SARS-CoV-2 spike receptor-binding domain is internalized and promotes protein ISGylation in human induced pluripotent stem cell-derived cardiomyocytes

Figures

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  Figure 1. Internalization of SARS-CoV-2 S-RBD/ACE2 complex into the human iPSC-CMs. (a) Time course of monolayer differentiation into the cardiomyocytes. Differentiated cardiomyocytes were replated on day 14 after differentiation for further analysis. The iPSC-CMs were incubated with purified His-tagged SARS-CoV-2 S-RBD protein for 48 h before immunostaining and western blotting on day 28 after differentiation. (b)Whole cell lysates were extracted from the iPSCs and iPSC-CMs on day 28 after differentiation and analyzed by western blotting using the indicated antibodies. Original blots are presented in Supplementary Fig. S5. (c) The iPSC-CMs were fixed and immunostained with the indicated antibodies on day 28 after differentiation. Nuclei were detected by Hoechst staining. (d) The iPSC-CMs were incubated with 1250 ng/mL purified His-tagged SARS-CoV-2 S-RBD protein for 48 h before immunostaining on day 28 after differentiation with the indicated antibodies. White arrows show the accumulation of S-RBD at the periphery of the iPSC-CMs co-localized with ACE2. (e) The iPSC-CMs were incubated with 1250 ng/mL purified His-tagged SARS-CoV-2 S-RBD protein for 48 h before immunostaining on day 28…

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  Figure 2. Internalization of SARS-CoV-2 S-RBD-sfGFP into the iPSC-CMs through endolysosomal pathway. (a) ACE2-WT-iPSC-CMs were incubated with SARS-CoV-2 S-RBD-sfGFP for 48 h before immunostaining on day 28 after differentiation with the indicated antibodies. (b) Whole cell lysates were extracted from ACE2-WT-iPSC-CMs and ACE2-KO-iPSC-CMs on day 28 after differentiation and analyzed by western blotting using the indicated antibodies. Both iPSC-CMs were incubated with 1,200 ng/mL SARS-CoV-2 S-RBD-sfGFP for 48 h prior to western blotting. Original blots are presented in Supplementary Fig. S5. (c) Quantified S-RBD-sfGFP expression levels were normalized by GAPDH expression in ACE2-WT-iPSC-CMs and ACE2-KO-iPSC-CMs (n = 3). Data are presented as the mean ± SD. Statistical differences were calculated using the Student’s t-test. ***p < 0.001. (d) Time-lapse imaging of ACE2-WT-iPSC-CMs after S-RBD-sfGFP treatment. S-RBD-sfGFP-positive iPSC-CMs were detected at approximately 10 min after S-RBD-sfGFP treatment (white arrows). (e) Time-lapse imaging of ACE2-WT-iPSC-CMs after S-RBD-sfGFP treatment. The S-RBD-sfGFP signals disappeared completely 48 h after the removal of S-RBD-sfGFP in ACE2-WT-iPSC-CMs…

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  Figure 3. Live cell imaging of the endocytosis of SARS-CoV-2 S-RBD-sfGFP in the iPSC-CMs. (a) Time course of immunostaining and live cell imaging after replating the iPSC-CMs. Differentiated cardiomyocytes were replated on day 14 after differentiation and transduced with AAV2-DsRed-RAB5A at 1.0–2.0 × 104 vg/cell on day 21, then incubated with SARS-CoV-2 S-RBD-sfGFP for 48 h before immunostaining or 10 min before live cell imaging on day 28 after differentiation. (b) ACE2-WT-iPSC-CMs transduced with AAV2 encoding DsRed-RAB5A were incubated with SARS-CoV-2 S-RBD-sfGFP for 48 h before immunostaining on day 28 after differentiation with the indicated antibodies. (c) ACE2-WT-iPSC-CMs were incubated with SARS-CoV-2 S-RBD-sfGFP for 10 min before live-cell imaging on day 28 after differentiation and observed by confocal microscopy. The areas enclosed within the white squares are enlarged in the right panel. (d) ACE2-WT-iPSC-CMs transduced with AAV2 encoding DsRed-RAB5A were incubated with SARS-CoV-2 S-RBD-sfGFP for 10 min before live-cell imaging on day 28 after differentiation and observed by confocal microscopy. Each dot indicated by a white or yellow arrow shows the same signal of S-RBD-sfGFP…

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  Figure 4. Upregulation of pro-inflammatory cytokines after S-RBD treatment depending on ACE2. (a) Time course of quantitative real-time PCR array analysis after replating the iPSC-CMs. Differentiated cardiomyocytes were replated on day 14 after differentiation and incubated with 1,800 ng/mL S-RBD-sfGFP and control-GFP for 48 h before quantitative real-time PCR array analysis on day 28 after differentiation. (b) Heat map of the selected differentially expressed genes that were upregulated in ACE2-WT-iPSC-CMs treated with S-RBD-sfGFP compared to control- and ACE2-KO-iPSC-CMs treated with S-RBD-sfGFP. The color scale denotes relative gene expression across groups (high red and low green). (c) The mRNA expression levels of IL1A, IL1B, CXCL1, CXCL8, and TNF were normalized by GAPDH expression and validated using quantitative real-time PCR. ACE2-WT-iPSC-CMs were treated with S-RBD-sfGFP for 48 h at low (600 ng/mL) and high (1,800 ng/mL) doses. ACE2-KO-iPSC-CMs were then treated with high doses of S-RBD-sfGFP. Relative expression levels were normalized to the expression levels in each iPSC-CM treated with control-GFP (n = 4–6 independent biological replicates). Data are presented as the mean ±…

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  Figure 5. Transcriptional profiling of the iPSC-CMs subpopulations by scRNA-seq. (a) Time course of scRNA-seq after replating the iPSC-CMs. Differentiated cardiomyocytes were re-plated on day 14, after differentiation and incubated with 1800 ng/mL S-RBD-sfGFP and control-GFP for 48 h before scRNA-seq on day 28, after differentiation. (b) The iPSC-CMs treated with S-RBD-sfGFP and control-GFP (S-RBD-sfGFP = 2315 cells, GFP = 2731 cells; n = 5,046 total cells) were clustered using the k-means method (six clusters are indicated by color) and visualized using t-SNE. Circles indicate cardiomyocyte clusters (TNNT2 positive; C1, C2, C3, and C4), including the proliferative cardiomyocyte cluster (TNNT2 positive /MKI67 positive; C4), and triangles indicate differentiated non-cardiomyocyte cell clusters (i.e., all other TNNT2 negative clusters). (c) The same t-SNE as B is colored according to the transcript abundance of the cardiomyocyte marker cardiac troponin T (TNNT2). (d) Violin plots show the normalized transcript abundance of cell-type marker genes in each cluster. Boxplots are presented as medians and interquartile ranges, and whiskers represent the 5th and 95th percentiles. CM =…

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  Figure 6. Protein ISGylation in the iPSC-CMs after S-RBD treatment. (a) The mRNA expression levels of IFI6, ISG15, IFITM3, SOCS3, and USP18 were normalized by GAPDH expression and validated using quantitative real-time PCR. ACE2-WT and ACE2-KO iPSC-CMs were treated with 1800 ng/mL S-RBD-sfGFP for 48 h. Relative expression levels were normalized to the expression levels in each iPSC-CM treated with control GFP (n = 5–10 independent biological replicates). Data are presented as medians and interquartile ranges. Statistical differences were calculated using the Kruskal–Wallis test followed by Dunn’s test for multiple comparisons. *p < 0.05, **p < 0.01. (b) The mRNA expression levels of HERC5, HERC6, and EFP were normalized by GAPDH expression and validated using quantitative real-time PCR. ACE2-WT and ACE2-KO iPSC-CMs were treated with 1800 ng/mL S-RBD-sfGFP for 48 h. Relative expression levels were normalized to the expression levels in each iPSC-CM treated with control GFP (n = 5–10 independent biological replicates). Data are presented as medians and interquartile ranges. Statistical differences were calculated using the Kruskal–Wallis test followed by Dunn’s test for multiple comparisons.…

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  Figure 7. Graphical summary of the main results. S-RBD was internalized into the human iPSC-CMs via ACE2 by the endolysosomal pathway. The internalized S-RBD induced an innate immune response, upregulated IFN-responsive genes (IFI6, ISG15, and IFITM3) in mature cardiomyocytes, and promoted protein ISGylation in the iPSC-CMs.