Single-cell analysis uncovers that metabolic reprogramming by ErbB2 signaling is essential for cardiomyocyte proliferation in the regenerating heart
- Hubrecht Institute-KNAW and University Medical Center Utrecht, Netherlands
- Weizmann Institute of Science, Israel
- University Medical Center Utrecht, Utrecht University, Netherlands
- Duke University Medical Center, United States
- Vrije Universiteit Amsterdam, Netherlands
- Ulm University, Germany
- Max Planck Institute of Immunobiology and Epigenetics, Germany
- VU University Medical Center, Netherlands
- University Medical Center Utrecht, Netherlands
Figures
Figure 1 with 4 supplements
Single-cell mRNA sequencing identifies different cardiomyocyte-populations in the injured zebrafish heart.
(a) Schematic of cryoinjury procedure on adult TgBAC(nppa:mCitrine) fish and immunohistochemistry on section of injured TgBAC(nppa:mCitrine) heart 7 dpi. Overview image on left and zoom-in of boxed …
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Figure 1—source data 1
Single-cell mRNA sequencing data from cryoinjured TgBAC(nppa:mCitrine) zebrafish hearts at 7 dpi.
Raw reads are represented per gene (rows) and single cell (columns).
- https://cdn.elifesciences.org/articles/50163/elife-50163-fig1-data1-v2.xlsx
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Figure 1—source data 2
List of the clusters in the adult injured heart (raw data see SD1), as identified by StemID and the associated cells per cluster.
- https://cdn.elifesciences.org/articles/50163/elife-50163-fig1-data2-v2.xlsx
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Figure 1—source data 3
List of differentially expressed genes between cardiomyocytes clusters 2 and 7 of the adult heart dataset.
Only genes with a p-value<0.05 are listed. Per gene, the log2 fold change, adjusted p-value (padj) and associated gene name are given. GO-terms for genes upregulated between clusters 2 and 7 (p<0.01) are listed in separate excel sheets.
- https://cdn.elifesciences.org/articles/50163/elife-50163-fig1-data3-v2.xlsx
Figure 1—figure supplement 1
TgBAC(nppa:mCitrine) expression recapitulates endogenous nppa gene expression.
(a) Design of the bacterial artificial chromosome (BAC) used for generation of the transgenic line TgBAC(nppa:mCitrine). (b) Transgenic mCitrine expression in the heart in relation to RFP expression …
Figure 1—figure supplement 2
Single-cell mRNA sequencing identifies different cell-populations in the injured zebrafish heart.
tSNE maps visualizing log2-transformed read-counts of genes with high expression in cardiomyocytes (mt-co1, tnnc1a, tnnt2a), in fibroblasts (fn1b), in endothelial cells (cdh5) and immune cells (ctsd)…
Figure 1—figure supplement 3
Cluster 7 cells display highest nppa:mCitrine expression.
(a) tSNE map of the cardiomyocyte clusters derived from the single-cell mRNA-sequencing. (b) mCitrine fluorescence levels of the nppa:mCitrine FACS sorted cells from injured hearts that were used …
Figure 1—figure supplement 4
Enhanced expression of genes elevated in cluster 7 versus cluster 2 is injury induced.
(a) In situ hybridizations for border zone genes nppb, mustn1, nppa and vmhc in zebrafish hearts at seven dpi compared to uninjured hearts. (b) In situ hybridizations for glycolysis genes hk1 and ldh…
Figure 2
Single-cell transcriptome of border zone cardiomyocytes resembles that of embryonic cardiomyocytes.
(a) Cartoon to illustrate the experimental procedure for single cell analysis of embryonic and adult cardiac cells. (b) tSNE map of combined adult (red) and embryonic datasets (light blue). (c) tSNE …
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Figure 2—source data 1
Single-cell mRNA sequencing data from Tg(cmlc2:GFP) zebrafish hearts at 2 days post fertilization.
Raw reads are represented per gene (rows) and single cell (columns).
- https://cdn.elifesciences.org/articles/50163/elife-50163-fig2-data1-v2.csv
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Figure 2—source data 2
List of the clusters from the embryonic heart data (SD5) identified by StemID and the associated cells for each cluster.
- https://cdn.elifesciences.org/articles/50163/elife-50163-fig2-data2-v2.xls
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Figure 2—source data 3
Combined single-cell mRNA sequencing data from embryonic and adult hearts.
Raw reads represented per gene (rows) and single cell (columns).
- https://cdn.elifesciences.org/articles/50163/elife-50163-fig2-data3-v2.csv
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Figure 2—source data 4
List of pairwise differentially expressed genes between all cardiomyocyte clusters from the combined embryonic and adult datasets.
Only genes with a p-value<0.01 are listed. Per gene, the log2 fold change, adjusted p-value (padj) and associated gene name are given. GO-terms for genes upregulated between clusters 5 and 6 (p<0.01) are listed in separate excel sheets.
- https://cdn.elifesciences.org/articles/50163/elife-50163-fig2-data4-v2.xls
Figure 3 with 1 supplement
Pseudo time analysis reveals dedifferentiation and metabolic changes in border zone cardiomyocytes.
(a) Bar plot of StemID scores for the cardiomyocyte clusters (clusters #2, 1, 4 and 7) calculated by the formula: number of significant links for each cluster multiplied by the median transcriptome …
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Figure 3—source data 1
List of genes that are differentially upregulated in the respective modules identified in the pseudo timeline analysis (related to Figure 3c).
- https://cdn.elifesciences.org/articles/50163/elife-50163-fig3-data1-v2.xlsx
Figure 3—figure supplement 1
Induction of hexokinase1 expression precedes expression of myomesin1b.
In situ hybridization for the glycolysis gene hexokinase1 (a,b) and the embryonic cardiac gene myomesin1b (c,d) on sections of injured zebrafish hearts at 3 dpi (a,c) and 7 dpi (b,d). While hk1 …
Figure 4 with 1 supplement
Border zone cardiomyocytes undergo a metabolic switch from mitochondrial OXPHOS to glycolysis.
(a) Succinate dehydrogenase (SDH) enzyme staining on a seven dpi heart section with injury area separated by dashed line. Quantification of SDH activity in remote zone (RZ), border zone (BZ) and …
Figure 4—figure supplement 1
Energy metabolism genes are differentially expressed between cluster 7 and cluster 2 cells.
(a) Plot showing differentially expressed genes between cluster 7 versus cluster 2 cells. Differentially expressed genes (p-value<0.05) are highlighted in red (upregulated in cluster 7 cells) and …
Figure 5
2-Deoxy glucose impairs cardiomyocyte proliferation.
(a) Experimental design for the 2-DG injections to inhibit glycolysis in injured zebrafish hearts. (b) Confocal image of injured zebrafish hearts at seven dpi either injected with PBS or 2-DG …
Figure 6 with 1 supplement
Nrg1/ErbB2 signaling induces glycolysis genes in zebrafish and mouse.
(a) Cartoon showing experimental procedure to induce cardiomyocyte specific Nrg1 expression in zebrafish. Panels show in situ hybridization for hexokinase 1 (hk1) and lactate dehydrogenase a (ldha) …
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Figure 6—source data 1
Oligonucleotide sequences for real-time PCR analysis.
- https://cdn.elifesciences.org/articles/50163/elife-50163-fig6-data1-v2.xlsx
Figure 6—figure supplement 1
Hypoxia sensor phd3:GFP expression does not correlate with expression of the glycolytic gene ldha.
(a) Overview picture of phd3:GFP heart 7dpi co-stained for expression of ldha by fluorescent ISH. (b) Zoom-ins of trabecular border zone cardiomyocytes. Note high ldha expression and low expression …
Figure 7 with 1 supplement
Glycolysis inhibitors impair mitogenic effect of ErbB2 activation in cardiomyocytes.
(a) Cartoon showing the experimental procedure to analyse the effects of glycolysis inhibitors (2-DG and lonidamine) on cardiomyocyte proliferation. (b) Immunofluorescence analysis on P7 cardiac …
Figure 7—figure supplement 1
Activation of ErbB2 signaling in murine MI model induces glycolytic gene expression while repressing mitochondrial genes.
(a) Cartoon showing experimental procedure. Myocardial infarction (MI) was induced by left anterior descending coronary artery ligation at the same day when Dox was removed to induce cardiomyocyte …
Author response image 1
Different read cut-offs do not alter clustering pattern, while altering number of included cells.
(A) Distribution of reads per cell. Green line represents 3,500 reads, red line indicates the mean number of reads per cell (10,443). (B) tSNE maps generated with different read cutoffs. Read …
Author response image 2
GO-term analysis on differentially expressed genes in cardiomyocyte clusters.
Representative GO-terms for cardiomyocyte clusters. GO-terms in upper table are obtained from differentially expressed genes between adult clusters 2 and 7 (p<0.01). Lower table GO-terms are …
Author response image 3
Glycolytic gene expression in the embryonic zebrafish heart.
Expression of eno1a (a, b, c, d), hk1 (e, f, g, h) and ldha (i, j, k, l) during zebrafish development at 28hpf (a, e, i), 48 hpf (b, f, j) and 5dpf (c, d, g, h, k, l) respectively. Outline of the …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Danio rerio) | Tupfel Long Fin (TL) | ZIRC | ZDB-GENO-990623–2 | |
| Genetic reagent (Danio rerio) | Tg(myl7:dsRED)s879Tg | Chi et al., 2008 | ZDB-FISH-150901–3078 | |
| Genetic reagent (Danio rerio) | Tg(myl7:GFP)twu34Tg | Huang et al., 2003 | ZDB-FISH-150901–212 | |
| Genetic reagent (Danio rerio) | Tg(phd3:GFP)sh144 | Santhakumar et al., 2012 | ZDB-FISH-150901–26851 | |
| Genetic reagent (Danio rerio) | Tg(gata4:EGFP)ae1 | Heicklen-Klein and Evans, 2004 | ZDB-FISH-150901–14762 | |
| Genetic reagent (Danio rerio) | TgBAC(nppa:mCitrine) | This paper | More info on generation of this line can be obtained from the Materials and methods section ‘Transgenic zebrafish lines and cryoinjury’. | |
| Cell line (Mus musculus) | TetRE-caErbb2 X MYH6-tTA X MYH6-cre ROSA26-tdTomato | D'Uva et al., 2015 | More info on how to obtain primary cell line can be obtained from Materials and methods sections ‘Transgenic mouse lines and animal procedures’ and ‘Pharmacological inhibition of glycolysis’. | |
| Biological sample (Mus musculus) | TetRE-caErbb2 X MYH6-tTA hearts | D'Uva et al., 2015 from: Xie et al., 1999 and Yu et al., 1996. | The Jackson Laboratory, stock no. 010577; | |
| Biological sample (Danio rerio) | Tg(cmlc2:CreER)pd10 x Tg(β-act2:BSNrg1)pd107hearts | Gemberling et al., 2015 | ZDB-FISH-150901–25249 x ZDB-FISH-150901–25354 | |
| Biological sample (Danio rerio) | Tg(gata4:EGFP)ae1hearts | Lin et al. 2009 | ZDB-FISH-150901–14762 | |
| Biological sample (Danio rerio) | Tg(phd3:GFP)sh144hearts | Santhakumar et al., 2012 | ZDB-FISH-150901–26851 | |
| Antibody | Anti-AuroraB kinase (mouse monoclonal) | BD Transduction Laboratories | #611082, RRID:AB_2227708 | 1:200 |
| Antibody | Anti-Ki67 (Rabbit monoclonal) | Cell Marque | #275R | 1:200 |
| Antibody | Anti-MCT4 (rabbit polyclonal) | Santa Cruz | #SC50329, RRID:AB_2189333 | 1:200 |
| Antibody | Anti-PCNA (mouse monoclonal) | DAKO | #M0879, RRID:AB_2160651 | 1:800 |
| Antibody | Anti-GFP (chicken polyclonal) | Aves | #GFP-1010, RRID:AB_2307313 | 1:1000 |
| Antibody | Anti-Mef2C (rabbit polyclonal) | Santa Cruz/Biorbyt | #SC313, RRID:AB_631920 / #orb256682 | Both 1:1000 |
| Chemical compound, drug | 2-Deoxyglucose | Sigma-Aldrich | #D6134 | 1 mg/g or 3 mM |
| Chemical compound, drug | Lonidamine | Sigma-Aldrich | L4900 | 80 uM |
| Chemical compound, drug | 2NBDG | Caymanchem | #11046 | 400 uM |
| Chemical compound, drug | AG1478 | Selleck Chemical | S2728 | 5M, from 10 mM stock in DMSO |
| Chemical compound, drug | Doxycycline | Harlan Laboratories | TD02503 | |
| Chemical compound, drug | Trizol | Life technologies | #15596026 | |
| Chemical compound, drug | Fast SYBR Green Master Mix | Applied Biosystems | #4385612 | |
| Chemical compound, drug | Collagenase type II | Gibco | 17101015 | 0.1% |
| Chemical compound, drug | TrypLE Express Enzyme (1x), phenol red | Gibco | 12605036 | |
| Software, algorithm | FIJI | Schindelin et al., 2012 | Version 2.0.0, RRID:SCR_002285 | |
| Software, algorithm | RaceID2/StemID | Grün, D. et al. De Novo Prediction of Stem Cell Identity using Single-Cell Transcriptome Data. Cell Stem Cell 19, 266–277 (2016). | RRID:SCR_017242 | |
| Software, algorithm | Rstudio | Rstudio | RRID:SCR_000432 | Version 1.2.1335 |
| Software, algorithm | Imaris | Bitplane | RRID:SCR_0007370 | V9.3.1 |
| Software, algorithm | Gene Set Enrichment Analysis | Genepattern, Broad Institute | RRID:SCR_003199 | # of permutations = 1000 |
| Commercial assay, kit | Superscript III First strand synthesis system | Thermo Fisher Scientific | #18080051 | Input 200 ng RNA |
| Commercial assay, kit | MiRNeasy | Qiagen | 217004 | |
| Commercial assay, kit | High capacity cDNA Reverse Transcription kit | Applied Biosystems | 4374966 | Input 1 ug RNA |
| Commercial assay, kit | Neonatal Dissociation kit | Miltenyi Biotec | 130-098-373 | |
| Commercial assay, kit | TruSeq small RNA primers | Illumina | 20005613 |
