Transcriptional Effects of Candidate COVID-19 Treatments on Cardiac Myocytes

Figures

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  FIGURE 1. Differences in the response of cardiac myocytes to drug treatments assessed by RNA-seq. (A) Various concentrations of each drug were added to hiPSC-CMs for 24 h, and cell viability was determined using the MTT assay. Absorbance at 570 nm was measured using a plate reader. All the cell viability assays were performed at least three times. L/R/I: 25 μm L/R + 0.17, 1.7, or 17U interferon. Two-group comparisons were performed using Student’s two-tailed t-test. Data are represented as mean with all error bars indicating ± s.e.m. ***P ≤ 0.001 compared to DMSO control. (B) Principal component analysis of RNA-seq data from hiPSC-CMs treated with the five drugs and control samples. CQ (blue): chloroquine; HCQ (light purple): hydroxychloroquine; LR (dark blue): lopinavir/ritonavir; LRI (pink): lopinavir/ritonavir + interferon-β; Rem (yellow): remdesivir. (C) Absolute numbers of differentially expressed genes downregulated (green: Log2 fold change ≤ 0.5) and upregulated (red: Log2 fold change ≥ 0.5) after treatments compared to control. (D) Shared differentially expressed genes between LR, LRI, and Rem. (E,F) Differential expression of genes shown in volcano plots in panel (E), LR vs.…

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  FIGURE 2. Gene Ontology (GO) functional enrichments for candidate COVID-19 treatments. (A) CQ vs. control. (B) HCQ vs. control. (C) LR vs. control. (D) LRI vs. control. (E) Rem vs. control. FDR ≤ 0.05. CQ: chloroquine; HCQ: hydroxychloroquine; LR: lopinavir/ritonavir; LRI: lopinavir/ritonavir + interferon-β; Rem: remdesivir.

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  FIGURE 3. Global map of Gene Ontology (GO) functional enrichments after LR, LRI, and Rem treatment. Heatmap based on TPM-normalized RNA-seq data (orange: high expression, purple: low expression) for differentially expressed genes between control and LR, LRI, or Rem (FDR ≤ 0.05, | Log2 fold change ≥ 0.5|). Below, black lines indicate genes associated with the respective GO category. TPM: Transcripts Per Kilobase Million. LR: lopinavir/ritonavir; LRI: lopinavir/ritonavir + interferon-β; Rem: remdesivir.

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  FIGURE 4. Expression of ER stress associated genes with LR, LRI, and Rem treatment. (A) Clustered heatmap of a panel of selected ER stress associated genes showing the calculated Log2 fold change after LR, LRI, and Rem treatment compared to control (FDR ≤ 0.05, | Log2 fold change| ≥ 0.5). LR: lopinavir/ritonavir; LRI: lopinavir/ritonavir + interferon-β; Rem: remdesivir. (B) Gene expression was determined by qRT-PCR using RNA from hiPSC-CMs plated at 1 × 106 cells/well on 6-well culture dishes and treated for 24 h with LR, LRI, Rem, or vehicle control (DMSO). (C) Mapping of differential gene expression data onto a representation of the ER stress response pathway. Each box contains a color-coded representation of the Log2 fold change of the gene in the following sample order: CQ, HCQ, LR, LRI, Rem. CQ: chloroquine; HCQ: hydroxychloroquine. Two-group comparisons were performed using Student’s two-tailed t-test. Data are represented as mean with all error bars indicating ± s.e.m. **P ≤ 0.01 and ***P ≤ 0.001 compared to DMSO control.

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  FIGURE 5. Calcium handling-associated genes and genes associated with sarcomere organization with LR, LRI, and Rem treatment. (A) Clustered heatmap of a panel of 100 selected genes associated with calcium handling and sarcomere organization showing the calculated Log2 fold change after LR, LRI, and Rem treatment compared to control (FDR ≤ 0.05, |Log2 fold change| ≥ 0.5). LR: lopinavir/ritonavir; LRI: lopinavir/ritonavir + interferon-β; Rem: remdesivir. (B) Expression of myosin light and heavy chains was determined by qRT-PCR using total RNA from hiPSC-CMs plated at 1 × 106 cells/well on 6-well culture dishes and treated for 24 h with LR, LRI, or Rem. (C) Expression of selected troponin, tropomyosin, and actin genes was determined by qRT-PCR using total RNA from hiPSC-CMs plated at 1 × 106 cells/well on 6-well culture dishes and treated for 24 h with LR, LRI, or Rem. Two-group comparisons were performed using Student’s two-tailed t-test. Data are represented as mean with all error bars indicating ± s.e.m. *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001 compared to DMSO control.