Single-Cell RNA Sequencing and Quantitative Proteomics Analysis Elucidate Marker Genes and Molecular Mechanisms in Hypoplastic Left Heart Patients With Heart Failure

Figures

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  FIGURE 1. The workflow of this study.

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  FIGURE 2. Characterization of cell cluster compositions in HLH and normal cardiomyocytes derived from iPSCs. (A) Identification of highly variable genes between different cardiomyocytes. Red dots indicate highly variable genes, and black dots indicate non-variable genes. (B) The t-SNE plot of cardiomyocyte populations. (C) Visualization of the top 20 marker genes for different cell clusters. (D–F) Pseudotime analysis results. Different colors express different states of cell clusters. The shade of the color indicates the sorting of the cells according to the pseudotime value. Each dot represents a cell, and cells with similar states are clustered together. Each branch point represents a decision point of a possible biological process.

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  FIGURE 3. Functional enrichment analysis of marker genes in the pseudotime clusters. GO and KEGG annotation enrichment analyses of marker genes in pseudotime cluster 1 (A,B), pseudotime cluster 2 (C,D), and pseudotime cluster 3 (E,F). GO includes biological processes (circle), cellular components (triangle), and molecular functions (rectangle).

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  FIGURE 4. Data-independent acquisition quantitative proteomics analysis of HLH-NHF, HLH-HF, and control plasma samples. (A) Correlation analysis of plasma proteins between the three groups of HLH-NHF, HLH-HF, and control samples. (B) The top 10 GO term enrichment analysis results of DEproteins in the three groups of HLH-NHF vs. control, HLH-HF vs. control, HLH-NHF vs. HLH-HF. (C) A heat map visualizing the top 20 DEproteins in the three groups. (D) The top 15 KEGG map enrichment analysis results of DEproteins in the three groups. (E) The COG term enrichment analysis results of DEproteins in the three groups.

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  FIGURE 5. Validation of hub genes from scRNA-seq and proteomics for HLH. (A) A PPI network for DEproteins between HLH-NHF and the control. (B) A PPI network for DEproteins between HLH-HF and the control. (C) A PPI network for DEproteins between HLH-NHF and HLH-HF. Each node represents a DEprotein, and the lines between nodes represent interactions between DEproteins. The darker the color, the greater the fold change of a DEprotein. (D) Venn diagram visualizing three hub genes (MMP2, COL5A1, and B2M) by the intersection of pseudotime markers and DEproteins in HLH-NHF vs. control, HLH-HF vs. control, and HLH-NHF vs. HLH-HF. (E) Validation of the hub genes in the four groups of control LV, HLH-LV, control RV, and HLH-RV from the GSE77798 dataset. (F) Results of color Doppler echocardiography among the left ventricular ejection fraction (EF) and short rate of left ventricle short axis (FS). (G) The results of color Doppler echocardiography corresponding to the left ventricular size and main pulmonary artery diameter. (H) RT-qPCR validation of the hub genes in the control, HLH-NHF, and HLH-HF groups. (I) Western blot validation of the hub genes in the control, HLH-NHF, and HLH-HF groups. #p <…