Comparison of HFpEF fibrotic signatures

Collaborators: Laura Wienecke and Florian Leuschner.

Background: Inflammation, fibrosis and metabolic stress critically promote heart failure with preserved ejection fraction (HFpEF). Exposure to high-fat diet and nitric oxide synthase inhibitor N[w]-nitro-l-arginine methyl ester (L-NAME) recapitulate features of HFpEF in mice. To identify disease specific traits during adverse remodeling, we performed single-cell RNAseq (scRNAseq) of interstitial cells in murine HFpEF. Diastolic dysfunction and fibrosis were accompanied by an activation of cardiac fibroblast and macrophage subsets. Comparison with scRNAseq datasets of murine heart failure with reduced ejection fraction (HFrEF) identified a specific HFpEF fibroblast disease signature, characterized by e.g. overexpression of basement membrane genes. While myo- and matrifibroblast activation is known to be crucial for cardiac fibrosis in HFrEF, we found this cell-state switch to be less important in HFpEF. Disease specific fibroblast signatures were corroborated in human myocardial bulk transcriptomes and main markers confirmed at protein level in murine samples. Lipoproteinlipase Angiopoietin-like 4 was identified to indicate HFpEF mediated fibroblast activation and may serve as a potential biomarker for disease progression. Taken together, our results provide a characterization of the interstitial cellular landscape of murine HFpEF, including specific characteristics of fibroblast activation.

Methods: Whole single cell RNA sequencing with 10x genomics and Illumina

Analysis Workflow

Processing, QC and integration

1. sample wise QC run_samplewise_processing.R
2. first sample integration harmony_integration_across_sample.R
3. Cluster marker identification and manual labeling annotate_celltypes.R
4. filtering of clusters and reintegration round1 round2
5. calculate distance between pseudobulk profiles sample_distance.R
6. calculate cell proportion changes with label permutation differntial_cell_proportions.R

Cellstate definitions and Differntial gene expression

1. harmony_integration_per_celltype.R
2. annotate_cellstates.R
3. fibroblast_state_interpretation.R
4. HfpEF DEA per celltype with downsampling DEA_per_celltype.R

Functional analysis

1. Enrichment of Gene Ontology terms GO_enrich.R
2. pathway analysis and cytokine footprinting run_cytosig_progeny.R
3. TF activity estimation run_dorothea.R
4. Ligand Receptor analysis run_liana.R
5. Ligand-network analysis run_nichenet.R

Fibroblast integration with different studies

1. Process_study_MI.R
2. Process_study_AngII.R
3. Harmony based integration of three studies with cells filtered integration_fibroblasts.R + integration_fibroblasts_filtered.R
4. fibroblast_state_interpretation_integrated.R
5. composition_comparison.R

Fibroblast signature comparisons

1. Fibroblas Marker gene detection in each study DEA_fibs_all_studies.R 2 GSEA, progeny, dorothea on signatures fib_signature_interpretation.R
2. compare overlap and correlation between signatures compare_study_degs.R
3. Fibroblast marker are mapped back to each study map_study_DEG_to_integrated_cluster.R
4. relate statemarker and signatures compare_statemarker_and_deg.R

Human bulk comparison

1. Process Human HFpEF bulk_human_process.R
2. compare bulk signatures

A workflowr project.