Neutrophil peptidylarginine deiminase 4 deficiency influences cardiac fibroblast phenotypes and the immune response to cardiac pressure overload in aging

Abstract Deficiency of peptidylarginine deiminase 4 (PAD4), a key enzyme for neutrophil extracellular trap (NET) formation, protects from age-related cardiac fibrosis and pressure overload-induced fibrosis in young mice. To explore mechanisms underlying this cardioprotection, we investigated the effect of neutrophil PAD4 deficiency on the transcriptome of aged murine heart cells and the response of the aged heart to pressure overload. Single nucleus RNA sequencing was performed on ventricular nuclei isolated from two year old neutrophil-specific PAD4 knockout (MRP8Cre+PAD4fl/fl) mice and PAD4fl/fl controls. Young (8–14 weeks) and aged (17–19 months) male mice underwent ascending aortic banding using age-adapted O-rings to obtain ~50% stenosis. Early cardiac immune response was assessed on day 3 by spectral flow cytometry and cardiac fibrosis was evaluated at two weeks using Sirius Red staining. The proportions of the major cardiac cell types were comparable between MRP8Cre+PAD4fl/fl and PAD4fl/fl hearts. Fibroblast subclustering revealed 6 substates with distinct distribution patterns between genotypes. In aged PAD4fl/fl hearts, ECM-modulating fibroblasts were the dominant subtype (65.9%). By contrast, in MRP8Cre+PAD4fl/fl mice, this population was equally represented alongside a more reactive subset of fibroblasts (39.2 and 39.4%, respectively), with marker genes encoding ion channel receptors and proteins involved in downstream signaling and oxidative stress response. The main communication axis of collagen-related signaling involved fibroblasts and immune cells. Pressure overload increased γδ T-cell proportions in aged hearts (13.5% to 40% (PAD4fl/fl), 10.9% to 27.1% (MRP8Cre+PAD4fl/fl)). Aged PAD4fl/fl mice showed reduced neutrophil frequencies compared to young controls (9% vs 19.8%), whereas aged MRP8Cre+PAD4fl/fl mice resembled their younger counterparts (17.8% vs 16.9%). Reduced presence of neutrophils may reflect altered recruitment or prior NET release. No difference in cardiac collagen content was observed between aged MRP8Cre+PAD4fl/fl and PAD4fl/fl animals after banding (2.8% vs 2.6%). We hypothesize that neutrophil PAD4-deficient mice, with lower baseline collagen due to cardioprotection in aging, demonstrate a more pronounced fibrotic response upon pressure overload. This is supported by the presence of distinct collagen patches in cardiac tissue, which are typically observed after pressure overload in young mice. We show that neutrophil PAD4-deficient aged mice have higher proportions of cardiac fibroblasts with a reactive phenotype, which is also reflected in their pattern of fibrotic remodeling after pressure overload, paralleling the stress response of young mice. Furthermore, we highlight that both aging and neutrophil PAD4-deficiency shape the immune response after pressure overload. These data suggest a more prominent role for γδ T-cells and reduced neutrophil involvement in pressure overload-induced fibrotic remodeling during aging.