Circulating Immune Cell Signature Analysis in HFpEF Across Species

Figures

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  Figure 1.. Comparing the immune cell signatures of heart failure with preserved ejection fraction (HFpEF), heart failure with reduced ejection fraction (HFrEF), and obesity in humans. A, Peripheral blood mononuclear cells (PBMCs) were isolated from blood samples drawn from controls (Ctrl; n=7) and patients with heart failure (HF) with reduced (n=8) or preserved (n=6) ejection fraction and then subjected to single-cell RNA sequencing (scRNA-seq; cohort 1). The uniform manifold approximation and projection (UMAP) plot shows the 18 clusters of PBMCs identified by FindClusters. B, Different immune cell markers were used for annotation of the clusters to the cell types: platelets, monocytes, B cells, T cells, natural killer (NK) cells, and progenitor cells (Ctrl, n=7; HFrEF: n=8; and HFpEF: n=6). A dot plot shows the percentage of cells expressing the gene and the scaled average expression level. Scaled average expression refers to Z score normalized gene expression values. This standardization centers the values on 0 and scales the variance to 1. C, UMAP plots show the final annotation of single-cell clusters to immune cell types in PBMCs from Ctrl (n=7) and patients with heart failure (HFrEF:…

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  Figure 2.. Comparing the immune cell signatures of heart failure with preserved ejection fraction (HFpEF) in humans and mice. A, C57BL/6N mice were fed either a standard (chow) or a high-fat diet (HFD; 60% fat, 20% protein, and 20% carbohydrates). HFD animals additionally received the endothelial nitric oxide synthase (eNOS) inhibitor Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME) via drinking water (0.5 g/L). After 11 weeks, HFD+L-NAME animals were treated for 4 weeks with vehicle or nitro-oleic acid (NO2-OA; 50:50 mix of (E)-9- and 10-nitrooctadec-9-enoic acid) via osmotic minipumps3 (cohorts 3+4). For simplicity, chow-fed mice will be referred to as controls, mice subjected to the HFpEF-inducing regimen as HFpEF and those receiving the HFpEF-inducing regimen plus NO2-OA treatment as HFpEF+NO2-OA. B, Left ventricular ejection fraction (LVEF), (C) left atrial (LA) area, and (D) ratio of early diastolic mitral inflow velocity to early diastolic mitral annulus velocity (MV E/e´) are shown for control (n=10), HFpEF (n=10), and HFpEF+NO2-OA (n=10) mice. Bar graphs are presented as mean±SEM (B–D: ordinary 1-way ANOVA with the Tukey multiple comparisons test, with a single pooled…

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  Figure 3.. Nitro-oleic acid (NO2-OA) treatment reduces the inflammatory response of human immune cells. A, Schematic illustration of experiments performed on the functional mechanism of NO2-OA: peripheral blood mononuclear cells (PBMCs) were isolated from healthy blood donors and then treated with vehicle or NO2-OA (1 or 3 µmol/L) for 2.5 hours. Furthermore, PBMCs were either left untreated or treated with IL (interleukin)-6 (10 ng/mL), TNF-α (tumor necrosis factor alpha; 10 ng/mL), IL-1β (10 ng/mL), or a combination of IL-6 (10 ng/mL) and TNF-α (10 ng/mL). Quantitative polymerase chain reaction (qPCR)/bulk RNA sequencing (n=3; 4.5-hour cytokine treatment), qPCR (n=3–4 [19 hours, n=3]; 1–24-hour cytokine treatment), qPCR (n=10; 4.5-hour cytokine treatment), and IB (immunoblotting)/IF (immunofluorescence) (15-minute cytokine treatment) were performed. For IF experiments, THP1 cells (human monocytic cell line) were used instead of primary human PBMCs. B, RNA expression of IL1B (interleukin-1 beta) in PBMCs after pretreatment with NO2-OA (1 or 3 µmol/L) for 2.5 hours and subsequent cytokine treatment (each cytokine: 10 ng/mL) for 4.5 hours measured by qPCR (n=4, cell culture). C, RNA…

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  Figure 4.. Validation of identified differentially expressed genes (DEGs) in a human heart failure with preserved ejection fraction (HFpEF) cohort. A, Using FindMarkers (logfc.threshold=0.5, min.pct=0.05, and only.pos=TRUE), differentially expressed genes (DEGs) between control obese and control lean, heart failure with reduced ejection fraction (HFrEF) lean and control lean, HFrEF obese and control lean, HFpEF lean and control lean, and HFpEF obese and control lean were identified (cohort 1). A Venn diagram shows the number of significantly upregulated genes (Wilcoxon rank-sum test, Bonferroni-corrected P<0.05) in each disease separated by body mass index (BMI: lean: <30 kg/m2 and obese: >30 kg/m2) and overlaps. B and C, Dot plots (B: cohort 1, human, single-cell RNA sequencing (scRNA-seq); C: cohort 3, mouse, scRNA-seq) display selected genes of A (Venn diagram, only ↑ HFpEF), percentage of cells expressing each gene scaled by the maximal percentage per gene (dot size), and scaled average expression among each gene (dot color). Scaled average expression refers to Z score normalized gene expression values. This standardization centers the values on 0 and scales the variance to 1. Legend…