Single-cell multiomics guided mechanistic understanding of Fontan-associated liver disease

Abstract

The Fontan operation is the current standard of care for single-ventricle congenital heart disease. Individuals with a Fontan circulation (FC) exhibit central venous hypertension and face life-threatening complications of hepatic fibrosis, known as Fontan-associated liver disease (FALD). The fundamental biology and mechanisms of FALD are little understood. Here, we generated a transcriptomic and epigenomic atlas of human FALD at single-cell resolution using multiomic snRNA-ATAC-seq. We found profound cell type-specific transcriptomic and epigenomic changes in FC livers. Central hepatocytes (cHep) exhibited the most substantial changes, featuring profound metabolic reprogramming. These cHep changes preceded substantial activation of hepatic stellate cells and liver fibrosis, suggesting cHep as a potential first "responder" in the pathogenesis of FALD. We also identified a network of ligand-receptor pairs that transmit signals from cHep to hepatic stellate cells, which may promote their activation and liver fibrosis. We further experimentally demonstrated that activins A and B promote fibrotic activation in vitro and identified mechanisms of activin A's transcriptional activation in FALD. Together, our single-cell transcriptomic and epigenomic atlas revealed mechanistic insights into the pathogenesis of FALD and may aid identification of potential therapeutic targets.

Fig. 1.. The single-cell transcriptomic atlas of human FALD.

(A and B) UMAP of the snRNA-ATAC-seq transcriptome data of the control (A, n=2) and FC (B, n=4) livers. The total numbers of high-quality nuclei containing both the transcriptome and epigenome data are labeled, as are transcriptome-based cellular identities. (C and D) Violin plots (C) and feature plots (D) illustrating cluster-specific marker gene expression of each cell type in the FC livers (log₂[fold change]>0.25 and percentage of positive cells>25%).

Fig. 2.. Cell type-specific liver transcriptional remodeling in human FALD.

(A) Volcano plots showing differentially expressed genes (DEGs) in correlated cell types of control (n= 2) and FC (n= 4) livers. Numbers of upregulated (blue) and downregulated (red) genes in the FC livers are labeled. (B) Enriched pathways of genes significantly upregulated in central hepatocytes (cHeps) and hepatic stellate cells (HSCs) of FC livers by gene ontology (GO) analysis. (C) Violin plots showing representative genes significantly (P<1x10⁻⁵ and log₂[fold change]>0.58) upregulated in cHeps and HSCs of FC livers. (D) Immunohistochemistry of Catalase using control and FC liver sections.

Fig. 3.. The single-cell liver epigenomic atlas of human FALD.

(A and B) UMAP of the snRNA-ATAC-seq epigenome data of the control (A, n=2) and FC (B, n=4) livers. Cell identities based on their epigenomes and linked transcriptomes are labeled. (C) Aggregated ATAC signal (peaks) of representative cell type-specific genes in each cell type of FC livers. The gene structures are shown at the bottom, all with 5’ on the left.

Fig. 4.. Cell type-specific liver epigenetic remodeling in human FALD.

(A) Volcano plots showing differential ATAC peaks in correlated cell types of control (n=2) and FC (n=4) livers. Numbers of upregulated (blue) and downregulated (red) ATAC peaks in the FC livers are labeled. (B) Enriched pathways of genes linked to ATAC peaks more accessible in FC cHeps by gene ontology (GO) analysis. (C) Single-cell level joint gene expression and chromatin accessibility analysis uncovering chromatin regions important for ADH4 gene expression. The higher the score, the stronger correlation between each linked chromatin region and ADH4 expression. (D) Enriched DNA sequence motifs (P<6.3x10⁻²⁰ and log₂[fold enrichment]>1.35) and associated transcription factors of chromatin regions linked to increased metabolic gene expression in cHeps.

Fig. 5.. cHep to HSC signaling promotes HSC activation and liver fibrosis in FALD.

(A) Ligand-receptor pairs in FALD revealed by snRNA-ATAC-seq data. The bigger the circle and the thicker the line, the more ligand-receptor pairs included. (B) Violin plots showing gene expression of ligands INHBA, INHBB, INHBC and receptors ACVR1B, ACVR2A, and ACVR2B in different cell types of control (n=2) and FC (n=4) livers. (C) ACTA2 and COL4A1 expression by qPCR in MRC-5 cells treated with PBS (as control), 10ng/ml TGFβ, and 10, 30, and 100 ng/ml of Activin A, B, or C for 72 hours. n=3 biological replicates for each treatment. Error bars are standard error of the mean. *p<0.05 vs control. (D) ACTA2 and βACTIN (as loading control) protein abundance in MRC-5 cells treated with PBS, 10ng/ml TGFβ, or 100 ng/ml of Activin A, B or C for 72 hours. (E) Alignment of aggregated FALD snATAC-seq (this study), published NRF1 ChIP-seq (21), and H3K27Ac ChIP-seq (22) signals showing that an accessible region in the promoter of human INHBA gene is bound by NRF1 and H3K27 in this region is acetylated. (F) NRF1 and ZNF148 but not PPARα activate INHBA promoter in a luciferase reporter assay (n=3 biological replicates). Error bars are standard error of the mean. **p<0.01 vs control.

Fig. 6.. Cell type-specific changes between FALD and MASH.

Venn diagrams showing unique and overlapping upregulated genes in MASH (n=9) (27) and FALD (n=4), in central hepatocytes (cHeps) and peripheral hepatocytes (pHeps) (A), HSCs (B), lymphocytes, ECs, and cholangiocytes (C). Number of genes are underlined. Enriched pathways (P<1x10⁻⁵ and log₂[fold change]>0.58) of unique and overlapping genes based on GO analysis are presented. EC and cholangiocyte overlapping genes showed no enriched pathways.