The SARS-CoV-2 recombinant S protein interacts with cardiac PCs and induces functional alterations
(A) S protein interaction with PC receptors. Western blotting analysis of PCs (n=2 patients) exposed to the S protein (SPIKE) or PBS vehicle (VEH) for 1 h. The bands corresponding to the 6×-His-Tag recognise the His-tagged S protein. The purified S protein was used as a positive control. (B,C) Dose–response migration wound closure assay. A scratch was created in confluent PCs and images taken at baseline. Cells were incubated with increasing concentrations of S protein or vehicle for 24 h and final images were recorded. The surface of wound closure was calculated as % of the baseline area and expressed as fold-change vs vehicle. n=6 patients’ PCs. (D) Cell viability. Live cell imaging of PCs after 6 and 24 h incubation with the S protein (1 μg/ml – 5.8 nM). In green, Calcein-AM shows the cytoplasm of live cells. The red fluorescence of EthD-III indicates the nuclei of dead cells (not detected). Saponin treatment, used as a positive control for dead cells, shows the nuclear staining of EthD-III in the absence of Calcein-AM. Images are representative of one patient. The assay was done in n=3 patients’ PCs. (E) Cell proliferation. PCs were exposed to the S protein (1 μg/ml – 5.8 nM) or vehicle for 24 h, in the presence of EdU. Proliferation was measured as the % of EdU+ cells and data expressed as fold-change vs vehicle. Immunostaining shows EdU+ cells in green, nuclei (DAPI) in blue. n=8 patients’ PCs. (F) Matrigel assay. CAECs and cocultures of CAEC + PCs were incubated on the top of Matrigel for 5 h, in the presence of the S protein (1 μg/ml – 5.8 nM) or vehicle. PCs were labelled with the red fluorescent tracker CM-Dil to assess the interaction with ECs (small inserts). Graphs report the total tube length per imaging field, expressed as fold-change vs CAEC vehicle. n=6 patients’ PCs. All graphs report individual values and means ± SEM. **P<0.01, ****P<0.0001.