Single-cell transcriptomics defines heterogeneity of epicardial cells and fibroblasts within the infarcted murine heart

  1. Julia Hesse
  2. Christoph Owenier
  3. Tobias Lautwein
  4. Ria Zalfen
  5. Jonas F Weber
  6. Zhaoping Ding
  7. Christina Alter
  8. Alexander Lang
  9. Maria Grandoch
  10. Norbert Gerdes
  11. Jens W Fischer
  12. Gunnar W Klau
  13. Christoph Dieterich
  14. Karl Köhrer
  15. Jürgen Schrader

  • Curated by eLife

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    Evaluation Summary:

    The article by Hesse et al. defines heterogeneity of epicardial cells and fibroblasts in a murine model of cardiac injury to analyze the resulting populations through single cell RNA sequencing. Spatial confirmation of associated markers is performed using in-situ RNA hybridization. The work provides new insights into the heterogeneity of epicardial stromal and activated cardiac stromal cells post-injury.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

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Abstract

In the adult heart, the epicardium becomes activated after injury, contributing to cardiac healing by secretion of paracrine factors. Here, we analyzed by single-cell RNA sequencing combined with RNA in situ hybridization and lineage tracing of Wilms tumor protein 1-positive (WT1 + ) cells, the cellular composition, location, and hierarchy of epicardial stromal cells (EpiSC) in comparison to activated myocardial fibroblasts/stromal cells in infarcted mouse hearts. We identified 11 transcriptionally distinct EpiSC populations, which can be classified into three groups, each containing a cluster of proliferating cells. Two groups expressed cardiac specification markers and sarcomeric proteins suggestive of cardiomyogenic potential. Transcripts of hypoxia-inducible factor (HIF)-1α and HIF-responsive genes were enriched in EpiSC consistent with an epicardial hypoxic niche. Expression of paracrine factors was not limited to WT1 + cells but was a general feature of activated cardiac stromal cells. Our findings provide the cellular framework by which myocardial ischemia may trigger in EpiSC the formation of cardioprotective/regenerative responses.

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  1. Version published to 10.7554/elife.65921 on eLife
  2. eLife

    Author Response:

    Reviewer 2:

    Hesse et al. implemented a murine model of cardiac ischemia to study two populations, epicardial stromal cells (EpiSC) and activated cardiac stromal cells (aCSC). Furthermore, uninjured cardiac stromal cells were used as a control. An isolation method for EpiSC was used by applying a gentle shear force to the cardiac surface. The authors show heterogeneity in the Epi-SC populations. Certain markers were confirmed by in-situ hybridization. Furthermore, molecular programs within these subsets were explored. A comparison between EpiSC and aCSCs cells (and EpiSC and uninjured CSCs cells) was performed, which showed differences in expression of multiple genes namely HOX, HIF1 and cardiogenic factor genes. A WT1 population was marked by tdTomato, confirming the localization of expression to a cell population. There are however specific weaknesses. First, a major concern is regarding clarity of the experimental conditions and sample purity. Data is not robustly presented showing differences across conditions, namely between uninjured CSCs and activated CSCs. Furthermore, the purity of isolating EpiSC was not explored, along with the anticipated overlap of cells between aCSC and EpiSC. Specifically, the in-situ findings do not clarify the subject of purity. For example, EpiSC-3 (Pcsk6) is a large population in the scRNA-seq shown in Fig 1; however, this gene is also expressed in the myocardium. There is an attempt to perform EpiSC and aCSC comparison analysis in Figure 3; however without clarity the expected overlap, these data are hard to interpret. Furthermore, cluster-based approaches for comparing population fractions can be problematic due to the inherent stochasticity of sampling. Lastly, there is no actual lineage tracing over time, but rather marking of WT1 cells with tdTomato. The RNA velocity analysis is not particularly robust with the number of expressed genes driving these results, rather than the author's conclusion of developmental potential.

    As to the clarity of experimental conditions and sample purity, a detailed protocol and comprehensive validation data of our cell isolation procedure have been previously published (Owenier et al. 2020, Cardiovasc Res, Apr 1;116(5):1047-1058), including analyses of cell yield, viability, and purity.

    As to the differences between uninjured CSC and activated CSC, there are already elaborate studies published that specifically address the activation of CSC after MI on single-cell level (Farbehi et al. 2019, eLife, Mar 26;8:e43882; Forte et al. 2020 Cell Rep Mar 3;30(9):3149-3163.e6). Therefore, this comparison was deliberately not included into our manuscript but instead we focused on the novel scRNAseq data of the post-MI epicardium.

    As to the RNA-in situ-hybridization experiments, hybridization data certainly do not clarify the purity of the EpiSC preparation. These experiments were aimed to confirm the presence and the location of selected cellular markers in the post-MI epicardium. Specifically, we concluded that cells expressing Pcsk6 are present in the epicardium, which is consistent with the Pcsk6-expressing EpiSC population in the scRNAseq data set; we did not claim Pcsk6 to be a unique marker for the epicardium.

    As to the potential overlap between EpiSC and aCSC, the issue of sample purity and estimation of populations sizes in the heart has been extensively discussed previously (Owenier et al. 2020, Cardiovasc Res, Apr 1;116 (5):1047-1058). Our study included 13,796 EpiSC and 24,470 aCSC which is sufficient to reduce data artefacts by the inherent stochasticity of sampling.

    As to the tracing of WT1+ cells, the labelling of WT1+ cells was induced 5 and 3 days prior to MI and they were traced 5 days after MI to analyze their contribution to the epicardial stromal cell populations formed during the acute injury response. Since the explorative power of WT1+ lineage tracing, RNA velocity and number of expressed genes as measure for developmental potential each by itself may have limitations, we would like to emphasize that our main conclusions are drawn by combining the evidence from the different sets of analyses. In order to further clarify data representation and interpretation we will carefully revise the respective text passages of our manuscript to avoid misunderstandings.

  3. eLife

    Reviewer #3 (Public Review):

    The article by Hesse, Owenier et al entitled "Single-cell transcriptomics 1 defines heterogeneity of epicardial cells and fibroblasts within the infarcted heart" will be of interest to the readers of heart regeneration, as it helps in understanding how epicardial cells contribute to heart regeneration following myocardial Infarction. Hesse, Owenier et al. investigate the role of epicardial stromal cells(EpiSC) after arterial ligation induced myocardial infarction (MI) in mouse. They perform single-cell RNASeq (scRNASeq) on isolated, FAC sorted, epicardial stromal cells, activated cardiac stromal cells (aCSC) from infarcted hearts and control cardiac stromal cells (CSC). The authors find 11 cell clusters of EpiSC. They confirmed the spatial localization of the different clusters by in situ hybridization and performed Gene ontology studies to understand the biological processes affected by those clusters. They found that those clusters fall into three major functional groups, as follows: 1) Wt1 expressing and cardiogenic factor expressing, 2) chemokine expressing and HOX genes expressing and 3) cardiogenic factor expressing. Interestingly there are two identified groups which express different cardiogenic factors 1) Wt1 positive with cardiogenic factors MESP1, WNT11, ISL11, TBX5, GATA 4,5,6 and the other group 3) Wt1-with Nkx2.5, BMP2 and BMP4. Authors show that multiple clusters are enriched in Hif1a, Hif1a related genes and glycolysis related genes which are known to be downstream of Wt1 cells. To further understand the hierarchical development of the EpiSC cells, the authors performed pseudo time-series analysis using RNA Velocity analysis on Wt1 reporter mice and find three different groups. Interestingly Wt1+ cells did not convert into other cell types. They further performed ligand receptor analysis to find interactions between different cell types. The authors implemented scRNAseq for aCSC and find cell clusters ECM rich cells, fibroblasts, interferon expressing cells, and cycling fibroblasts/myofibroblasts. They further compared the transcriptional profiles of EpiSC with the aCSC. They found gene sets, which are specific for EpiSC, and genes that are specific for aCSCs. Specifically, they found that Hif1a, glycolysis responsive genes, and cardiac contractile proteins were highly expressed in EpiSCs. Furthermore, the authors showed that the transcriptional profile of EpiSC, aCSC and CSC are different.

    These data add an important knowledgebase to the understanding of the transcriptional landscape of the Epicardial stromal cells and would help identifying specific pathways/transcriptional genes which are activated during myocardial infarction.

  4. eLife

    Reviewer #2 (Public Review):

    Hesse et al. implemented a murine model of cardiac ischemia to study two populations, epicardial stromal cells (EpiSC) and activated cardiac stromal cells (aCSC). Furthermore, uninjured cardiac stromal cells were used as a control. An isolation method for EpiSC was used by applying a gentle shear force to the cardiac surface. The authors show heterogeneity in the Epi-SC populations. Certain markers were confirmed by in-situ hybridization. Furthermore, molecular programs within these subsets were explored. A comparison between EpiSC and aCSCs cells (and EpiSC and uninjured CSCs cells) was performed, which showed differences in expression of multiple genes namely HOX, HIF1 and cardiogenic factor genes. A WT1 population was marked by tdTomato, confirming the localization of expression to a cell population. There are however specific weaknesses. First, a major concern is regarding clarity of the experimental conditions and sample purity. Data is not robustly presented showing differences across conditions, namely between uninjured CSCs and activated CSCs. Furthermore, the purity of isolating EpiSC was not explored, along with the anticipated overlap of cells between aCSC and EpiSC. Specifically, the in-situ findings do not clarify the subject of purity. For example, EpiSC-3 (Pcsk6) is a large population in the scRNA-seq shown in Fig 1; however, this gene is also expressed in the myocardium. There is an attempt to perform EpiSC and aCSC comparison analysis in Figure 3; however without clarity the expected overlap, these data are hard to interpret. Furthermore, cluster-based approaches for comparing population fractions can be problematic due to the inherent stochasticity of sampling. Lastly, there is no actual lineage tracing over time, but rather marking of WT1 cells with tdTomato. The RNA velocity analysis is not particularly robust with the number of expressed genes driving these results, rather than the author's conclusion of developmental potential.

  5. eLife

    Reviewer #1 (Public Review):

    In the manuscript "Single-cell transcriptomics defines heterogeneity of epicardial cells and fibroblasts within the infarcted heart", the authors isolated epicardial stromal cells (EpiSC) and cardiac interstitial/stromal cells (termed active CSCs) from the same I/R heart and identified transcriptionally distinct subpopulation of EpiSCs via 10x genomics technology. They also performed transcriptome profile comparison between EpiSCs and aCSCs. This manuscript shows rigorous scientific investigation. Their isolation protocol is supported by their previous publication. Method section documented in detail of step-wise QC process of bioinformatics analysis. In summary, the analysis identified 11 clusters of EpiSC, some of which overlap with the well-established epicardial marker WT1 with confirmed in situ anatomical localization. When compared to aCSC, the two groups showed clear different function/states as expected. In the lineage tracing model, RNA velocity predicts cell hierarchy, cell-cell communication between populations, as well as cell cycle activity. Overall this manuscript provides a significant degree of information that can be helpful to the field.

  6. eLife

    Evaluation Summary:

    The article by Hesse et al. defines heterogeneity of epicardial cells and fibroblasts in a murine model of cardiac injury to analyze the resulting populations through single cell RNA sequencing. Spatial confirmation of associated markers is performed using in-situ RNA hybridization. The work provides new insights into the heterogeneity of epicardial stromal and activated cardiac stromal cells post-injury.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

  7. Version published to 10.1101/2021.01.26.428270v1 on bioRxiv