The respiratory illness triggered by severe acute respiratory syndrome virus-2 (SARS-CoV-2) is often particularly serious or fatal amongst patients with pre-existing heart conditions. Previously, we reported new insights into the mechanisms of SARS-CoV-2 entry into the heart and defined promising targets for antiviral interventions for COVID-19 patients with pre-existing heart conditions or patients with co-morbidities. Although the mechanisms underlying SARS-CoV-2 is CoV-2-related cardiac damage guided by inflammation and oxidative stress, the entry pathways are still debated. Therefore, in this manuscript we sought to determine 1) which heart cells are targeted by SARS-CoV-2, 2) the mechanisms of virus entry into the heart, and 3) how inflammation and oxidative stress promote the viral entry into endothelial cells.

Methods and results: We used serum from SARS-CoV-2 infected patients and non-infected subjects, also tissues from left ventricular (LV) patients that died as a result of pneumonia/acute respiratory distress syndrome or multi-organ failure. Based on confocal microscopy and transmission electron microscopy (TEM), we saw that SARS-CoV-2 reaches the heart via EVs and exosomes. In addition, we found an oxidized microenvironment as a result of SARS-CoV-2 infection as seen from heavily oxidized proteins in endothelial cells and cardiomyocytes and in particular mitochondrial proteins using confocal microscopy and OxICAT method coupled with mass spectrometry (MS) that allows the precise quantification of oxidative thiol modifications in hundreds of different proteins in a single experiment. The OxICAT revealed also highly oxidized cysteins in various proteins involved in exosomal traffic. In addition, we found a close proximity/interaction (less than 40nm) of vesicles and SARS-CoV-2 components in serum and cardiac tissue of severely infected patients. As a result, oxidized microenvironment causes alterations of protein localization and expression, enzyme activity, inflammation, oxidative stress, and oxidized vesicles. Subsequently, we wanted to measure the consequences of the oxidized microenvironment and oxidized exosomes on endothelial and mitochondrial function. Endothelial cell culture experiments were conducted to study the effects of SARS-CoV-2 patient-derived exosomes on mitochondrial function, inflammatory, apoptotic, and glycolytic events. Our results showed increased inflammasomes upon treatment of endothelial cells with SARS-CoV-2 patient-derived exosomes supporting the "cytokines storm" hypothesis and this was also accompanied by mitochondrial dysfunction as seen from reduced basal respiration and ATP production.

Conclusion: Our study provides new insights into the mechanisms of SARS-CoV-2 entry into the heart and defines promising targets for antiviral interventions for COVID-19 patients with pre-existing heart conditions or patients with co-morbidities.