellular vesicles (EVs) are a prominent alternative route of virus dissemination. Our results further hint at the potential of EVs to affect macroenvironment causing alterations of protein functionality and expression, enzyme activity, inflammation, and oxidative stress, ultimately causing an impairment of mitochondrial function. We propose that EVs are an important mediator of cellular stress signals and viral particles during infection, suggesting their potential to trigger severe cardiomyocyte damage and subsequent cell death.