Different Transcriptomic Response to T. cruzi Infection in hiPSC-Derived Cardiomyocytes From Chagas Disease Patients With and Without Chronic Cardiomyopathy

Figure 1. Results from infection assay of PBMCs by flow cytometry with GFP-expressing Y strain. (A) The infection rate over time points was analyzed through the percentage of GFP+ events in Monocytes, T and B Lymphocytes. (B) The proliferation rate over time points was analyzed through the GFP intensity across times points in Monocytes, T and B Lymphocytes. (C) The 48/24 hpi GFP intensity ratio was used to calculate the proliferation between 24 and 48 hpi in Monocytes, T and B Lymphocytes. (n = 5 CCC; 5 IND and 5 Non-infected controls). The symbol *** means P ≤ 0.001.

Figure 2. Results from hiPSC-CM infection assay with GFP-expressing Y strain. (A) hiPSC-CM were infected with T. cruzi Y strain modified to express GFP in the amastigote form. Cell nuclei were stained with DAPI (blue) (Left panel bars = 40µm). (B) Time course (3, 6, 24 and 48 hpi) analysis of infection in hiPSC-CM from IND and CCC patients. Nuclei and amastigotes were counted in a custom script and the Parasite/Nucleus ratio was compared along time points. (n = 3 CCC and 2 IND). n.s., not significant.

Figure 3. RNA-seq results. (A) PCA plot showing the variability among CCC and IND replicates at 0 and 24 hpi (CCC replicates = 6 CCC_0hpi, 6 CCC_24hpi. IND replicates = 4 IND_0hpi and 4 IND_24hpi). (B) Hierarchical clustering of replicates. (C) Volcano plot showing the L2FC distribution of DEGs in the interaction term output.

Figure 4. Results from GSEA with Hallmark terms. (A) GSEA plots showing the rank distribution of DEGs in Hallmark gene sets and the association with each phenotype (red bars – CCC; blue bars – IND). (B) Reactome plot with the NES for the pathways associated to each group. (C) Hallmark gene sets associated with each phenotype. ES, Enrichment Score; NES, Normalized enrichment score; FDR, False-discovery rate.

Figure 5. Heat maps showing the normalize expression of significant DEGs across conditions in their respective Hallmark gene set. Samples were normalized by the mean counts. Below each heat map the boxplots show the level of gene expression at every time point (0 or 24 hpi) for each group (CCC or IND).

Figure 6. HLA expression pathways in IND and CCC cardiomyocytes. (A) DEGs were plotted against KEGG antigen processing and presentation pathway and both MHC I and MHC II pathways had a positive association with IND (green – IND association; red – CCC association). (B) Heat maps showing the normalized expression of HLA Class I and Class II genes across all conditions. *DEGs with statistical significance.