NRF2 activation in the heart induces glucose metabolic reprogramming and reduces cardiac dysfunction via upregulation of the pentose phosphate pathway

Figures

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  Figure 1. Cardiac-specific activation of NRF2 induces metabolic reprogramming in the heart. A) mRNA levels of Keap1 and NRF2 targets B) glutathione-S-transferase α2 (Gsta2), C) glutathione-disulphide reductase (Gsr), D) nicotinamide adenine dinucleotide phosphate-quinone-oxidoreductase 1 (Nqo1) in the hearts of csKeap1KO and WT control mice. n ≥ 3/group. E) Principal component analysis of targeted metabolomic profile of csKeap1KO vs. WT hearts, n ≥ 4/group. F) Heat map showing hierarchical clustering of metabolite abundance for the top 60 features in WT vs. csKeap1KO hearts. Data are presented as mean ± SEM. **P < 0.01, ***P < 0.001, ****P < 0.0001 and ns, not significant by unpaired Student’s t-test.

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  Figure 2. Effect of constitutive activation of NRF2 in the heart on 13C enrichment of metabolites after ex vivo Langendorff perfusion of [U-13C]glucose. A) Schematic representation of [U-13C]glucose carbon labelling through glycolysis, PPP, and TCA cycle (filled circles indicate 13C-labelled carbons, and empty circles indicate 12C carbons). B) 13C-glucose incorporation into upper glycolytic metabolites: G6P and F6P (mass isotopologue distribution, M.I.D.), C) 13C-glucose incorporation into PPP metabolites: R5P and S7P (M.I.D.), D) 13C-glucose incorporation into lower glycolytic metabolites: 2/3PG and lactate (M.I.D.), E) 13C-glucose incorporation into TCA cycle metabolites: citrate, alpha-ketoglutarate, malate, and fumarate (M.I.D.) in WT vs. csKeap1KO heart following ex vivo Langendorff perfusion of [U-13C]glucose, n = 3/group. Mass isotopologue distribution refers to the relative abundance (in percentage) of isotopically labelled molecules with different masses within a given metabolite. m + 0, refers to unlabelled isotopologue, m + 1 to isotopologues containing one 13C atom, m + 2 to isotopologues with two 13C atoms, etc.34 Data are presented as mean ± SEM. *P < 0.05, ***P < 0.001,…

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  Figure 3. Effects of chronic pressure overload on csKeap1KO hearts. A) Representative M-mode echocardiography images of WT and csKeap1KO mice 6 weeks after AAB or sham surgery. B and C) Ejection fraction (EF) and left ventricular (LV) end-systolic volume (LVESV) in WT and csKeap1KO mice subjected to AAB or sham surgery, n ≥ 4/group. D) HW/BW, n ≥ 6/group. E) Cardiomyocyte cross-sectional area measured in heart sections of sham or AAB-operated WT and csKeap1KO hearts, n ≥ 4 hearts/group. F and G) mRNA levels of hypertrophic makers, Anp and Bnp, n ≥ 5/group. H) Representative images of heart sections of WT or csKeap1KO mice 6 weeks after sham or AAB surgery, stained with Picrosirius red. Scale bar: 50 µm. I) Mean data for fibrosis in heart sections from WT and csKeap1KO mice, n ≥ 4 hearts/group. J) TUNEL-positive cells per square millimetre of heart tissue in sections of WT and csKeap1KO hearts; n ≥ 3 hearts/group. Representative immunoblots (K) and quantification data (L) for changes in key PPP enzymes 6-phosphogluconate dehydrogenase (Pgd) and Transaldolase1 (Taldo1) in sham or AAB-operated WT and csKeap1KO hearts, n = 6. Data are presented as means ± SEM. *P < 0.05, **P < 0.01, ***P <…

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  Figure 4. NRF2-mediated upregulation of the PPP prevents stress-induced cardiomyocyte death. A) mRNA levels of Keap1, G6pd, Pgd, and Tkt measured in NRVMs transfected with either control scrambled siRNA or siKeap1. n ≥ 5/group. B) Schematic representation of glycolysis and PPP highlighting the PPP enzymes (G6PD and PGD) inhibited by 6-AN. C) Cell viability measured as the percentage of 7AAD-positive over Hoechst-positive cells (7AAD+/Hoechst+) in NRVM transfected with siScrambled or siKeap1, untreated or treated for 24 h with different combinations of isoproterenol and/or 6-AN. Four independent experiments, n ≥ 550 cells/group. D) Representative immunoblot showing efficient knockdown of G6PD in NRVM transfected with siG6pd alone or in combination with siKeap1. Tubulin was used as a loading marker. E) Percentage of 7AAD+/Hoechst+ calculated in NRVMs transfected with siScrambled or siKeap1 and with or without siG6pd, and treated as indicated; n = 4, ≥600 cells. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001 and ns, not significant by unpaired Student’s t-test or by two-way ANOVA, followed by Tukey’s multiple comparisons test.

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  Figure 5. Stress-induced cardiomyocyte DNA damage is limited by NRF2 and the PPP. A) mRNA levels of G6pd, and B) Pgd in NRVM transduced with adenovirus expressing either control scrambled shRNA or siNrf2 and untreated or treated with isoproterenol. n = 5/group. C) Quantification of cell viability measured as the percentage of 7AAD+/Hoechst+ cells. Four independent experiments, n ≥ 500 cells/group. D) Representative immunofluorescence images of NRVM transduced with adenovirus expressing control shRNA or shNRF2 untreated or treated with 100 µmol/L isoproterenol for 8 h. Cells were stained with antibodies for α-actinin, γ-H2A.X, and DAPI. Scale bar: 10 µm. E) Percentage of γ-H2A.X positive cells were divided into three groups depending on the number of γ-H2A.X foci per nucleus: low DNA damage (0–5 foci/nucleus), moderate DNA damage (5–15 foci/nucleus) and high DNA damage (>15 foci/nucleus). NRVM were infected with adenovirus expressing control shRNA or shNRF2, untreated or treated with isoproterenol for 8 h. Four independent biological replicates, n ≥ 50 cells per condition. F) Representative confocal images of NRVM infected with adenovirus expressing shNRF2 and untreated or treated for 8 h…

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  Figure 6. csKeap1KO hearts show better function and less DNA damage after MI. A–D) Echocardiography data for EF, LV end-diastolic volume (LVEDV), LV end-systolic volume (LVESV) and heart rate (HR) in WT and csKeap1KO mice subjected to MI or sham surgery; n ≥ 5/group]. E) HW/BW ratio values for WT and csKeap1KO mice, 4 weeks after MI, n = 8/group. F) Representative confocal images of cardiac sections from remote or border zone of csKEAP1KO or WT heart after MI and stained with WGA, DAPI, and for γ-H2A.X. G) Percentage of γ-H2A.X positive cardiomyocytes in the border zone or remote zone; n = 5 hearts/group. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 and ns, not significant by two-way ANOVA, followed by Tukey’s multiple comparisons test.