NOX1 mediates metabolic heart disease in mice and is upregulated in monocytes of humans with diastolic dysfunction

Figures

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  Figure 1. (A, B) Left ventricular structure assessed by echocardiography from the parasternal short axis view (M-mode). Mice were randomized to HFHS or CTD at 4 weeks of age and injected once with STZ or vehicle after 3 weeks on diet. Echocardiography was performed at 8- to 12-week intervals, comparisons between groups were done by fitting a R-Studio linear mixed effect model. (n = 16 for WT-CTD, 16 for WT-HFHS, 16 for KO-CTD, 17 for KO-HFHS) (A) Total wall thickness (LVAW; d + LVPW; d); (B) Left ventricular mass as calculated based on the corrected cube formula 1.053 × [(LVID; d; + LVPW; d + LVAW; d)3—LVID; d3] × 0.8. CTD, control diet; d, diastolic; HFHS, high-fat high-sucrose diet; LVAW, left ventricular anterior wall; LVID, left-ventricular internal diameter; LVPW, left ventricular posterior wall; STZ, streptozotocin. (C–E) Diastolic function and left-ventricular end-diastolic pressure. (C) Peak reverse longitudinal strain rate by echocardiography from the parasternal long-axis view (B-mode) in WT and KO mice at 44 weeks after STZ/vehicle injection and on respective diet (n = 16 for WT-CTD, 16 for WT-HFHS, 16 for KO-CTD, 17 for KO-HFHS); (D) Left ventricular end-diastolic pressures…

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  Figure 2. Heart weight, cardiomyocyte size, fibrosis, vessel density, and nitrotyrosinylation 44 weeks after STZ/vehicle injection. (A) Post-mortem heart weight normalized to tibia length (n = 16 for WT-CTD, 16 for WT-HFHS, 16 for KO-CTD, 17 for KO-HFHS); (B) Wheat germ agglutinin (WGA) and isolectin B (IB4) staining, scale bar: 20 μm; (C) Quantification of cardiomyocyte cross-sectional area based on WGA staining; (D) Representative microscope images from picrosirius red staining, scale bar: 50 μm; (E) Quantification of vessel density based on IB4 staining; (F) Representative nitrotyrosine staining of the myocardium, scale bar: 500 μm; insert scale bar: 20 μm. (G) Quantification of nitrosative stress based on nitrotyrosine staining (NT3). For quantifications, groups were compared using two-way ANOVA with Sidak test, *P < 0.05, **P < 0.01,****P < 0.0001; (B–G: n = 10 for WT-CTD, 8 in B–E and 9 in F–G for WT-HFHS, 9 for KO-CTD, 9 for KO-HFHS).

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  Figure 3. Endothelial activation based on VCAM-1 and ICAM-1 expression and cardiac inflammatory response by staining of Mac-2-, IL-1β-, and NLRP3 positive cells 44 weeks after STZ/vehicle injection. (A–D) Representative microscopic images for myocardial vessels stained with VCAM-1 and ICAM-1, scale bar: 20 μm. (E–H) Quantification of VCAM-1 and ICAM-1 expression in coronary arteries and venules. (I–K) Representative microscopic images for inflammatory cells stained with Mac-2, IL-1β, and NLRP3, scale bar: 20 μm; (L–N) Quantification of Mac-2-, IL-1β-, and NLRP3 positive cells per heart section. Groups were compared using two-way ANOVA with Sidak test, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, (n = 10 for WT-CTD, 9 for WT-HFHS, 9 for KO-CTD, 9 for KO-HFHS).

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  Figure 4. Nox1 expression in different cell types ex vivo and upon LPS or PMA stimulation in vitro. (A, left panel) Flow cytometry plot representing an example of the gating strategy used to sort CD31+ and CD45+ cells from WT mouse hearts. Full gating strategy is shown in Supplementary material online, Figure S6. (A, right panel) CD31+ and CD45+ cells were sorted from hearts of WT mice, and Nox1 mRNA expression was assessed by RT-qPCR. Each data point represents a pool of cells sorted from 5 to 6 mice, from three independently performed experiments. Data are shown as mean ± SEM. Comparison was made using unpaired t-test. (B, left panel) Flow cytometry plot representing an example of the gating strategy used to sort CD11b+CD64+ (CD64+) and CD64- subpopulations from WT mouse hearts. (B, right panel) CD11b+CD64+ (CD64+) and CD64- cells were sorted from WT mouse hearts and Nox1 mRNA levels were assessed by RT-qPCR. Each data point represents a pool of five mice, from two independently performed experiments. nd, not detectable. Data are shown as mean ± SEM. (C–F) Expression of NOX1 mRNA in non-stimulated cells and after 6 h of stimulation with LPS or PMA in different cell types; bEnd.3, mouse…