HIF expression and signaling in human cardiac macrophages during ischemic cardiomyopathy. (A) Gross appearance of remote (RT) and peri-infarct (INF) regions sampled for flow cytometry. (B) Gating strategy for cardiac macrophages and peripheral blood mononuclear cells (PBMCs). Cardiac macrophages and PBMCs were first gated on live, single cells and identified as CD14⁺CD64⁺ cells. (C) Expression of HIF-1⍺ and HIF-2⍺ in cardiac macrophages and PBMCs. FMO was used as a negative staining control. (D) Gross appearance of INF region sampled for single-cell RNA-sequencing analyses. Transcriptomic analysis was performed on 1,149 cells using the 10x Genomics platform. (E) Identification of eight unique clusters by t-distributed stochastic neighbor embedding dimensionality reduction analysis. EC, endothelial cell; SMC, smooth muscle cell. (F) Heatmap of the 20 most differentially expressed genes within each cluster. (G) Feature plots representing single-cell gene expression of canonical macrophage markers (Cd68, C1qa, Lyve1, Mertk, and Csfr1) identified cluster 3 as a macrophage cluster. (H) Pathway enrichment of differentially expressed genes in the macrophage cluster (cluster 3) was determined using gProfiler. Enrichment is expressed as the −log[P] and is adjusted for multiple comparisons. (I) Violin plots of Hif1 and Epas1 (Hif2) expression and inflammatory cytokine and chemokine gene expression shown to be HIF dependent in primary human macrophages during hypoxia treatment. RT, remote tissue; FSC-A, forward scatter area; FSC-W, forward scatter width; SSC-A, side scatter area; SSC-W, side scatter width; EC, endothelial cell; NK, natural killer; UMAP, Uniform Manifold Approximation and Projection for Dimension Reduction.