Background: Non-classical monocytes (NCM) are patrolling on the endothelium to maintain vascular homeostasis. Single-cell RNA sequencing recently revealed specific gene expression patterns in circulating NCM of heart failure patients, affecting genes involved in antigen processing and presentation, inflammatory response and negative regulation of apoptosis. However, the causal role of NCM in cardiac homeostasis, remodelling and heart failure remains elusive. Thus, the aim of this study was to determine the phenotype and function of NCM in angiotensin II (AngII)-induced cardiac remodelling.

Methods: Male and female C57BL/6J wildtype (WT) and mice deficient for NCMs due to Nr4a1 super-enhancer knockout (Nr4a1SE^-/-) were subjected to chronic AngII (1.5 µg/g/day) infusion for 7 days. Baseline analyses were performed in young and aged mice. Leukocyte subsets were examined via flow cytometry in blood, heart, and lymphoid organs. Further assessment of cardiac inflammation as well as fibrosis and hypertrophy were performed via qPCR and histology.

Results: In young WT and Nr4a1SE^-/- baseline mice, we noted significantly elevated circulating NCM counts and relative proportions of NCM in female compared to male mice (39% versus 8% of all monocytes). Flow cytometry of baseline mice lacking Nr4a1SE confirmed selective absence of Ly6C^lo NCM in blood and spleen. Following 7 days of AngII infusion, the cardiomyocyte size was increased, with comparable diameters measured in heart sections of WT and NCM-deficient mice. Nr4a1SE^-/- female mice showed less interstitial fibrosis (P=0.006) but pronounced perivascular fibrosis around coronary arteries and outward vascular remodelling when compared to WT mice. This phenotype was accompanied by enhanced numbers of cardiac leukocytes, mainly lymphocytes and Ly6C^hi monocytes in the left ventricle, while Ly6C^hi counts were reduced in the blood and splenic reservoir, suggesting an increased cardiac infiltration of inflammatory monocytes.

Conclusion: We may speculate that selective loss of NCM impairs vascular homeostatic maintenance, thereby promoting immune cell extravasation and possibly endothelial-to-mesenchymal transition, which might explain the observed particular fibrosis pattern. Further analyses employing immunostaining, transcriptomic analysis and echocardiography are currently ongoing to better understand the disease- and sex-specific role of NCM in this model of AngII-induced cardiac remodelling.