Altered bone marrow niche forms central innate immune memory driving cardiac dysfunction

Figures

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  Fig. 1. HF-experienced MSCs affect HSCs to promote cardiac dysfunction. a Schematic of the co-transplantation strategy of healthy cKit+ BM cells enriched with HSPCs and MSCs from post-sham (C-MSCs) or post-TAC (T-MSCs) male mice. b Representative fluorescence images of a femur 4 weeks after the co-transplantation. tdTomato signals from donor C-MSCs or T-MSCs are shown in red. c Echocardiographic assessment of the left ventricular ejection fraction and left ventricular end-diastolic volume 6 months after the co-transplantation. n = 10, 9 male mice for C-MSC and T-MSC groups, respectively. Two-tailed unpaired Student’s t-test. Exact P value: P = 0.0004 (two-tailed unpaired Student’s t-test; t = 4.445, df = 17). d Expression of cardiac fibrosis marker genes 3 months after the transplantation. n = 10 in each group. Two-tailed unpaired Student’s t-test (from two independent experiments). e Histological analysis of fibrotic areas identified using Picro-Sirius red staining. The same mice used for the echo studies in c were used. Representative histological images are shown in Supplementary Fig. 3d. Two-tailed unpaired Student’s t-test. n = 10 mice for the C-MSC group and n = 9 mice for the…

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  Fig. 2. TAC suppresses MSC HBEGF-to-HSC signaling. a UMAP plot of Lepr+ MSCs from male mice (n = 3 control; n = 8 TAC) 4 weeks after surgery. b Marker gene expression in each cluster. Additional marker genes are shown in Supplementary Fig. 7. c Heatmap showing expression of known MSC subpopulation markers. d UMAP plot of LSKs from the same male mice used for MSC scRNA-seq. e NicheNet analysis of inferred ligand-receptor interactions between MSCs (ligand producing) and LSKs (receptor-expressing). f Differential gene expression analysis of MSCs. Upregulated and downregulated genes (FDR < 0.05) are shown in red and blue, respectively. Cytokine and growth factor genes are highlighted in dark red, and those with FDR < 0.05 are labeled. Differential expression was assessed using edgeR with two-sided tests, and P values were adjusted for multiple comparisons using the Benjamini–Hochberg method (FDR < 0.05). g PROGENy pathway enrichment analysis. Positive pathway scores indicate increased pathway activity in post-TAC HSCs compared with controls. Top 8 pathways are shown. h Proportions of cells within each MSC cluster in control (CTRL) and TAC groups. Differences in proportions were analyzed…

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  Fig. 3. Hbegf downregulation in MSCs promotes myeloid skewing and cardiac dysfunction. a Expression of Hbegf in lentivirally generated NIH-3T3 stable cell lines overexpressing murine Hbegf and Cre recombinase following transfection with the AAV-FLEX–shHbegf transgene plasmid. n = 6 per group. Exact P value: P < 0.0001 (two-tailed unpaired Student’s t-test; t = 10.95, df = 10). b Schematic of the experimental workflow. Lepr-Cre male mice received intravenous AAV administration followed by TAC. c Fractions of primitive HSPC subpopulations. Exact P values: LT-HSC, P = 0.0220; MPP1, P = 0.8770; MPP2, P = 0.7289; MPP3, P = 0.0024; MPP4, P = 0.0006 (two-tailed unpaired Student’s t-tests after logit transformation; n = 7 scramble, n = 6 shHbegf). d Representative flow cytometry plots showing LSK populations. e Numbers of granulocyte/monocyte progenitor (GMP) per femur. f Numbers of CCR2hi and CCR2lo cardiac macrophages normalized to heart weight (cells per milligram of heart tissue). g Representative cardiac sections stained with Picrosirius Red/Fast Green; collagen fibers appear red, and the non-collagen components green. h Fibrosis area in cardiac tissue. i Left ventricular ejection fractions…

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  Fig. 4. Activation of the adipogenic program in MSCs. a Heatmap showing the scaled average regulon activity scores of the top 5 regulons specific to each cluster in the control and post-TAC datasets. Rows represent the TFs that control the regulons, which are hierarchically clustered. Based on the dendrogram, regulons were divided into six groups exhibiting distinct activity patterns. b Top 10 hallmark gene sets enriched in TAC versus control MSCs, ranked based on normalized enrichment score (NES). LEPR+ MSCs were isolated from male mice 4 weeks after either sham or TAC operation. n = 2 for sham, n = 3 for TAC. Gene sets were ranked by normalized enrichment score, and statistical significance was determined by gene set enrichment analysis (two-sided) with Benjamini–Hochberg-adjusted P values. c GSEA enrichment plot of the adipogenesis Hallmark gene set. d Oil red O staining of MSC cultures derived from bone marrow cells of control and TAC 4 week mice cultured in the adipogenic medium for 7 days. n = 6 mice per group. Data are from three independent experiments with similar results. e Representative histological sections of the first coccygeal segment from control and post-TAC mice.…

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  Fig. 5. Human HBEGF+ MSCs and BM adiposity in HF. a Clustering of human LEPR+ MSCs. b Expression of the marker genes in human LEPR+ MSCs. Violin plot of the full marker genes corresponding to Supplementary Fig. 7b is shown in Supplementary Fig. 20a. c Representative axial CT images of the 7th thoracic vertebra (T7) from a healthy control subject (left) and a patient with HF (right). Bone marrow regions with CT attenuation below 0 HU, corresponding to fat, are shown in red, and regions with attenuation at or above 0 HU are shown in green. d Percentage areas of CT values below 0 within the range from −200 to 1000 in CT values on coronary CT images of the thoracic vertebral BM in HF patients and age- and sex-matched healthy controls. Two-tailed unpaired Student’s t-test. n = 30 for control, n = 15 for heart failure. Box-and-whisker plots show the median, the 25th and 75th percentiles (box bounds), and the minimum and maximum values (whiskers). e Schematic model summarizing the proposed mechanism, comparing the healthy state (left) with heart failure (right). Under homeostasis, Notch–HES1 signaling in Lepr⁺ mesenchymal stromal cells of the bone marrow niche maintains the HBEGF-expressing…