Introduction. Cardiovascular diseases (CVD) remain the leading cause of death globally, claiming approximately 40% of all fatalities. Age is a major risk factor for the development of CVD. One of the hallmarks of aging is the accumulation of somatic DNA mutations. Thus, non-malignant mutations in the hematopoietic stem cells (HSC) can lead to the expansion of the mutant cells in a process referred to as clonal hematopoiesis of indeterminate potential (CHIP). Mutations in epigenetic regulators like DNA methyltransferase 3 alpha (DNMT3A) are correlated with an adverse prognosis in patients with heart failure (HF). Although recent reports suggest increased inflammation in DNMT3A CHIP carriers, the paracrine interactions between cells harboring CHIP mutations and other cardiac cell types are unknown and were investigated in the current study.

Results. We combined transcriptomic data from human single-cell RNA sequencing of peripheral blood mononuclear cells of HF patients with (n=5) and without DNMT3A CHIP mutations (n=4) and single nuclei RNA sequencing of cardiac tissue samples (healthy - n=13, HF – n=3) and assessed potential cellular interactions with the DB CellChat software. Cardiac fibroblasts were identified as major putative interaction partners of DNMT3A CHIP monocytes. To assess how DNMT3A CHIP monocytes interfere with cardiac fibroblasts, we used co-culture assays of DNMT3A-silenced macrophages and cardiac fibroblasts. Silencing of DNMT3A in macrophages increased fibroblast activation, as evidenced by the induction of smooth muscle actin expression (p<0.05), augmented cellular contractility (p<0.05), and cellular migration (p<0.05). Moreover, we tested the secretome from DNMT3A-silenced macrophages in 3D vascularized cardiac organoids. In this setting, we showed a significant reduction in contractility of cardiac organoids (p<0.05) and an increase in fibrosis markers(p<0.05).

Taking a step further, we detected an increase in cardiac interstitial fibrosis (p<0.05) but not the scar size in the mice carrying a human DNMT3A CHIP mutation subjected to the left anterior descending artery ligation. Cardiac magnetic resonance imagining of HF patients with reduced ejection fraction (HFrEF) proved significantly increased diffuse myocardial fibrosis (p<0.05) in DNMT3A CHIP-carriers (n=6) compared to the age-matched non-carriers (n=32).

Among the significantly enriched interactions between DNMT3A CHIP monocytes and fibroblasts, we identified HB-EGF - EGFR signaling pathway as a unique interaction pathway. We confirmed that HBEGF is upregulated in monocytes of HF patients with DNMT3A CHIP mutations (p<0.05) on single-cell level. HB-EGF was significantly increased in the serum of HFrEF patients with DNMT3A CHIP mutations (p<0.01). Additionally, HB-EGF was upregulated on gene and protein levels upon DNMT3A silencing in monocytes. Recombinant HB-EGF was sufficient to induce cardiac fibroblast activation in vitro (p<0.05). Inhibition of the EGFR signaling pathway in cardiac organoids partially rescued the effects of DNMT3A-silenced monocytes on contractility and fibrosis (p<0.05).

Conclusions. DNMT3A CHIP mutations in circulating monocytes augment macrophage-to-cardiac fibroblast interactions in patients with HF. The secretome of DNMT3A-silenced monocytes leads to activation of fibroblasts, partially through EGFR signaling. Resulting cardiac interstitial fibrosis might contribute to the poor prognosis of HF CHIP-carrier patients.