Journal: Cardiovascular Research

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

doi: 10.1093/cvr/cvae250

Figure Lengend Snippet: 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.

Article Snippet: Cells were transduced with adenoviral vectors (Welgen Inc., Worcester, MA) expressing a short hairpin sequence targeted against NRF2 (shNRF2) or a non-targeting sequence (shCtr) at MOI 500.

Techniques: Activation Assay, Control

Journal: Cardiovascular Research

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

doi: 10.1093/cvr/cvae250

Figure Lengend Snippet: Effect of constitutive activation of NRF2 in the heart on 13 C enrichment of metabolites after ex vivo Langendorff perfusion of [U- 13 C]glucose. A ) Schematic representation of [U- 13 C]glucose carbon labelling through glycolysis, PPP, and TCA cycle (filled circles indicate 13 C-labelled carbons, and empty circles indicate 12 C carbons). B ) 13 C-glucose incorporation into upper glycolytic metabolites: G6P and F6P (mass isotopologue distribution, M.I.D.), C ) 13 C-glucose incorporation into PPP metabolites: R5P and S7P (M.I.D.), D ) 13 C-glucose incorporation into lower glycolytic metabolites: 2/3PG and lactate (M.I.D.), E ) 13 C-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- 13 C]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 13 C atom, m + 2 to isotopologues with two 13 C atoms, etc. Data are presented as mean ± SEM. * P < 0.05, *** P < 0.001, **** P < 0.0001 and ns, not significant by one-way ANOVA followed by Bonferroni multiple comparison test for mass isotopologue comparisons.

Article Snippet: Cells were transduced with adenoviral vectors (Welgen Inc., Worcester, MA) expressing a short hairpin sequence targeted against NRF2 (shNRF2) or a non-targeting sequence (shCtr) at MOI 500.

Techniques: Activation Assay, Ex Vivo, Comparison

Journal: Cardiovascular Research

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

doi: 10.1093/cvr/cvae250

Figure Lengend Snippet: 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.

Article Snippet: Cells were transduced with adenoviral vectors (Welgen Inc., Worcester, MA) expressing a short hairpin sequence targeted against NRF2 (shNRF2) or a non-targeting sequence (shCtr) at MOI 500.

Techniques: Transfection, Control, Western Blot, Knockdown, Marker

Journal: Cardiovascular Research

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

doi: 10.1093/cvr/cvae250

Figure Lengend Snippet: 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 with isoproterenol alone or in combinations with nucleosides or NADPH. Cells were stained with antibodies for α-actinin, γ-H2A.X and DAPI. Scale bar: 10 µm. G ) Summary and quantification of the percentage of cells with more than five γ-H2A.X positive foci/nucleus. NRVM were infected, treated, and stained as described in ( E ). Three to four biological replicates, n ≥ 40 cells/group. Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01 and ns, not significant by one-way ANOVA, followed by Tukey’s multiple comparisons test.

Article Snippet: Cells were transduced with adenoviral vectors (Welgen Inc., Worcester, MA) expressing a short hairpin sequence targeted against NRF2 (shNRF2) or a non-targeting sequence (shCtr) at MOI 500.

Techniques: Transduction, Expressing, Control, shRNA, Immunofluorescence, Staining, Infection