Redox-dependent purine degradation triggers postnatal loss of cardiac regeneration potential

Figures

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  Fig. 1. Metabolomic and transcriptomic analyses of the postnatal mouse and opossum heart (A) A scheme depicting our strategy to identify metabolic pathway(s) that are evolutionary conserved, independent of postnatal environmental changes, and temporally correlated with cardiomyocyte cell cycle arrest. (B) Principal component analysis (PCA) of the comparative metabolome data. (C) Volcano plot of the increased and decreased metabolites in the P14 mouse heart compared with those in P1 mouse heart. Labeled metabolites are commonly increased/decreased in the postnatal opossum heart. (D) Volcano plot of the increased and decreased metabolites in the P56 opossum heart compared with those in the P14 opossum heart. Labeled metabolites are commonly increased/decreased in the postnatal mouse heart. (E) Enrichment pathway analysis of the commonly altered metabolites in the postnatal mouse and opossum heart. (F) Integrated analysis of transcriptome (P0 vs P14) and metabolome data (P1 vs P14) corrected from the postnatal mouse heart. Transcriptome data were reported in Cardoso-Moreira, M. et al. [19]. (G) Heatmap representation of the purine metabolism-related metabolites in the postnatal mouse (left)…

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  Fig. 2. Postnatal alteration of the pentose phosphate pathway in the mouse heart (A) Heatmap representation of the pentose phosphate pathway (PPP)-related metabolites in the P1 and P14 mouse heart. (B) Time-course quantification of PPP-related metabolites in the postnatal mouse heart. (C) Time-course analysis of glucose-6-phosphate dehydrogenase (G6PD) activity in the postnatal mouse heart. (D–F) Mass spectrometry analysis of (D) oxidized glutathione (GSSG), (E) reduced glutathione (GSH), and (F) ratio of the GSH and GSSG in the postnatal mouse heart. (G) A schematic representation of the postnatal changes in the PPP, and branching metabolic pathways including the generation of NADPH, de novo nucleotide biosynthesis, and returning back to glycolysis. Metabolites colored in red were increased, and those colored in blue were decreased, and those colored in gray showed no significant change in the P14 heart compared with P1. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

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  Fig. 3. Administration of NAC reduces the PPP in the postnatal mouse heart (A) A scheme depicting the protocol for a glutathione precursor, N-acetyl cysteine (NAC), injection to neonatal mice. (B) PCA of the metabolomic profiles of the heart harvested from control (red dots) and NAC-treated mice (green dots). (C) Heatmap representation of PPP-related metabolites in the heart of NAC-treated mice at P14. (D) Quantitative data of PPP-related metabolites in the heart of NAC-treated mice. (E) G6PD activity in the heart of NAC-treated mice at P14. (F) A schematic representation of the PPP in the P14 heart from NAC-treated mice. Metabolites colored in blue were decreased, and those colored in gray showed no significant change upon NAC treatment in the P14 heart compared with control. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

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  Fig. 4. Redox-dependent xanthine synthesis in the postnatal heart (A) Enrichment pathway analysis of the metabolomic profiles of the heart harvested from control and NAC-treated mice at P14. (B) Volcano plot of the metabolomic profiles of the heart from control and NAC-treated groups. Labeled metabolites were significantly increased (red) or decreased (blue) in the NAC-treated group compared with control. (C) Heatmap representation of the purine metabolism-related metabolites in the heart from control and NAC-treated groups at P14. (D) Quantitative data of purine metabolism-related metabolites in the heart from control and NAC-treated groups at P14. (E) A schematic representation of the adenine nucleotide degradation pathway in the heart from NAC-treated mice. Metabolites colored in blue were decreased, and those colored in gray showed no significant change upon NAC treatment in the P14 heart compared with control. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

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  Fig. 5. Xanthine oxidase inhibition extends postnatal cardiomyocyte proliferative window (A) A scheme depicting the protocol of allopurinol administration to neonatal mice. (B) Uric acid levels in the heart from control and allopurinol treated mice at P14. (C) Heart weight per body weight of the P14 heart harvested from control and allopurinol-treated groups. (D) Cardiomyocyte cell size evaluated by the imaging of WGA-stained sections of the heart from control and allopurinol-treated mice. A scale bar indicates 50 μm. (E) Representative images of the immunostaining using antibodies against 8-hydroxyguanosine (8OHG, a marker for oxidative DNA damage), a cardiomyocyte marker α-actinin, and DAPI. Oxidative DNA damage per cardiomyocytes was evaluated by counting the average number of nuclear foci of 8OHG signals in cardiomyocytes. Scale bars indicate 10 μm. (F) Representative images of immunostaining with an anti-phospho-histone H3 (pH3) antibody co-stained with an antibody against cardiac troponin T (cTnT) and DAPI (upper left, white arrowheads indicate pH3+ mitotic cardiomyocytes, a scale bar indicates 50 μm) and confocal z-stack imaging of a pH3+ mitotic cardiomyocyte (bottom left, a…

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  Fig. 6. Xanthine oxidase inhibition extends the postnatal window for heart regeneration capacity (A) A scheme depicting the protocol for daily allopurinol administration, induction of myocardial infarction (MI) to neonatal mice. (B) A representative image of z-stack imaging of the pH3+ cardiomyocytes (left, arrowheads indicate pH3+ mitotic cardiomyocytes) and quantification of cardiomyocyte mitosis at 7 days post injury (dpi). A scale bar indicates 20 μm. (C) Representative images of heart sections stained with WGA (left, a scale bar indicates 50 μm) and quantification of cardiomyocyte cell size at 21 dpi (right). (D) Representative images of Masson's-Trichrome staining of heart sections and ratio of fibrotic areas and non-fibrotic areas. Analysis was conducted at 21 dpi. A scale bar indicates 1 mm. (E) Echocardiography measurements of ejection fraction (EF), fractional shortening (FS), left ventricular internal diameter diastole (LVIDD), and left ventricular internal diameter systole (LVIDS) at 21 dpi. (F) Representative images of the visualization of cytosolic ROS with CellROX Deep Red after 2-day culture with or without allopurinol and quantification of the cytosolic ROS. (For…