1Universitätsklinikum Frankfurt Med. Klinik III - Kardiologie, Angiologie Frankfurt am Main, Deutschland; 2Goethe Universität Frankfurt am Main Institute of Cardiovascular Regeneration Frankfurt am Main, Deutschland; 3Goethe Universität Frankfurt am Main Institute of Cardiovascular Regeneration and Department of Cardiology Frankfurt am Main, Deutschland; 4Universitätsklinikum Frankfurt Kardiovaskuläre Bildgebung Frankfurt am Main, Deutschland; 5Goethe Universität Frankfurt am Main Zentrum für Molekulare Medizin, Institut für Kardiovaskuläre Regeneration Frankfurt am Main, Deutschland
Background:
The prevalence of heart failure with preserved ejection fraction (HFpEF) is rising worldwide and accounts for half of all heart failure cases. In contrast to heart failure with reduced ejection fraction (HFrEF), approved medical therapies for HFpEF are scarce, and there is an unmet clinical need for novel treatment options. Maladaptive cardiac fibrosis is central to the pathology of HFpEF. The contribution of myeloid cells to the development of HFpEF in humans has not been studied in detail so far.
Methods:
Single-nuclei RNA sequencing (snRNA-seq) of cardiac biopsies from patients with HFpEF was performed and merged with a publicly available dataset of healthy hearts. Single-nuclei transcriptomes were analyzed by R. For gene ontology analysis, Metascape was used. Ligand-receptor analysis was conducted by the CellChat package for R. For in-vitro studies self-assembling human iPSC-derived cardiac organoids were used. HFpEF in human iPSC-derived cardiac organoids was induced using a high-fat (200µM Palmitate) and L-NAME (2.1 mM)-supplemented medium. Genes of interest were analyzed by qPCR.
Results:
We performed single-nuclei RNA sequencing of cardiac tissue from patients with HFpEF and the resulting data were merged with a publicly available dataset of healthy hearts (69918 nuclei in total). Macrophages were annotated using published myeloid cell markers. Differentially expressed gene analysis of resident macrophages revealed a significant upregulation of a total of 1638 genes in HFpEF (cut-off: adjusted p-value<0,05). Gene ontology analysis showed upregulation of “signal transduction by growth factors”, “PDGFRB pathway”, VEGFA-VEGFR2” and “adaptive immune system” as key regulated pathways. Among the upregulated genes, proinflammatory receptors like Toll-like receptors and Interleukin receptors (IL6R, IL10RA/B) were present. In addition, we found an upregulation of the growth factors PDGFB, PDGFC, IGF1 and FGF13. For maladaptive fibrosis, cell-cell communication between various cell types in the heart, including immune cells and fibroblasts, is mandatory. Therefore, we performed in-silico ligand-receptor analysis. The top predicted ligand-receptor pairs between macrophages and fibroblasts were macrophage-derived PDGFs and their corresponding receptors on fibroblasts. To gain further insight into the role of macrophage-derived PDGFs in cardiac fibrosis in HFpEF, we treated self-assembling human iPSC-derived cardiac organoids with recombinant PDGF-B and C. Both induced significant upregulation of collagens (COL1A1, COL3A1, COL4A1) and the myofibroblasts markers (ACTA2) (p-value for all genes p<0,05). To validate our findings from the snRNA-seq of human HFpEF, we induced HFpEF in iPSC-derived cardiac organoids using a high-fat and L-NAME supplemented medium. HFpEF organoids showed overexpression of PDGFB (p<0.05) and a trend to more expression of PDGFC. In a drug-repurposing approach, we used Nintedanib, a clinically approved inhibitor of the critical pathways derived from our single-cell sequencing (PDGF, FGF and VEGF). Treating HFpEF organoids with Nintedanib significantly reduced markers of hypertrophy, fibrosis and inflammation.
Conclusions:
Our data, for the first time, dissect human HFpEF on a single cell level. We identified the PDGF-PDGFR axis as a central mediator of macrophage-fibroblast interactions in HFpEF. Treating cardiac organoids with Nintedanib ameliorated cardiac fibrosis, hypertrophy and inflammation.