Heart failure-specific cardiac fibroblasts contribute to cardiac dysfunction via the MYC–CXCL1–CXCR2 axis

Figures

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  Fig. 1. MYC expression in CFs of failing hearts. a, Experimental scheme using mice exposed to pressure overload. 0, day of TAC; 2 weeks, 2 weeks after TAC; 12 weeks, 12 weeks after TAC. On the day of TAC and 2 weeks and 12 weeks after TAC, non-cardiomyocytes were collected from the heart and scRNA-seq was performed. b, t-SNE visualization of unsupervised clustering of non-cardiomyocytes. Cells (dots) are colored according to the cell clusters and annotated using well-known marker genes. CE, capillary endothelial cell; MΦ, macrophage; SM, smooth muscle cell; AE, arterial endothelial cell; DC, dendritic cell; VE, venous endothelial cell; ME, MKI67 positive endothelial cell; NC, nerve cell; LE, lymphatic endothelial cell; BC, B cell; TC, T cell. c, Feature plot showing fibroblast marker gene expression on t-SNE. d, t-SNE reclassified CFs into six subpopulations according to their gene expression (left, colored according to the cell clusters; right, colored according to the time after TAC). The table shows the cell ratios of six clusters at each time course. e, GO analysis of genes specifically expressed in cluster 4, HF-specific CFs. f, Violin plot showing the expression of Myc and Tcf21…

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  Fig. 2. MYC overexpression in CFs showed significant deterioration of cardiac function in TAC mice. a, Experimental scheme for generating cardiac fibroblast-specific MYC-OE mice. 0, day of TAC; 12 weeks, 12 weeks after TAC. b, Western blot analysis of MYC and GAPDH expression in WT control, WT-TAC and OE-TAC mouse hearts. The experiment was repeated independently two times with similar results. c, Echocardiographic assessment of control and OE mice from before (0 week) to 12 weeks after TAC. d, Bar plots show cardiac function evaluated by echocardiography in control and OE mice before and after pressure overload (0–12 weeks). Means and standard errors are shown (TAC0w, control n = 6 and OE n = 5; TAC2w, 5 and 5; TAC4w, 6 and 5; TAC8w, 5 and 5; TAC12w, 4 and 5). Tcf21 iCre–R26StopFLMYC mice were used as control. *P < 0.05; P = 0.0014 (TAC12w); significance was determined using the two-sided Holm–Sidak test for mean comparisons. e, Relative mRNA expression of HF markers measured using RT-qPCR. Data are shown as mean ± s.d. *P < 0.05; P = 0.024 (control (n = 6) versus WT-TAC (n = 3), Nppa), P = 0.036 (WT-TAC versus OE-TAC (n = 5), Nppa), P = 0.0095 (control (n = 6) versus WT-TAC (n = 4),…

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  Fig. 3. MYC deletion in CFs ameliorates TAC-induced cardiac dysfunction. a, Experimental scheme for generating cardiac-fibroblast-specific Myc KO mice. 0, day of TAC; 12 weeks, 12 weeks after TAC. b, Relative mRNA expression of Myc in the heart as measured via RT-qPCR. Data are shown as mean ± s.d. *P < 0.05; P < 0.001 (control (n = 10) versus WT-TAC (n = 7)), P = 0.13 (control versus KO-TAC (n = 11)); significance was determined using the two-sided Mann–Whitney test for mean comparisons. c, Representative images of the echocardiographic assessment of control and KO mice before and after TAC (20 weeks). d, The bar plots show cardiac function evaluated by echocardiography in control and KO mice before and after pressure overload (0–20 weeks). Data are shown as mean ± s.e. (TAC0w, control n = 5 and KO n = 6; TAC2w, 4 and 6; TAC4w, 5 and 6; TAC8w, 4 and 6; TAC12w, 4 and 5; TAC14w, 4 and 5; TAC16w, 4 and 5; TAC20W, 3 and 4). Tcf21 iCre-MYC flox mice were used for control. *P < 0.05; P = 0.018 (TAC8w), P = 0.018 (TAC12w), P = 0.007 (TAC14w), P = 0.018 (TAC16w), P < 0.001 (TAC20w); significance was determined using the two-sided Holm–Sidak test for mean comparisons. e, Relative mRNA expression…

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  Fig. 4. Comparison of cardiac gene expressions of control, WT-TAC and KO-TAC mice. a, Principal component analysis (PCA) visualization showing global gene expression in WT control, WT-TAC and KO-TAC mice (n = 3 each). b, Heat map showing the expression levels of differentially expressed genes in WT control, WT-TAC and KO-TAC mice. Left: differentially expressed genes in WT and WT-TAC mice. Right: differentially expressed genes between the WT-TAC and KO-TAC mice. c, GO analysis of differentially expressed genes. Genes were upregulated in WT-TAC mice compared with those in WT control mice. The red bars indicate GO terms associated with genes expressed in cardiomyocytes. The white bars represent other GO terms not specifically related to cardiomyocyte-expressed genes. d, GO analysis of differentially expressed genes. Genes were downregulated in KO-TAC mice compared with those in WT-TAC mice. The red bars indicate GO terms associated with genes expressed in cardiomyocytes. The white bars represent other GO terms not specifically related to cardiomyocyte-expressed genes. e, Top: GSEA of genes associated with MYC targets. Plots were prepared using the HALLMARK_MYC_TARGETS_V1 dataset…

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  Fig. 5. MYC-induced secreted factors in CFs upregulate HF marker genes in cardiomyocytes. a, Experimental setup. Culture medium conditioned by MYC overexpression in CFs using a retroviral vector was transferred to cultured neonatal rat cardiomyocytes (CM). b, Relative mRNA expression of HF markers in cardiomyocytes cultured in medium conditioned by MYC or GFP overexpression in CF. Data are shown as mean ± s.d. *P < 0.05; P = 0.036 (Nppa, GFP n = 3, MYC n = 5), P = 0.024 (Nppb, GFP n = 3, MYC n = 6), P = 0.024 (Myh7, GFP n = 3, MYC n = 6); significance was determined using the two-sided Mann–Whitney test for mean comparisons. c, Venn diagram of genes upregulated in WT-TAC mice versus WT control mice, downregulated in KO-TAC mice versus WT-TAC mice in RNA-seq data and upregulated in HF-specific CFs (cluster 4) versus other cluster 1–3 CFs in scRNA-seq data. A total of 21 common genes are listed. d, Top: genome browser view showing Cxcl1 gene expression in WT control, WT-TAC and KO-TAC mice. Bottom: relative mRNA expression of CXCL1 as measured by RT-qPCR (n = 4 each). Data are shown as mean ± s.d. *P < 0.05; P = 0.029 (control versus TAC), P = 0.029 (TAC versus KO); significance was…

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  Fig. 6. MYC–CXCL1–CXCR2 signaling contributes to HF pathogenesis. a, Western blot analysis of CXCR2 expression in the negative control (NC), positive control (PC) and CM. Top: the NC is mouse embryonic fibroblasts, the PC is a human embryonic kidney cell line transfected with a Cxcr2 expression vector and the CM are cardiomyocytes of neonatal rats. Bottom: the NC is undifferentiated P19CL6 (mouse embryonic carcinoma) cells, the PC is a human embryonic kidney cell line transfected with a Cxcr2 expression vector and the CM are cardiomyocytes of mice. The experiment was repeated independently three times with similar results. b, Relative mRNA expression of HF markers in cardiomyocytes after addition of the medium conditioned by MYC overexpression in CFs, with and without a neutralizing antibody against CXCR2 (n = 3 each). Data are shown as mean ± s.d. *P < 0.05; P < 0.001 (GFP (n = 6) versus MYC (n = 10), Nppa), P = 0.0027 (MYC versus MYC plus anti-CXCR2 (n = 5), Nppa), P < 0.001 (GFP (n = 7) versus MYC (n = 10), Nppb), P = 0.0027 (MYC versus MYC plus anti-CXCR2 (n = 5), Nppb), P < 0.001 (GFP (n = 12) versus MYC (n = 9), Myh7), P = 0.0040 (MYC versus MYC plus anti-CXCR2 (n = 5), Myh7);…

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  Extended Data Fig. 1. Single-cell RNA-seq of non-cardiomyocytes in the murine heart. Related to Fig. 1. a: t-distributed stochastic neighbour embedding (t-SNE) visualisation of unsupervised clustering of non-cardiomyocytes. The cells (dots) are coloured according to the cell clusters, and the numbers represent cluster numbers. b: Non-cardiomyocytes classification based on gene expression using t-SNE. Non-cardiomyocytes were defined as cells expressing each marker gene. Marker genes include Car4 (capillary endothelium), Cd14 (macrophage), Acta2 (smooth muscle cells), Efnb2 (arterial endothelium), Cd80 (dendritic cells), Vwf (vein endothelium), Mki67 (Mki67 positive endothelium), Plp1 (nerves), Mmrn1 (lymphatic endothelium), Cd79a (B cells), and Cd3g (T cells). c: Violin plot depicting HF-Fibro (cluster 4) expression of Postn but not Acta2 or Chad. d: Violin plot showing the expression of transcription factors in six clusters. e: FACS analyses of PDGFRα and MYC expression in sham (TAC0w), TAC4w, TAC6w, and TAC 8w hearts.

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  Extended Data Fig. 2. Generation of cardiac fibroblast-specific MYC overexpression mice. Related to Fig. 2. a: Bar plots illustrating left ventricle chamber size and wall thickness by echocardiography in control and OE mice pre- and post-pressure overload (TAC0w, n = 6 [control] and 5 [OE]; TAC2w, 5 and 5; TAC4w, 6 and 5; TAC8w, 5 and 5; TAC12w, 4 and 5). Tcf21 iCre-R26StopFLMYC mice were used as controls. b: Kaplan–Meier survival curves for control and OE mice after TAC were compared using the Gehan-Breslow-Wilcoxon test (n = 10 [control] and 20 [OE]). Source data

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  Extended Data Fig. 3. Generation of cardiac fibroblast-specific MYC knockout mice. Related to Fig. 3. a: FACS analyses of MYC-expressed fibroblasts in MYC KO hearts of TAC 12w. Negative control, secondary antibody alone. b: Bar plots showing left ventricle chamber size and wall thickness by echocardiography in control and MYC KO mice pre- and post-pressure overload (TAC0w, n = 5 [control] and 6 [KO]; TAC2w, 4 and 6; TAC4w, 5 and 6; TAC8w, 4 and 6; TAC12w, 4 and 6; TAC14w, 4 and 5; TAC16w, 4 and 5; TAC20w, 3 and 4). Tcf21 iCre-MYC flox mice were used as controls. *, p < 0.05; p = 0.018 (LVDs, TAC20w); significance was determined using the two-sided Holm-Sidak test for mean comparisons. Source data are provided as a Source Data file. Source data

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  Extended Data Fig. 4. RNA-seq of WT control mice, WT-TAC mice, and KO-TAC mice. Related to Fig. 4. a: Principal component analysis visualisation (PC1-PC2) showing global gene expression in hearts of WT control, WT-TAC, and KO-TAC mice (n = 3 each). b: Gene ontology (GO) analysis of differentially expressed genes. Left, Genes downregulated in WT-TAC mice vs. WT control mice. Right, Genes upregulated in KO-TAC mice vs. WT-TAC mice. c: GSEA of fibrosis-associated genes, indicating upregulation in WT-TAC mice vs. WT control mice and no significant difference between KO and WT-TAC mice.

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  Extended Data Fig. 5. Identifying secreted factors, such as CXCL1, as a direct target of MYC. Related to Fig. 5. a: GO analysis bar graph for 21 candidate genes, revealing Cxcl1 and Tnfsf9 as intercellular signalling molecules. b, Relative mRNA expression of Cxcl1 and Tnfsf9 in CFs overexpressing MYC compared to that in the control (GFP). Data are shown as mean ± SD. *, p < 0.05; p = 0.0079 (CXCL1); significance was determined using the two-sided Mann-Whitney test for mean comparisons. Source data are provided as a Source Data file. c: ChIP-qPCR showing MYC at Cdh1, as a negative control, and Snail1, as a positive control, in MYC-overexpressing CF. IgG, nonspecific IgG control; MYC, anti-MYC antibody. d: Relative mRNA expression of the HF marker Myh7 after stimulation with CXCL1, measured by RT-qPCR. Data are shown as mean ± SD. *, p < 0.05, n.s., not significant; p = 0.26 (Control n = 6, CXCL1 n = 3); significance was determined using the two-sided Mann-Whitney test for mean comparisons. Source data are provided as a Source Data file. Source data

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  Extended Data Fig. 6. Effect of CXCR2 neutralising antibody and targeted knockdown of CXCL1 in CFs and CXCR2 in cardiomyocytes. Related to Fig. 6. a: Experimental scheme of TAC mice injected with a neutralising antibody against CXCR2. b: Bar plots showing left ventricle chamber size and wall thickness measured by echocardiography in control and anti-CXCR2 mice after TAC. Mean and standard error are shown (TAC0w, n = 8 [control] and 7 [anti-CXCR2]; TAC2w, 6 and 6; TAC4w, 6 and 6; TAC8w, 6 and 7; TAC12w, 8 and 7; TAC14w, 5 and 6; TAC16w, 7 and 6). *, p < 0.05; p = 0.040 (LVDs, TAC16w); significance was determined using the two-sided Holm-Sidak test for mean comparisons. Source data are provided as a Source Data file. c: Bar plots showing left ventricle chamber size and wall thickness measured by echocardiography in control and shCXCR2 mice after TAC. Mean and standard error are shown (n = 6 [control] and 5 [shCXCR2] for TAC0w, TAC2w, TAC4w, TAC6w, TAC8w, and TAC10w). *, p < 0.05; p = 0.015 (LVDd, TAC10w), p = 0.0020 (LVDs, TAC10w); significance was determined using the two-sided Holm-Sidak test for mean comparisons. Source data are provided as a Source Data file. d: Bar plots showing cardiac function,…