Mettl3-mediated m6A modification of Fgf16 restricts cardiomyocyte proliferation during heart regeneration
- Key Laboratory of Regenerative Medicine of Ministry of Education, Department of Developmental & Regenerative Biology, Jinan University, China
- College of Basic Medical Sciences, Shandong First Medical University & Shandong Academy of Medical Science, China
- Department of Cardiovascular Surgery, Guangdong Cardiovascular Institute, Guangdong Provincial People's Hospital & Guangdong Academy of Medical Sciences, China
- Department of Neuroanatomy, Institute of Anatomy, University of Bonn, Germany
- Stem Cell and Regeneration TRP, School of Biomedical Sciences, Chinese University of Hong Kong, China
- Department of Physiology, Wonju College of Medicine, Yonsei University, Republic of Korea
- Key Laboratory of Regenerative Medicine of Ministry of Education, Institute of Aging and Regenerative Medicine, China
Figures
Figure 1 with 2 supplements
Expression patterns of m6A methylases and demethylases during heart regeneration in neonatal mice.
(A) Quantification of m6A methylases (Mettl3 and Mettl14) and demethylases (Alkbh5 and Fto) expression in neonatal hearts at the indicated time points after birth (n=3 hearts). (B and C) …
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Figure 1—source data 1
Western blot for Figure 1B showing Mettl3 expression.
- https://cdn.elifesciences.org/articles/77014/elife-77014-fig1-data1-v1.zip
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Figure 1—source data 2
Western blot for Figure 1B showing β-actin expression.
- https://cdn.elifesciences.org/articles/77014/elife-77014-fig1-data2-v1.zip
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Figure 1—source data 3
Western blot for Figure 1H showing Mettl3 expression.
- https://cdn.elifesciences.org/articles/77014/elife-77014-fig1-data3-v1.zip
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Figure 1—source data 4
Western blot for Figure 1H showing Mettl14 expression.
- https://cdn.elifesciences.org/articles/77014/elife-77014-fig1-data4-v1.zip
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Figure 1—source data 5
Western blot for Figure 1H showing β-actin expression.
- https://cdn.elifesciences.org/articles/77014/elife-77014-fig1-data5-v1.zip
Figure 1—figure supplement 1
Mettl3 expression and m6A modification levels in the remote areas of injured neonatal heart at 5 dpr.
Apical resection was performed in neonatal mice at p3, followed by cardiac tissues isolation from remote areas at 5 dpr for Western blotting assay and m6A level examination. (A) Western blotting …
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Figure 1—figure supplement 1—source data 1
Western blot for Figure 1—figure supplement 1A showing Mettl3 expression.
- https://cdn.elifesciences.org/articles/77014/elife-77014-fig1-figsupp1-data1-v1.zip
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Figure 1—figure supplement 1—source data 2
Western blot for Figure 1—figure supplement 1A showing β-actin expression.
- https://cdn.elifesciences.org/articles/77014/elife-77014-fig1-figsupp1-data2-v1.zip
Figure 1—figure supplement 2
M6A modification and Mettl3 expression levels in CMs and nCMs.
(A) M6A levels in CMs and nCMs isolated from neonatal heart at p1 (n=4). (B) qPCR validation of Mettl3 expression in CMs and nCMs isolated from neonatal hearts at 5 dpr following apical resection at …
Figure 2 with 1 supplement
Mettl3 suppresses the proliferation of primary cardiomyocytes in vitro.
(A and B) Primary cardiomyocytes were transfected with siMettl3 and siNC for 48 hr, followed by qPCR (A, n=3) and western blotting (B) validation. (C and D) Primary cardiomyocytes were transfected …
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Figure 2—source data 1
Western blot for Figure 2B showing Mettl3 expression.
- https://cdn.elifesciences.org/articles/77014/elife-77014-fig2-data1-v1.zip
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Figure 2—source data 2
Western blot for Figure 2B showing β-actin expression.
- https://cdn.elifesciences.org/articles/77014/elife-77014-fig2-data2-v1.zip
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Figure 2—source data 3
Western blot for Figure 2D showing Mettl3 expression.
- https://cdn.elifesciences.org/articles/77014/elife-77014-fig2-data3-v1.zip
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Figure 2—source data 4
Western blot for Figure 2D showing β-actin expression.
- https://cdn.elifesciences.org/articles/77014/elife-77014-fig2-data4-v1.zip
Figure 2—figure supplement 1
Effects of Mettl3 on the proliferation of H9c2 cells.
(A) siMettl3-induced silence of Mettl3 mRNA in H9c2 cells was confirmed by qPCR (n=5). (B–D) H9c2 cells were transfected with siMettl3 or siNC for 48 hr, followed by EdU labeling or cell counting …
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Figure 2—figure supplement 1—source data 1
Western blot for Figure 2—figure supplement 1F showing Mettl3 expression.
- https://cdn.elifesciences.org/articles/77014/elife-77014-fig2-figsupp1-data1-v1.zip
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Figure 2—figure supplement 1—source data 2
Western blot for Figure 2—figure supplement 1F showing β-actin expression.
- https://cdn.elifesciences.org/articles/77014/elife-77014-fig2-figsupp1-data2-v1.zip
Figure 3 with 3 supplements
Mettl3 knockdown promotes cardiomyocyte proliferation in neonatal hearts at 5 dpr.
(A) Schematic of AAV9-shMettl3 virus injection designed to knock down Mettl3 in neonatal hearts. (B) qPCR validation of Mettl3 in the AAV9-injected hearts at 0 dpr (n=3 hearts). (C–E) Primary …
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Figure 3—source data 1
Western blot for Figure 3C showing Mettl3 expression.
- https://cdn.elifesciences.org/articles/77014/elife-77014-fig3-data1-v1.zip
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Figure 3—source data 2
Western blot for Figure 3C showing β-actin expression.
- https://cdn.elifesciences.org/articles/77014/elife-77014-fig3-data2-v1.zip
Figure 3—figure supplement 1
Time course of AAV9 system infection in neonatal mice heart.
(A) A diagram of the AAV9 system harboring a reporter gene (EGFP) expression cassette. (B) Schematic of AAV9 virus injection experiment designed to verify the infective efficiency in neonatal …
Figure 3—figure supplement 2
Mettl3 knockdown promotes cardiomyocyte proliferation in the remote area in injured heart at 5 dpr.
(A and B) Representative images (A) and quantification (B) of Ki67+ cardiomyocytes in the remote zone are shown. (C and D) Representative Z-stack confocal images (C) and quantification (D) of EdU+ …
Figure 3—figure supplement 3
Effects of Mettl3 deficiency on cardiomyocyte proliferation in neonatal mice without injury at p8.
(A) Schematic of AAV9-shMettl3 virus injection designed to knock down Mettl3 in homeostatic neonatal hearts without injury. (B) Quantification of heart weight (HW) to body weight (BW) ratio (n=5 …
Figure 4 with 2 supplements
Knockdown of Mettl3 accelerates heart regeneration in neonatal mice at 14 dpr.
(A) Schematic of AAV9 virus injection, apex resection, and EdU-pulse injection, followed by sample collection at 14 dpr. (B and C) Representative masson’s trichrome staining images of cardiac apex (B…
Figure 4—figure supplement 1
Effects of Mettl3 deficiency on heart regeneration in postnatal mice without injury at p17.
(A) Schematic of AAV9-shMettl3 virus injection and EdU-pulse injection in homeostatic postnatal hearts without injury. (B) Quantification of heart weight (HW) to body weight (BW) ratio (n=5 hearts …
Figure 4—figure supplement 2
Mettl3 knockdown promotes heart regeneration upon apex resection at p7.
(A) Schematic of AAV9-shMettl3 virus injection, apex resection, and sample collection. (B and C) Representative images (B) and quantification (C) of western blotting assay using heart tissues at p7 …
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Figure 4—figure supplement 2—source data 1
Western blot for Figure 4—figure supplement 2B showing Mettl3 expression.
- https://cdn.elifesciences.org/articles/77014/elife-77014-fig4-figsupp2-data1-v1.zip
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Figure 4—figure supplement 2—source data 2
Western blot for Figure 4—figure supplement 2B showing β-actin expression.
- https://cdn.elifesciences.org/articles/77014/elife-77014-fig4-figsupp2-data2-v1.zip
Figure 5 with 3 supplements
Overexpression of Mettl3 suppresses heart regeneration in neonatal mice upon injury.
(A) Schematic of AAV9-Mettl3 virus injection, apex resection, and sample collection in neonatal mice. (B and C) Representative images (B) and quantification (C) of western blotting for Mettl3 …
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Figure 5—source data 1
Western blot for Figure 5B showing Mettl3 expression.
- https://cdn.elifesciences.org/articles/77014/elife-77014-fig5-data1-v1.zip
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Figure 5—source data 2
Western blot for Figure 5B showing β-actin expression.
- https://cdn.elifesciences.org/articles/77014/elife-77014-fig5-data2-v1.zip
Figure 5—figure supplement 1
Overexpression of Mettl3 in postnatal hearts with AAV9 injection at p27.
(A) qPCR validation of Mettl3 mRNA expression in postnatal hearts at 27 dpr. Data are presented as the mean ± SEM (n=5 hearts per group), ***p<0.001 versus control. p values were determined by …
Figure 5—figure supplement 2
No proliferating cardiomyocytes was detected in heart at 27 dpr.
(A) Sections from apical (upper panels) and remote (lower panels) ventricles were subjected to Ki67 and cTnT double staining. Representative images of cardiomyocytes in apical (upper panels) and …
Figure 5—figure supplement 3
Effects of Mettl3 overexpression on heart regeneration in postnatal mice without injury at p31.
(A) Schematic of AAV9-Mettl3 virus injection and EdU-pulse injection in homeostatic postnatal hearts without injury. (B) Quantification of heart weight (HW) to body weight (BW) ratio (n=5 hearts per …
Figure 6 with 1 supplement
Mettl3 deficiency promotes heart regeneration in postnatal mice injured at p7.
(A) Schematic of AAV9 virus injection at p1, myocardium infarction (MI) induction at p7, and histological analysis at 5 and 21 dpM. (B and C) Representative WGA staining images (B) and …
Figure 6—figure supplement 1
Mettl3 expression in postnatal and adult hearts upon myocardium infarction injury.
(A) Western blotting validation of Mettl3 expression in the injured heart at 5 dpM following MI injury performed in postnatal mice at p7. (B) Western blotting validation of Mettl3 expression in the …
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Figure 6—figure supplement 1—source data 1
Western blot for Figure 6—figure supplement 1A showing Mettl3 expression.
- https://cdn.elifesciences.org/articles/77014/elife-77014-fig6-figsupp1-data1-v1.zip
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Figure 6—figure supplement 1—source data 2
Western blot for Figure 6—figure supplement 1A showing β-actin expression.
- https://cdn.elifesciences.org/articles/77014/elife-77014-fig6-figsupp1-data2-v1.zip
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Figure 6—figure supplement 1—source data 3
Western blot for Figure 6—figure supplement 1B showing Mettl3 expression.
- https://cdn.elifesciences.org/articles/77014/elife-77014-fig6-figsupp1-data3-v1.zip
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Figure 6—figure supplement 1—source data 4
Western blot for Figure 6—figure supplement 1B showing β-actin expression.
- https://cdn.elifesciences.org/articles/77014/elife-77014-fig6-figsupp1-data4-v1.zip
Figure 7
Mettl3 deficiency promotes heart regeneration in adult mice upon myocardium infarction injury.
(A) Schematic of AAV9 virus injection 4 weeks prior to myocardium infarction (MI), followed by histological analysis at 14 and 28 dpM. (B) Representative images of reporter gene (EGFP) expression …
Figure 8 with 8 supplements
Mettl3-mediated m6A post-transcriptionally regulates Fgf16 during neonatal heart regeneration.
(A and B) Primary cardiomyocytes were transfected with siFgf16 and siNC for 48 hr, followed by Ki67 (red) and cTnT (green) double staining. Representative images (A) and quantification (B) of Ki67+ …
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Figure 8—source data 1
Western blot for Figure 8J showing Mettl3 expression.
- https://cdn.elifesciences.org/articles/77014/elife-77014-fig8-data1-v1.zip
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Figure 8—source data 2
Western blot for Figure 8J showing β-actin expression.
- https://cdn.elifesciences.org/articles/77014/elife-77014-fig8-data2-v1.zip
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Figure 8—source data 3
Western blot for Figure 8K showing Fgf16 expression.
- https://cdn.elifesciences.org/articles/77014/elife-77014-fig8-data3-v1.zip
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Figure 8—source data 4
Western blot for Figure 8K showing β-actin expression.
- https://cdn.elifesciences.org/articles/77014/elife-77014-fig8-data4-v1.zip
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Figure 8—source data 5
Western blot for Figure 8L showing Fgf16 expression.
- https://cdn.elifesciences.org/articles/77014/elife-77014-fig8-data5-v1.zip
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Figure 8—source data 6
Western blot for Figure 8L showing β-actin expression.
- https://cdn.elifesciences.org/articles/77014/elife-77014-fig8-data6-v1.zip
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Figure 8—source data 7
Western blot for Figure 8M showing Fgf16 expression.
- https://cdn.elifesciences.org/articles/77014/elife-77014-fig8-data7-v1.zip
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Figure 8—source data 8
Western blot for Figure 8M showing β-actin expression.
- https://cdn.elifesciences.org/articles/77014/elife-77014-fig8-data8-v1.zip
Figure 8—figure supplement 1
Variations of m6A-tagged transcripts in neonatal heart in response to injury.
(A) Predominant consensus motif GGACU was detected basing on m6A-seq in both the sham and injured hearts at 5 dpr. (B) Distribution of m6A peaks along the 5’ untranslated region (5’UTR), coding …
Figure 8—figure supplement 2
Apex resection upregulates levels of m6A-tagged transcripts in neonatal hearts.
(A) Venn diagrams showing the overlap of m6A-tagged transcripts between two replicates in sham and 5dpr. (B) Total MeRIP bound reads from MeRIP-seq. (C) Venn diagrams of m6A-tagged transcripts …
Figure 8—figure supplement 3
PCA, volcano plot, and GSEA analysis from RNA-seq data.
(A) Principle component analysis (PCA) plot based on the RNA-seq data from control (sham) and injured hearts at 5 dpr. (B) Volcano plot for differentially expressed genes in the tests of neonatal …
Figure 8—figure supplement 4
Identification of cell cycle and proliferation-related target genes from the overlapped gene sets between m6A-tagged and RNA-upregulated genes.
(A) Venn diagrams showing the overlapped 333 genes between m6A-tagged and upregulated transcripts in neonatal hearts at 5dpr. (B) GO enrichment analyses of the overlapped 333 genes. (C) Heat map of …
Figure 8—figure supplement 5
Luciferase reporter assay for Mettl3-mediated Fgf16 expression.
(A) Map of reporter plasmid for subcloning wild-type or m6A consensus sequence mutant Fgf16 cDNA to fuse with Gaussia luciferase reporter gene. (B and C) Quantification of relative luciferase …
Figure 8—figure supplement 6
Effects of Fgf16 knockdown on Mettl3-mediated regulation of CM proliferation and heart regeneration.
(A and B) Primary cardiomyocytes were transfected with siMettl3 with or without siFgf16 for 48 hr, followed by Ki67 (red) and cTnT (green) double staining. Representative images (A) and …
Figure 8—figure supplement 7
Effects of Mettl14 and Fto on Fgf16 expression and cell proliferation in CMs.
(A–C) qPCR validation of Mettl14 (A), Fto (B), and Fgf16 (C) in primary cardiomyocytes treated with siMettl14 and/or siFto for 48 hr (n=3). (D and E) Representative images (D) and quantification (E) …
Figure 8—figure supplement 8
Mettl3 regulates Fgf16 expression in an Ythdf2-dependent manner.
(A) qPCR validation of Ythdf1 and Ythdf2 silencing in primary cardiomyocytes (n=3). (B) qPCR validation of Fgf16 in primary cardiomyocytes transfected with siYthdf1 or siYthdf2, respectively (n=3). …
Figure 9
Mutation of the consensus sequence of m6A modification in Fgf16 mRNA promotes heart regeneration.
(A) Schematic of AAV9 vectors for the expression of negative control (AAV9-NC), wild-type Fgf16 (AAV9-Fgf16), and mutant Fgf16 (AAV9-ΔFgf16). (B) Schematic of AAV9 virus injection, apex resection, …
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Figure 9—source data 1
Western blot for Figure 9C showing Fgf16 expression.
- https://cdn.elifesciences.org/articles/77014/elife-77014-fig9-data1-v1.zip
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Figure 9—source data 2
Western blot for Figure 9C showing β-actin expression.
- https://cdn.elifesciences.org/articles/77014/elife-77014-fig9-data2-v1.zip
Additional files
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Transparent reporting form
- https://cdn.elifesciences.org/articles/77014/elife-77014-transrepform1-v1.docx
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Supplementary file 1
Sequences of siRNAs used in this study.
- https://cdn.elifesciences.org/articles/77014/elife-77014-supp1-v1.docx
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Supplementary file 2
Primer sequences for real-time PCR analysis in mouse.
- https://cdn.elifesciences.org/articles/77014/elife-77014-supp2-v1.docx
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Supplementary file 3
Primer sequences for real-time PCR analysis in H9c2 cells.
- https://cdn.elifesciences.org/articles/77014/elife-77014-supp3-v1.docx
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Supplementary file 4
Full plasmid sequence of the pGL-RF vector.
- https://cdn.elifesciences.org/articles/77014/elife-77014-supp4-v1.docx
