Fig 1: Mydgf is necessary for heart regeneration and cardiomyocyte proliferation. (A and B) Masson's staining showed heart regeneration in Mydgf-KO mice after apical resection (AR) injury relative to wild-type (WT) at 21 days post resection (dpr). Statistical analysis revealed that heart regeneration was reduced in Mydgf-KO mice (n = 15 for Mydgf-KO mice and n = 10 for WT mice). Scale bars, 500 µm. (C) Representative images of echocardiography analysis in Mydgf-KO and WT mice at 21 dpr. (D) Echocardiography analysis of left ventricular ejection fraction (LVEF) in Mydgf-KO and WT mice at 21 dpr (n = 10 for Mydgf-KO mice and n = 9 for WT mice). *P < 0.05 and **P < 0.01 compared to WT by Student's t-test (B and D). (E) Schematic diagram showed the experimental design for F-K. (F and G) Immunostaining illustrated proliferative (pH3+, green, white arrows) cardiomyocytes (CMs) were decreased in Mydgf-KO mice relative to wild-type (WT) at 7 days post resection (dpr) (n = 3 per group). Scale bars, 20 µm. (H and I) Immunostaining illustrated proliferative (Ki67+, green, white arrows) CMs were decreased in Mydgf-KO mice relative to WT at 7 dpr (n = 3 per group). (J and K) Immunostaining illustrated proliferative (Aurkb+, green, white arrows) CMs were decreased in Mydgf-KO mice relative to WT at 7 dpr (n = 3 per group). *P < 0.05 and **P < 0.01 compared to WT by Student's t-test (G, I and K). (L and M) Example photomicrographs of isolated CMs from Mydgf-KO mice heart and quantification of ploidy at 14 dpr (n = 6 per group). *P < 0.05, **P < 0.01 and ***P < 0.001 compared to WT by two-way ANOVA with Bonferroni's multiple comparisons test (M). Scale bars, 20 µm. Values were presented as the mean ± S.E.M.
Fig 2: Assay for screening the effects of cell cycle modifying substances on CMs. a Scheme depicts the cross-breeding of αMHC-H2B-mCh mice with the CAG-eGFP-anillin proliferation indicator mouse. Depending on the cell cycle status, CMs (H2B-mCh+ nuclei) express eGFP-anillin in different subcellular localizations. b Fluorescence pictures of dissociated αMHC-H2B-mCh/CAG-eGFP-anillin double transgenic hearts (P2), transfected with the cell cycle-modifying miR-199 and a miR-NC. Scale bars 100 µm. c Quantification of eGFP-anillin expression in miR-treated CMs 72 h after transfection (n ≥ 3). d Examples of CMs with cytokinesis-indicating eGFP-anillin localizations (arrows). Scale bars 100 µm. e Staining of eGFP-anillin/H2B-mCh CMs with the proliferation marker AURKB Aurora B kinase. Note the overlap between the M-phase specific localization of eGFP-anillin (green) contractile ring and Aurora B kinase (white). Scale bar 10 µm. f Analysis of different eGFP-anillin localizations in CMs after miR-treatment (n ≥ 3). g Portion of binuclear CMs 72 h after miR-transfection (n ≥ 3)
Fig 3: Mydgf controls cardiomyocyte proliferation through c-Myc/FoxM1 pathway. (A) Schematic diagram showed the experimental design for B-F. (B) Western blot of p-Akt and cell cycle relative protein in PBS and Mydgf-treated cardiomyocytes (CMs). (C) Heat map of genes under cell cycle regulated networks. (D) Western blot of c-Myc and FoxM1 in primary CMs treated with PBS and Mydgf. (E) Western blot of c-Myc and FoxM1 in wild-type (WT) mouse hearts harvested at 1, 4, 7 dpr. (F) Western blot of p-Akt, c-Myc and FoxM1 of hearts harvested at 4, 7 days post resection (dpr) in wild-type (WT) or Mydgf-KO mouse hearts. (G and H) Immunostaining illustrated proliferative (pH3+, green, white arrows) primary CMs were decreased treated with siRNA-Akt, c-Myc and FoxM1 respectively prior to Mydgf treatment relative to siRNA-NC treatment group after 48 hours (n = 3 per group). (I and L) Immunostaining illustrated proliferative (Ki67+, green, white arrows) primary CMs were decreased treated with siRNA-Akt, c-Myc and FoxM1 respectively prior to Mydgf treatment relative to siRNA-NC treatment group after 48 hours (n = 3 per group). (K and L) Immunostaining illustrated proliferative (Aurkb+, green, white arrows) primary CMs were decreased treated with siRNA-Akt, c-Myc and FoxM1 respectively prior to Mydgf treatment relative to siRNA-NC treatment group after 48 hours (n = 3 per group). #P < 0.05 and ##P < 0.01 compared to Mydgf treatment by Student's t-test (H, J and L). (M) Western blot of p-Akt, c-Myc, FoxM1, Cyclin B1, Cyclin D1 and CDK1 in primary CMs transfected with different treatment. Values were presented as the mean ± S.E.M.
Fig 4: Mydgf is sufficient for cardiomyocyte proliferation and heart regeneration. (A) Schematic diagram showed the experimental design for B-G. (B and C) Immunostaining illustrated proliferative (pH3+, green, white arrows) primary cardiomyocytes (CMs) were increased in Mydgf-treated group relative to PBS-treated group after 16 hours (n = 3 per group). (D and E) Immunostaining illustrated proliferative (Ki67+, green, white arrows) primary CMs were increased in Mydgf-treated group relative to PBS-treated group after 16 hours (n = 3 per group). (F and G) Immunostaining illustrated proliferative (Aurkb+, green, white arrows) primary CMs were increased in Mydgf-treated group relative to PBS-treated group after 16 hours (n = 3 per group). *P < 0.05 and **P < 0.01 compared to PBS by Student's t-test (C, E and G). (H) Schematic diagram showed the experimental design for I-R. (I and J) Masson's staining showed heart regeneration in Mydgf-KO mice treated with Mydgf relative to PBS at 21 days post resection (dpr). Statistical analysis revealed that heart regeneration was induced in Mydgf-treated mice (n = 20 for Mydgf-treated mice and n = 15 for PBS-treated mice). Scale bars, 500 µm. (K) Representative images of echocardiography analysis in Mydgf-KO mice treated with Mydgf relative to PBS at 21 dpr. (L) Echocardiography analysis of left ventricular ejection fraction (LVEF) in Mydgf-KO mice treated with Mydgf relative to PBS at 21 dpr (n = 10 for Mydgf-treated mice and n = 9 for PBS-treated mice). *P < 0.05 and ***P < 0.001 compared to PBS-treated mice by Student's t-test (J and L). (M and N) Immunostaining illustrated proliferative (pH3+, green, white arrows) CMs were increased in Mydgf-KO neonatal mice treated with Mydgf relative to PBS at 7 dpr (n = 3 per group). (O and P) Immunostaining illustrated proliferative (Ki67+, green, white arrows) CMs were increased in Mydgf-KO neonatal mice treated with Mydgf relative to PBS at 7 days post resection (dpr) (n = 3 per group). (Q and R) Immunostaining illustrated proliferative (Aurkb+, green, white arrows) CMs were increased in Mydgf-KO neonatal mice treated with Mydgf relative to PBS at 7 dpr (n = 3 per group). Scale bars, 20 µm. *P < 0.05 compared to AR+PBS by Student's t-test (N, P and R). Values were presented as the mean ± S.E.M.
Fig 5: Mydgf promotes heart regeneration in adult mice. (A) Schematic diagram showed the experimental design for B-L. (B and C) Masson's staining elucidated the infarcted area in wild-type (WT) adult mice treated with PBS/Mydgf at 21 days post infarction (dpi). Statistical analysis of fibrotic area showed the infarcted size was significantly smaller in adult WT mice treated with Mydgf at 21 dpi relative to PBS (n = 25 for Mydgf-treated mice and n = 13 for PBS-treated mice). Scale bars, 500 µm. (D) Representative images of echocardiography analysis in adult WT mice treated with PBS/Mydgf at 21 dpi. (E) Echocardiography analysis of left ventricular ejection fraction (LVEF) in adult WT mice treated with PBS/Mydgf at 21 dpi (n = 19 for Mydgf-treated mice and n = 13 for PBS-treated mice). *P < 0.05 and **P < 0.01 compared to WT by Student's t-test (C and E). (F) Cumulative survival after MI in WT mice treated with 25 PBS and 20 Mydgf. *P < 0.05 compared to PBS-treated group by log-rank test. (G and H) Immunostaining illustrated proliferative (pH3+, green, white arrows) cardiomyocytes (CMs) were increased in WT adult mice treated with Mydgf relative to PBS at 7 dpi (n = 3 per group). (I and J) Immunostaining illustrated proliferative (Ki67+, green, white arrows) CMs were increased in WT adult mice treated with Mydgf relative to PBS at 7 dpi (n = 3 per group). (K and L) Immunostaining illustrated proliferative (Aurkb+, green, white arrows) CMs were increased in WT adult mice treated with Mydgf relative to PBS at 7 dpi (n = 3 per group). Scale bars, 20 µm. *P < 0.05 and **P < 0.01 compared to PBS-treated group by Student's t-test (H, J and L). Values were presented as the mean ± S.E.M.
Supplier Page from MilliporeSigma for Anti-Aurora B antibody produced in rabbit