Figure 8. TF cytoplasmic tail deletion attenuates ROS production and ERK1/2–TGF-β1 signaling–dependent cardiac fibrosis and improves cardiac function.

WT and TF^ΔCT mice were subjected to permanent LAD ligation versus sham surgery and investigated after 7 days and 4 weeks. (A) Confocal microscopy of myocardial cryosections. Representative images and quantification of MFI of CD45/gp91^phox double-positive cells. Scale bars: 75 μm. (B) Assessment of superoxide formation in infarcted myocardium by dihydroethidium-HPLC analysis. Representative chromatogram of 2-hydroxyethidium (2-HE), the oxidation product of DHE, and quantification normalized to weight of the infarcted tissue. Ordinary 1-way ANOVA, Šidák’s multiple-comparison test; n = 5–6 animals per group. (C) Protein expression analysis of monocytes isolated from NOX2^–/– animals and stimulated with an inflammatory cytokine cocktail containing IL-6, TNF-α, and CCL2 at a concentration of 20 ng/mL with and without hypoxia for 4 hours. Western blotting of p-ERK1/2 (normalized to ERK1/2) and activated TGF-β1 (normalized to GAPDH). Ordinary 1-way ANOVA, Šidák’s multiple-comparison test; n = 4 replicates per group (2–3 mice were pooled for each sample). (D) Western blot analysis of activated TGF-β1 (normalized to GAPDH) and p-SMAD2 (normalized to total SMAD2) in infarcted myocardium obtained from WT or TF^ΔCT mice after 7 days of MI. Representative blots and quantification of biological replicates. Ordinary 1-way ANOVA, Šidák’s multiple-comparison test; n = 5–7 animals per group. (E) Sirius red staining and deconvoluted images of fibrotic area on paraffin-embedded heart sections 4 weeks after permanent LAD ligation to induce IHF versus sham surgery. Representative images and quantification of fibrotic areas normalized to surface area. Ordinary 1-way ANOVA, Šidák’s multiple-comparison test; n = 5–7 animals per group. (F) Longitudinal echocardiographic studies over 4 weeks for LVEF (%) and LVEDV (μL) in parasternal long axis M-mode. Two-way ANOVA, Bonferroni’s multiple-comparisons test; n = 6–17 animals per group. (G) Kaplan-Meier survival analysis of permanently LAD-ligated versus sham-operated C57BL/6J and TF^ΔCT mice over 4 weeks. Log-rank (Mantel-Cox) test; n = 15–20 animals per group. Data are shown as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.