Selective loss of resident macrophage-derived insulin-like growth factor-1 abolishes adaptive cardiac growth to stress

Abstract

Hypertension affects one-third of the world's population, leading to cardiac dysfunction that is modulated by resident and recruited immune cells. Cardiomyocyte growth and increased cardiac mass are essential to withstand hypertensive stress; however, whether immune cells are involved in this compensatory cardioprotective process is unclear. In normotensive animals, single-cell transcriptomics of fate-mapped self-renewing cardiac resident macrophages (RMs) revealed transcriptionally diverse cell states with a core repertoire of reparative gene programs, including high expression of insulin-like growth factor-1 (Igf1). Hypertension drove selective in situ proliferation and transcriptional activation of some cardiac RM states, directly correlating with increased cardiomyocyte growth. During hypertension, inducible ablation of RMs or selective deletion of RM-derived Igf1 prevented adaptive cardiomyocyte growth, and cardiac mass failed to increase, which led to cardiac dysfunction. Single-cell transcriptomics identified a conserved IGF1-expressing macrophage subpopulation in human cardiomyopathy. Here we defined the absolute requirement of RM-produced IGF-1 in cardiac adaptation to hypertension.

Keywords: IGF-1; cardiac; cardiomyocyte hypertrophy; fate mapping; heart failure; hypertension; macrophages; organ growth; resident macrophages; scRNA-seq.

Figure 1.. Self-renewing resident cardiac macrophages expand numerically during hypertensive stress.

(A) Diagram of tamoxifen administration and AngII infusion in Cx3cr1^creERT2; Rosa26^Td (RM^Td) mice. (B) Representative flow cytometric analyses of blood CD11b⁺ CD115⁺ monocytes and cardiac CD11b⁺ CD64⁺ macrophages at 4 weeks and 8 weeks of age, as indicated. (C) Representative flow cytometric analyses of cardiac CD11b⁺ CD64⁺ macrophages gated on Td⁺ or Td⁻ cells, as indicated. (D-F) Mice were infused with AngII for 28 days (D28 AngII) and compared to mice that underwent sham surgery (Sham). (D) Quantification of aortic systolic and diastolic blood pressure from Millar catheterization (top), left ventricular ejection fraction (LVEF) from echocardiography, and heart weight to initial body weight ratio (bottom); n=8–15, pooled from two experiments. (E) Representative immunofluorescence images and quantification of cardiomyocyte cross-sectional area from Wheat Germ Agglutinin staining, scale bar = 25 μm; n=6–12, pooled from N=2. (F) Representative histochemical images and quantification of fibrotic area in cardiac tissue from Sirius Red staining, scale bar = 50 μm; n=8–9, pooled from two experiments. (G-I) RM^Td mice were infused with AngII for 4, 7 and 28 days (D4, D7, and D28 AngII). (G) Quantification of infiltrating Ly6C^hi CD11b⁺ monocytes and cardiac CD11b⁺ CD64⁺ macrophages with flow cytometry; n=5–6, pooled from two experiments. (H) Representative flow cytometry analyses (top) and quantification (bottom) of cardiac CD11b⁺ CD64⁺ macrophages gated on Td⁺ or Td⁻ cells, as indicated; n=4–6, pooled from two experiments. (I) Representative immunofluorescence images and quantification of Td⁺ cells per 20× field of view (FOV) in cardiac tissue sections from RM^Td mice as indicated, scale bar = 50 μm; n=4, pooled from two experiments. AngII: angiotensin II. *p < 0.1; ***p < 0.001; ****p < 0.0001. Data is shown as box and whisker plots (D-G, I) or as mean ± SEM (H). Analysis was performed using the unpaired t-test (D-F), and one-way ANOVA with multiple comparisons vs. Sham (G-I). See also Figure S1.

Figure 2.. Hypertensive stress induces the selective increase of fate-mapped TIMD4^hiMHC-II^lo resident cardiac macrophages.

Sorted resident cardiac macrophages (CD45⁺DAPI⁻CD11b⁺CD64⁺Td⁺) from Cx3cr1^creERT2; Rosa26^Td (RM^Td) mice following sham surgery (Sham; steady state), 4 days (D4 AngII; acute hypertension) and 28 days of AngII infusion (D28 AngII; chronic hypertension) were processed individually for scRNA-seq (10x Genomics). Two independent experiments were performed, each using 3–4 pooled animals per sample. Experiment 1 consist of Sham, D4 AngII and D28 AngII, and Experiment 2 consisted of Sham and D4 AngII. (A) UMAP projection of the combined analysis of Sham, D4 AngII and D28 AngII visualized together. (B) Feature plots illustrating expression of subset-defining genes. (C) Quantification of unique or shared differentially expressed genes (DEGs) of each cluster, with selected genes highlighted (min.pct = 0.2, logFC threshold = 0.2, adjusted p-value <0.05). (D) UMAP projection of Sham, D4 AngII and D28 AngII visualized separately (left). Quantification of the relative abundance of each cluster in each condition (right). (E) The proportion of resident cardiac macrophages (all subsets combined) that are proliferating in Sham, D4 AngII and D28 AngII as assessed by scRNA-seq. Sham and D4 AngII represent the average of Experiment 1 and Experiment 2, shown as mean ± SEM. (F) Representative flow cytometric analyses of cardiac Td⁺CD11b⁺CD64⁺ macrophages, as indicated from Sham, D4 AngII, D7 AngII and D28 AngII; green = TIMD4^hiMHC-II^lo; blue = TIMD4^hiMHC-II^hi; orange = TIMD4^loMHC-II^hi (left). Quantification of the abundance of each subset (right); n=4–8, pooled from two experiments. (G) The proportion of cardiac Td⁺CD11b⁺CD64⁺ macrophages in each subset that are proliferating (BrdU⁺) across conditions; n=4–8, pooled from two experiments. (H) Quantification of each Td⁺CD11b⁺CD64⁺ subset as absolute amount per heart (left) and normalized to per mg cardiac tissue (right) across conditions; n=4–8, pooled from two experiments. Representative immunofluorescence images (I) and quantification (J) of LYVE-1⁺Td⁺ cells (indicated with white arrowheads) per 40× field of view (FOV) in cardiac tissue sections from RM^Td mice as indicated, scale bar = 50 μm; n=3. AngII: angiotensin II; DEGs: differentially expressed genes; *** p-value<0.001, **** p-value<0.0001. Data is shown as mean ± SEM. Analysis was performed on pooled data in A-E and using two-way ANOVA with multiple comparisons vs. Sham (E-G). See also Figure S2.

Figure 3.. High resolution analysis of resident cardiac macrophages reveals nine transcriptional states and maintenance of reparative programs in acute and chronic hypertensive stress.

(A) High resolution clustering identifies nine resident cardiac macrophage states (S1–9) in Sham, D4 AngII and D28 AngII split by condition (UMAP projection on left). The relative abundance of each cell state (S1–9) across conditions (right). (B) Heatmap depicting the top 30 DEGs for each cell state (logFC threshold = 0.2, min.pct = 0.2, adjusted p-value <0.05). Colour bar denotes the high-resolution state (top; S1–9), low-resolution cluster (C1–3; middle), and condition (bottom). The number of DEGs in each cell state is shown (bottom). (C) Pathway enrichment analysis (gProfiler, GO Biological Processes) using DEGs for each cell state (S1–9). (D) DEGs (upregulated and downregulated combined) at D4 AngII (pink bars) or D28 AngII (blue bars) relative to Sham for each cell state (S1–9). (E) Percentage of DEGs that were unique to D4 or D28 AngII (relative to Sham) or shared in both conditions for selected cell states. (F) Percentage of DEGs that were shared or unique to each cell state at D4 or D28 AngII (relative to Sham), as indicated. Genes were considered shared if they were either upregulated at both D4 and D28 AngII or downregulated at both D4 and D28 AngII. (G) Pathway enrichment analysis (gProfiler, GO Biological Processes, KEGG) using DEGs upregulated at D4 AngII relative to Sham for selected cell states. (H) Pathway enrichment analysis (gProfiler, GO Biological Processes, KEGG) using DEGs upregulated at D28 AngII relative to Sham for selected cell states. (I) DEGs of total resident cardiac macrophages relative to monocytes were computed for Sham, D4 AngII and D28 AngII for both experiments separately. The overlap of these DEGs were used for pathway enrichment analysis (gProfiler, GO Biological Processes). (J) Feature plots of the expression of growth factors that are constitutively expressed in resident cardiac macrophages at steady state (Sham) and during hypertensive stress (D4/D28 AngII). AngII: angiotensin II. DEGs: differentially expressed genes. data is from two pool experiments, merged for analysis. See also Figure S3.

Figure 4.. Inducible depletion of tissue resident macrophages inhibits adaptive cardiomyocyte growth and leads to cardiac dysfunction during chronic hypertensive stress.

Cx3cr1^creERT2; Rosa26^Td/DTR (RM^Td-DTR) mice or RM^Td controls were infused with AngII for 7 or 28 days (D7 AngII or D28 AngII) concurrently with DT administration in all groups. (A) Diagram of tamoxifen, AngII and DT treatment in RM^Td-DTR mice. (B) Representative flow cytometric analyses and quantification of cardiac Td⁺CD11b⁺CD64⁺ macrophages from D7 AngII with DT treatment; n=4. (C-G) At D28 AngII with DT treatment, cardiac function and disease pathology were assessed. (C) Quantification of body weight loss shown as mean ± SEM; asterisks indicate statistical significance of each RM^Td-DTR group against respective RM^Td controls; n=3–6. (D) Indices of cardiac growth; heart weight to initial body weight ratio, left ventricle (LV) anterior wall thickness (measured by echocardiography) and LV mass (approximated with echocardiography); n=3–6. (E) Representative immunofluorescence images and quantification of cardiomyocyte cross-sectional area from Wheat Germ Agglutinin staining, scale bar = 50 μm; n=3–6. (F) Representative histochemical images and quantification of fibrotic area in cardiac tissue from Sirius Red staining, scale bar = 100 μm; n=3–6. (G) Echocardiographic assessment of cardiac function and remodeling; LV ejection fraction (LVEF), LV end systolic volume (ESV), LV end diastolic volume (EDV), LV internal diameter end diastole (LVIDd); n=3–6. AngII: angiotensin II; DT: diphtheria toxin; LV: left ventricle; * p-value<0.1 ** p-value<0.01 *** p-value<0.001, **** p-value<0.0001. Data is shown as box and whisker plots unless otherwise indicated. Analysis was performed using two-way ANOVA with multiple comparisons. See also Figure S4.

Figure 5.. Specific deletion of IGF-1 from tissue resident macrophages abolishes adaptive cardiomyocyte growth and leads to cardiac dysfunction during chronic hypertensive stress.

(A) Violin plot showing mRNA expression of Igf1 in resident cardiac macrophages and monocytes. (B) Quantification of IGF-1 protein in cardiac tissue (left; n=8–12, pooled from two experiments) and serum (right; n=3–6) through ELISA in RM^Td and RM^Td-DTR mice at D28 AngII with diphtheria toxin treatment, shown as mean ± SEM. (C-G) Cx3cr1^creERT2; Igf1^fl/fl (RM^ΔIgf1) mice were infused with AngII for 28 days (D28 AngII) and compared to control Igf1^fl/fl (Igf1 flox) mice. (C) Diagram of tamoxifen and AngII administration in RM^ΔIgf1 mice. (D) Indices of cardiac growth; left ventricular (LV) anterior wall thickness (measured by echocardiography) and LV mass (approximated with echocardiography); n=6–12, pooled from two experiments. (E) Representative immunofluorescence images and quantification of cardiomyocyte cross-sectional area from Wheat Germ Agglutinin staining, scale bar = 50 μm; n=6–12, pooled from two experiments. (F) Echocardiographic assessment of cardiac function and remodeling; LV ejection fraction (LVEF), LV end systolic volume (ESV), LV end diastolic volume (EDV), LV internal diameter end diastole (LVIDd); n=6–12, pooled from two experiments. (G) Quantification of fibrotic area in cardiac tissue from Sirius Red staining; n=3–6. AngII: angiotensin II; LV: left ventricle; * p-value<0.1 ** p-value<0.01 *** p-value<0.001, **** p-value<0.0001. Data is shown as box and whisker plots unless otherwise indicated. Analysis was performed using Wilcoxon Rank Sum test (A) or two-way ANOVA with multiple comparisons (D-G). See also Figure S5.

Figure 6.. IGF1-expressing cardiac macrophage subset is conserved in human heart failure.

Single-cell RNA sequencing (10x Genomics) was performed on human cardiac tissue from two patients: 5-month-old neonate undergoing corrective surgery (with normal cardiac function) and adult human end-stage heart failure from non-ischemic origins. (A) UMAP dimensionality reduction revealed two clusters of monocytes and three clusters of macrophages with distinct gene expression profiles. (B) UMAP projection split by patient sample (left) and the frequency of each cluster present in each sample (right). (C) Heatmap depicts the top 30 DEGs in each cluster (min.pct = 0.2, logFC threshold = 0.2, adjusted p value <0.05). (D) Pathway enrichment analysis (gProfiler, GO Biological Processes) using DEGs for each macrophage cluster (MF 1–3). (E) A murine resident cardiac macrophage gene signature was generated by computing the top DEGs in resident cardiac macrophages relative to monocytes (Figure S2). The machine-learning algorithm Garnett was used to classify cells in both human datasets individually using the gene signature generated from murine resident cardiac macrophages. Heatmap shows the percentage of cells from each cluster in each sample that were classified as resident cardiac macrophages due to similar gene expression profiles. (F) Feature plots depict the gene expression of LYVE1 and IGF1. Outlined region highlights the cluster which specifically and distinctly expresses these genes, and were the most enriched in the murine resident cardiac macrophage signature (E). MF: macrophage. See also Figure S6.