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YAP induces a neonatal-like pro-renewal niche in the adult heart

Nature Cardiovascular Research volume 3, pages 283–300 (2024)Cite this article

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Abstract

After myocardial infarction (MI), mammalian hearts do not regenerate, and the microenvironment is disrupted. Hippo signaling loss of function with activation of transcriptional co-factor YAP induces heart renewal and rebuilds the post-MI microenvironment. In this study, we investigated adult renewal-competent mouse hearts expressing an active version of YAP, called YAP5SA, in cardiomyocytes (CMs). Spatial transcriptomics and single-cell RNA sequencing revealed a conserved, renewal-competent CM cell state called adult (a)CM2 with high YAP activity. aCM2 co-localized with cardiac fibroblasts (CFs) expressing complement pathway component C3 and macrophages (MPs) expressing C3ar1 receptor to form a cellular triad in YAP5SA hearts and renewal-competent neonatal hearts. Although aCM2 was detected in adult mouse and human hearts, the cellular triad failed to co-localize in these non-renewing hearts. C3 and C3ar1 loss-of-function experiments indicated that C3a signaling between MPs and CFs was required to assemble the pro-renewal aCM2, C3+ CF and C3ar1+ MP cellular triad.

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Fig. 1: Combining single-cell and ST to assay in vivo cardiac renewal.
Fig. 2: scRNA-seq and ST integration identifies two distinct CM populations.
Fig. 3: aCM2 is induced by injury in the neonatal heart and co-localizes with C3-expressing CFs and C3ar1-expressing MPs.
Fig. 4: Signaling among CMs, CFs and MPs in YAP5SA hearts.
Fig. 5: C3 loss of function in AAV-YAP5SA mice leads to decreased cell cycle progression.
Fig. 6: Knockout of C3ar1 in AAV-YAP5SA-injected mice leads to decreased sarcomere disassembly and prolongation of cell cycle progression.
Fig. 7: Knockout of C3ar1 impairs neonatal cardiac renewal after MI.
Fig. 8: Knockout of C3 alters metabolism-related transcription and reduces CM proliferation in the neonatal heart.

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Data availability

All data generated or analyzed during this study are included in this published article and its supplementary information files. Source Data are provided with the manuscript. All raw and processed sequencing data are deposited at the National Center for Biotechnology Informationʼs Gene Expression Omnibus (GEO): GSE217828 and GSE152856. Previously published datasets used in this study include SCP498 deposited in the Broad Institute’s Single Cell Portal, ERP12313 deposited in the Human Cell Atlas Data Coordination Platform and GSE130699 and GSE163631 deposited in the GEO.

Code availability

In-house code for reproducing all bioinformatics analyses is available at GitHub (https://github.com/XL-Genomics/2023_YAP_Induced_Regenerative_Cardiac_Niche).

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Acknowledgements

This work is supported by the National Heart, Lung, and Blood Institute (HL127717, HL130804 and HL118761 to J.F.M.); the American Heart Association (849706 to S.L., 903651 to R.G.L. and 903411 to F.M.); the Don McGill Gene Editing Laboratory of the Texas Heart Institute (X.L.); and the Vivian L. Smith Foundation (J.F.M.).

We thank T. T. Tran of Baylor College of Medicine for assistance with animal surgical procedures, H. Zheng of Baylor College of Medicine for providing C3ar1-TDTomato mice and N. Stancel of the Scientific Publications Department of the Texas Heart Institute for editorial support. Figures 1a, 4e, 7a, 7d and 8a were created with BioRender.

Author information

Author notes

  1. These authors contributed equally: Rich Gang Li, Xiao Li.

Authors and Affiliations

  1. Cardiomyocyte Renewal Laboratory, Texas Heart Institute, Houston, TX, USA

    Rich Gang Li, Yuka Morikawa, Francisco J. Grisanti-Canozo, Jong Kim, Shijie Liu & James F. Martin

  2. McGill Gene Editing Laboratory, Texas Heart Institute, Houston, TX, USA

    Rich Gang Li, Xiao Li, Yi Zhao, Lin Liu & James F. Martin

  3. Department of Integrative Physiology, Baylor College of Medicine, Houston, TX, USA

    Francisco J. Grisanti-Canozo, Fansen Meng, Chang-Ru Tsai, Bing Xie, Elzbieta Klysik, Md Abul Hassan Samee & James F. Martin

  4. Center for Organ Repair and Renewal, Baylor College of Medicine, Houston, TX, USA

    James F. Martin

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Contributions

Conceptualization: R.G.L. and J.F.M. Formal analysis: R.G.L., X.L., F.J.C., Y.Z. and J.K. Investigation: R.G.L., X.L., Y.M., F.M., C.T., L.L., B.X. and E.K. Methodology: R.G.L., X.L., S.L., Y.M., F.M., M.H.S. and J.F.M. Writing (original draft): R.G.L. and J.F.M. Writing (review and editing): R.G.L., X.L., Y.M., F.J.C., F.M., S.L., B.X., M.H.S. and J.F.M.

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Correspondence to James F. Martin.

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Competing interests

J.F.M. is a co-founder of and owns shares in YAP Therapeutics. He is a co-inventor on the following patents associated with this study: patent no. US-20200206327-A1 (‘Hippo pathway deficiency reverses systolic heart failure post-infarction’), allowed, not issued yet, applicant: Baylor College of Medicine, inventor: James F. Martin; patent covers Figs. 4–6 and Extended Data Figs. 8 and 9 of the present manuscript; patent no. US-20220411798-A1 (‘Hippo and dystrophin complex signaling in cardiomyocyte renewal’), still in prosecution, applicants: Baylor College of Medicine and Texas Heart Institute, inventor: James F. Martin; patent covers Figs. 4–6 and Extended Data Figs. 8 and 9 of the present manuscript; and patent no. US-11459565-B2 (‘Hippo and dystrophin complex signaling in cardiomyocyte renewal’), granted, applicants: Baylor College of Medicine and Texas Heart Institute, inventor: James F. Martin; patent covers Figs. 4–6 and Extended Data Figs. 8 and 9 of the present manuscript. Y.M. is a co-inventor on the above-indicated patents associated with this study. The remaining authors declare no competing interests.

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Nature Cardiovascular Research thanks Sikander Hayat and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Spatial transcriptomics and scRNAseq quality control.

a, H&E staining of tissue sections showing no gross morphologic differences between control and YAP5SA hearts. Scale = 1000 µm. b, Spatial transcriptomics dataset (n = 1 per genotype) with the number of genes (median: control, 2358; YAP5SA, 3323) and counts (average: control, 12,804; YAP5SA, 15,101) captured per spot. c, Correlation of gene expression between control and YAP5SA hearts (R2 = 0.96). d, Differentially expressed genes between YAP5SA and control hearts with increased expression of representative Yap target genes Ccnd1, Anxa2, Sptan1, Ccl7, and Timp1 in YAP5SA hearts. e, Single-cell RNA-seq cell-type composition across replicates shows increased MPs in YAP5SA hearts (n = 3 per genotype). f, aCM1 proportion is decreased and aCM2 proportion is increased in YAP5SA compared to Control hearts (n = 3 per genotype). g, Pseudotime analysis using Slingshot trajectory inference showing potential transition from aCM1 to aCM2. h, Gene expression module modeled as a function of progression through the pseudotemporal trajectory. The 62 temporally correlated genes increasing during the transition from aCM1 to aCM2 are listed in Supplementary Table 2. Center lines in all box plots are shown as mean values and whiskers extended to a maximum of 1.5 x interquartile range beyond the boxes.

Extended Data Fig. 2 Validation of aCM2 marker genes.

a, GO analysis of upregulated genes in control murine aCM2 versus aCM1 showing increased structural gene expression, with the top GO terms and associated upregulated genes shown. b, GO analysis of downregulated genes in control murine CM2 versus control CM1 showing decreased metabolic processes, with the top GO terms and associated downregulated genes shown. c, Immunostaining showing Tβ4 (red) in the subendocardium and subepicardium of a wildtype control heart (left, scale = 100 μm). WGA (green) and DAPI (blue) stains were used for cell membrane and nuclei staining. Zoomed-in regions showing Tβ4 expression in cardiomyocytes of the subendocardium, no Tβ4 expression in the midmyocardium, and Tβ4 expression in the subepicardium (right, scale = 50 μm). d, Tβ4 expression is greatly increased in YAP5SA hearts (left, scale = 100 μm). High magnification images showing expression of Tβ4 in YAP5SA-activated CMs (FLAG positive, green) with sarcomere disassembly (CTnT, grey) (right, scale = 20 μm). e, aCM2 marker gene Lmcd1 (red) is expressed in some control subendocardial CMs (yellow arrows) and is increased in YAP5SA CMs. Scale = 50 μm. f, aCM2 marker gene Acta2 (red) is expressed in some control subendocardial CMs (yellow arrows) and is increased in YAP5SA CMs. Scale = 50 μm. g, Smooth muscle actin (αSMA), encoded by Acta2, is expressed in control subendocardial CMs (yellow arrowheads) and significantly increased in dedifferentiating YAP5SA CMs, identified by disorganized CTnT expression. Scale = 100 μm. h, High magnification images of transverse and longitudinal axes show colocalization of α-SMA with CMs undergoing sarcomere disassembly. Scale = 50 μm. Statistical significance for enriched GO terms was determined using a one-sided Fisher’s exact test, with an adjusted p value (Benjamini-Hochberg correction) < 0.05.

Extended Data Fig. 3 The adult human heart contains aCM2-like CMs.

a, Original annotation of human scRNA-seq data by Litviňuková et al.24 showing atrial and ventricular CMs (left). Subclustering of ventricular CMs revealed 14 distinct CM populations (right). b, Expression profile of CM2 marker genes in the 14 CM populations showed the highest expression in CM_8. The complement regulator CLU was also highly expressed in CM_8. c, Compared with the other CM populations, CM_8 showed the highest CM2 similarity score (***p < 0.001, one-way ANOVA with multiple comparisons, cell numbers in the 14 CM clusters range from 545 to 21,334). d, Volcano plot of differentially expressed genes between CM_8 and all other CM populations showing the upregulation of multiple CM2 marker genes. e, The top GO terms for upregulated genes in CM_8 were cytoskeletal organization terms, similar to those in CM2. f, Cellular origin composition of CM_8 showing that 9% of cells originated from the left ventricular apex, 24% from the left ventricle, 41% from the right ventricle, and 24% from the interventricular septum. g, Representative human heart ST control and MI samples from Kuppe et al.15 with annotations of remote, border, and ischemic zones based on Calcagno et al.25. h, aCM1 and aCM2 scores for the representative tissue sections showing an increase of aCM2 signature in border zones of MI hearts. i, Quantification of 18 hearts from Kuppe et al.15 shows aCM1 is downregulated in both border and ischemic zones, while aCM2 is upregulated in the border zone compared to ischemic and remote zones. C3+ CF and C3ar1+ MP scores are increased in the ischemic zone but not in the border zone compared to the remote zone. Statistical significance was determined using a two-sided pairwise t-test. Center lines in all box plots are shown as mean values and whiskers extended to a maximum of 1.5 × interquartile range beyond the boxes.

Extended Data Fig. 4 Spatial niche analysis of Control versus YAP5SA hearts.

a, Pairwise co-localization between the major cell types based on deconvolved proportions shows MP significantly co-localizes with EC2, CF and aCM2 but not with aCM1. b, Unsupervised clustering of spots in the ST data yielded seven spatial niches for each genotype. c, Spatial niches mapped onto Control and YAP5SA hearts. d, Control Niche 5 (C5) is abundant in aCM2, CF, and EC2, while YAP5SA Niche 3 and 5 (Y3 & Y5) contains higher amount of aCM2, CF, and MP than other niches. Asterisks indicate increased composition of a cell type in a niche compared with other niches (one-sided Wilcoxon rank sum test, adjusted (adj.) P < 0.01).

Extended Data Fig. 5 C3+ CFs express cardioprotective and antifibrotic genes and is conserved between mouse and human.

a, Immunofluorescence staining of CM marker α-actinin (green), C3 (red), nuclei counterstain DAPI (blue), and cell membrane marker wheat germ agglutinin (WGA, grey) shows expression of C3 in the subendocardium of control hearts. Scale = 50 μm. b, C3 (green) is colocalized with fibroblast marker vimentin (red). Scale = 20 μm. c, C3 (red) expression is increased in YAP5SA compared to Control hearts. Scale = 50 μm. d, Quantification of C3+ and vimentin+ cells in Control and YAP5SA hearts (n = 5 per genotype). e, Proportion of CFs expressing C3 in Control and YAP5SA hearts (n = 3 per genotype). f, Volcano plot (left) showing differentially expressed genes between C3-positive and C3-negative CFs and categorized as GO terms (right) showing increased expression of anti-fibrotic genes (Dcn, Igfbp4, and Cst3) and cardioprotective genes (Mt1, Mt2, and Fstl1) by C3-positive CFs. g, Ligand-receptor analysis from C3- or C3+ CFs to aCM1 and aCM2. VCAM, SEMA3, and VEGF pathway genes are upregulated in YAP5SA hearts. h, CFs from Litviňuková et al. and Tucker et al. shows a subpopulation of CFs with high C3 expression24,33. i, Volcano plot of differentially expressed genes between human C3+ CFs and C3- CFs shows the upregulation of many of the same genes as in the mouse, including FSTL1, DCN, CST3, and IGFBP4. GO analysis of genes upregulated in human C3+ versus C3- CFs shows extracellular matrix organization and immune activation terms, similar to the categories observed in murine C3+ CFs. Error bars indicate means ± s.e.m. Statistical significance was determined using a two-tailed Wilcoxon rank sum test. Center lines in all box plots are shown as mean values and whiskers extended to a maximum of 1.5 × interquartile range beyond the boxes.

Source data

Extended Data Fig. 6 aCM2 is induced following myocardial infarction.

a, UMAP projection of single-cell RNA sequencing data from Wang et al.35. b, The proportion of C3-expressing CFs increase after MI in both P1 and P8 hearts. c, P1 sham hearts have higher proportion of C3ar1-expressing MPs compared to P8 sham hearts. P1 MI induces a greater increase in C3ar1-expressing MPs compared to P8 MI. d, H&E staining of P1 sham and MI heart sections 3 days and 7 days post-surgery (P1 Sham D3, P1 MI D3, P1 Sham D7, P1 MI D7) used for ST in Cui et al.36.

Extended Data Fig. 7 C3ar1+ macrophages are anti-inflammatory and signal to cardiomyocytes.

a, Spatial colocalization probability of ligand-receptor gene pairs (Cx3cl1-Cx3cr1, Thbs1-Cd47, and Thbs1-Cd36) are increased in YAP5SA compared to Control hearts. b, UMAP projection of Control and YAP5SA myeloid cells. c, Most MP populations are increased in YAP5SA compared to Control hearts, with MP2 being most increased. d, MP2 macrophages are the most increased fraction in YAP5SA compared to control hearts. e, GO analysis of upregulated genes in C3ar1+ MPs versus C3ar1- MPs reveals terms associated with anti-inflammatory M2 MPs, such as anti-apoptosis, wound healing, and angiogenesis. Statistical significance was determined by one-sided Fisher’s exact test, with an adjusted p value (Benjamini-Hochberg correction) < 0.05. f, Differentially expressed genes include markers of M2 (Mertk, Mrc1, Il10, Maf, Cd163, Cd68, Cd36), cardiac growth (Igf1), anti-apoptosis (Gdf15), wound healing (F13a1), angiogenesis (Tnfsf12), and ECM degradation (Adam15). Statistical significance was determined by two-tailed Wilcoxon rank sum test. g, Ligand-receptor analysis from C3ar1+ MPs to aCM2 shows increased TNF, ADAM15, GDF, and IGF signaling. h, The expression of the MP ligand genes Igf1, Adam15, and Tnfsf12 are specific to C3ar1+ MPs increased in YAP5SA compared to Control. i, Colocalization of Igf1, Adam15, and Tnfsf12 expressing spots with C3ar1+ MPs in YAP5SA ST data. j, The expression of the aCM2 receptor genes Igf1r, Itgb1, and Tnfrsf12a are increased in YAP5SA compared to Control. k, Igf1r, Itgb1, and Tnfrsf12a expressing spots colocalize with aCM2 but not aCM1 in YAP5SA ST data. Statistical significance was determined by spatial colocalization testing (detailed in Methods).

Extended Data Fig. 8 AAV-GFP and AAV-YAP5SA are induced robustly in wildtype and C3−/− hearts.

a, Wildtype (WT) and C3−/− mice injected with AAV-GFP shows high transduction efficiency in cardiomyocytes, as seen in GFP expression (green). Wildtype (WT) and C3−/− mice injected with AAV-YAP5SA shows high transduction efficiency of YAP5SA, as evidenced by FLAG expression (red), in cardiomyocytes. Scale = 100 μm. b, Flow cytometry gating strategy for obtaining DAPI+ singlets (Non-CMs). c, Low magnification images showing increased sarcomere disassembly (CTnT, grey) in WT + AAV-YAP5SA compared to C3−/− + AAV-YAP5SA hearts. Scale = 50 μm.

Extended Data Fig. 9 AAV-GFP and AAV-YAP5SA are induced robustly in wildtype and C3ar1−/− hearts.

a, Wildtype (WT) and C3ar1−/− mice injected with AAV-GFP shows high transduction efficiency in cardiomyocytes, as seen in GFP expression (green). Wildtype (WT) and C3ar1−/− mice injected with AAV-YAP5SA shows high transduction efficiency of YAP5SA, as evidenced by FLAG expression (red), in cardiomyocytes. Scale bars = 100 μm. b, Representative images of S to G2 marker CCNA2 (red) shows a decrease in C3ar1−/− + AAV-YAP5SA CMs compared to WT + AAV-YAP5SA CMs. Scale bars = 100 μm. c, Survival of mice injected with AAV-GFP or AAV-YAP5SA. WT mice have reduced survival at D6 compared to C3ar1-/- and C3-/- mice injected with AAV-YAP5SA (53.3% vs. 93.3% and 100%). Statistical significance was determined using a chi-squared test.

Extended Data Fig. 10 Single-nucleus RNA sequencing of WT and C3−/− P2 MI 3 DPI hearts.

a, Integrated UMAP projection of WT MI and C3−/− MI hearts 3 DPI (n = 2 each group). b, Quantification of basal ATP production rate, comprised of mitochondrial and glycolytic ATP production, in cultured WT and C3−/− cardiac fibroblasts (n = 20 wells). c, Cardiomyocyte marker genes showing a proliferative cluster expressing Top2a, Mki67, and Pcna. d, Feature plots showing aggregated proliferative gene signatures and example genes expressed by the Prol. CM cluster. Statistical significance was determined using a two-sided t-test. Error bars indicate means ± s.e.m.

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Supplementary Tables 1–10

Supplementary Table 1 Top cell type markers for deconvolution. Supplementary Table 2 aCM1 to aCM2 trajectory correlated genes. Supplementary Table 3 aCM2 versus aCM1 differentially expressed genes. Supplementary Table 4 MI zone markers for annotating human MI ST data. Supplementary Table 5 aCM2 spots upregulated genes. Supplementary Table 6 Odds ratio of co-localization with aCM2. Supplementary Table 7 Spatial niche marker genes. Supplementary Table 8 C3ar1+ MP versus C3ar1− MP differentially expressed genes. Supplementary Table 9 C3KO MI versus WT MI differentially expressed genes in CF cluster 1. Supplementary Table 10 C3KO MI versus WT MI differentially expressed genes in all CM clusters.

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Li, R.G., Li, X., Morikawa, Y. et al. YAP induces a neonatal-like pro-renewal niche in the adult heart. Nat Cardiovasc Res 3, 283–300 (2024). https://doi.org/10.1038/s44161-024-00428-w

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