Clin Res Cardiol (2022). https://doi.org/10.1007/s00392-022-02002-5

Clonal hematopoiesis–driver DNMT3A mutations induce pro-inflammatory phenotype in macrophages and enhance macrophage-to-fibroblast interaction in the failing human heart
M. Shumliakivska1, W. Abplanalp1, G. Luxan1, B. Schuhmacher1, R. Schulze Bruening1, D. John1, S. Cremer2, A. M. Zeiher2, S. Dimmeler1
1Zentrum für Molekulare Medizin, Institut für Kardiovaskuläre Regeneration, Universitätsklinikum Frankfurt, Frankfurt am Main; 2Institute of Cardiovascular Regeneration and Department of Cardiology, Goethe Universität Frankfurt am Main, Frankfurt am Main;

Background:
Age-acquired somatic mutations in epigenetic regulators could lead to clonal expansion of hematopoietic cells (clonal hematopoiesis of indeterminate potential, CHIP). Mutations in the methylation regulator DNA methyltransferase 3 alpha (DNMT3A) were shown to be associated with poor prognosis in patients with heart failure (HF). The point of convergence for most heart muscle diseases is a myocardial fibrosis
that contributes to the HF development. Although studies suggest that DNMT3A CHIP driver mutations augment the inflammatory profile, the paracrine effects of CHIP immune cells on the cardiac fibroblasts remain to be elucidated. Here, we investigate mechanisms by which DNMT3A CHIP mutant macrophages facilitate the activation of fibroblasts and thus, the progression of heart fibrosis.

Methods:
We performed interaction analysis utilizing
transcriptomic data from single cell and nuclei RNA sequencing. We analysed circulating monocytes obtained from HF patients with and without DNMT3A CHIP mutations and fibroblasts obtained from myocardial tissue specimens. Using a bioinformatics tool, we identified unique interaction patterns between DNMT3A CHIP monocytes and fibroblasts. We explored the functional relevance of the genes predicted to mediate cellular communication by using siRNA-based loss of function studies. In vitro validation of potential interactions was performed by macrophages-to-fibroblasts and fibroblasts-to-macrophages co-culture experiments and further assessed by immunocytochemistry analysis, gene and protein expression measurements.  

Results:
Using
CellPhone DB software, we identified an upregulated activin A receptor type I (ACVR1B)/activin A signalling pathway as a monocytes/macrophages-to-fibroblasts specific interaction for DNMT3A CHIP carriers. Since activins belong to the TGF-ß superfamily and might be involved in fibrosis, we further investigated this interaction. We confirmed that ACVR1B is upregulated in monocytes of HF patients with loss-of-function DNMT3A CHIP mutations versus HF patients without these mutations. Additionally, DNMT3A variant allele frequency was positively correlated with ACVR1B gene expression levels. Importantly, silencing of DNMT3A in macrophages in vitro led to an increase in ACVR1B gene expression. Curiously, secretome from cardiac fibroblasts promoted pro-inflammatory, M1 skewing of DNMT3A-silenced macrophages in vitro. Mechanistically, activin A gene expression levels and protein secretion were increased by cardiac fibroblasts and partially induced a pro-inflammatory phenotype in DNMT3A-silenced macrophages upon co-culture. Conversely, co-culture with DNMT3A-silenced macrophages induced an increase in fibroblast activation, as evidenced by induction of smooth muscle actin and an increase in migration and contraction.

Conclusions:
DNMT3A CHIP mutations in circulating monocytes reciprocally augment macrophages-to-cardiac fibroblasts interactions in patients with HF.  Mechanistically, the ACVR1B/activin A signalling pathway appears to orchestrate an unique interaction between DNMT3A CHIP mutation carrier monocytes and cardiac fibroblasts. While DNMT3A CHIP monocytes/macrophages are sensitized to cardiac fibroblasts secretome and become more pro-inflammatory, conversely, the secretome of DNMT3A mutant cells leads to myofibroblastic activation of fibroblasts. These data support the hypothesis of a vicious cycle fueling cardiac fibrosis in HF progression of DNMT3A mutation carriers.


https://dgk.org/kongress_programme/jt2022/aP477.html