Depletion of non-classical monocytes abolishes sex differences in angiotensin II-induced cardiac fibrosis

Background: Sex differences in the clinical characteristics and pathophysiology of heart failure (HF) have been described in clinical studies, with women more prone to HF with preserved ejection fraction (EF), and men more prone to HF with reduced EF. However, sex differences in animal models of HF have often included only one sex, which may limit the potential for translational application. We therefore aimed to investigate the sex-specific differences in angiotensin II (AngII)-induced cardiac remodelling.

Methods: Male and female 10-week-old C57BL/6J wildtype (WT) and Nr4a1 super-enhancer 2 knockout (Nr4a1se_2^-/-) mice (PMID: 27814941), leading to selective non-classical monocytes (NCMs; Ly6C^lo) deficiency (n=4-7), were subjected to vehicle (saline) or AngII infusion for 7 and 28 days. Circulating and cardiac leukocyte subsets were examined via flow cytometry. Differentially expressed genes (DEGs) in sorted circulating Ly6C^hi and Ly6C^lo monocytes and cardiac endothelial cells (ECs) were assessed via bulk RNA sequencing with a sequencing depth of 10 million reads per sample. Assessment of cardiac inflammation and fibrosis was performed via qPCR and histology.

Results: In WT mice, we observed increased interstitial and perivascular fibrosis in the male AngII-treated group after 7 and 28 days (P=0.001), associated with increased pro-fibrotic gene (Col1, Col3, Ctgf, Ccl2, Il-6) expression as well as cardiac macrophage and fibroblast expansion compared to the female AngII-treated group. Notably, the number and relative proportion of NCMs in the heart increased in females (44% NCMs), but not in males (23% NCMs) in response to AngII. Transcriptomic profiling of circulating NCMs revealed a significant enrichment of DEGs related to antigen processing and presentation and T cell-mediated cytotoxicity in females with AngII infusion compared to males. The resistance to AngII-induced adverse cardiac remodeling was lost in female Nr4a1se_2^-/- mice lacking NCMs, showing a similar extent of cardiac fibrosis and pro-fibrotic gene expression compared to males. The extent of perivascular fibrosis was aggravated in Nr4a1se_2^-/- AngII-treated male and female mice compared to WT mice. Bulk RNA-seq of cardiac endothelial cells showed that NCM deficiency contributed to the enrichment of genes associated with vascular permeability, extracellular matrix assembly, cardiac epithelial-to-mesenchymal transition and increased response to oxidative stress.

Conclusion: Our findings suggest that the increased number of NCMs in WT females when subjected to AngII may help preserve cardiac endothelial integrity, limit inflammatory cell infiltration, and thus counterbalance AngII-induced cardiac fibrosis. The underlying mechanisms leading to a preferential cardiac NCM expansion in female, but not male mice subjected to chronic AngII infusion and implications for human pathophysiology deserve further investigation.