NADPH oxidase 4 (Nox4) is a major source of oxidative stress in the failing heart

The role of endogenous Nox4 in mediating various cardiac phenotypes in response to PO. c-Nox4^−/− mice and WT littermates were subjected to either TAC or sham operation. (A) LVW/TL and (B) LungW/TL in response to 4 wk TAC were determined. (C) Ejection fraction, an index of LV systolic function, was obtained echocardiographically. (D) LV myocyte cross-sectional area was evaluated histologically. (E) Apoptosis was evaluated by TUNEL staining. In A–E, bar graphs indicate mean ± SEM obtained from three to five experiments.

The level of oxidative stress in response to PO. c-Nox4^−/− mice and WT littermates were subjected to either TAC for 4 wk or sham operation. (A) Microsomal, cytosolic, mitochondrial, and nuclear fractions were prepared from WT and c-Nox4^−/− mouse hearts. NADH-dependent and SOD-inhibitable O₂⁻ release was measured by the lucigenin chemiluminescent method. (B) Effects of rotenone and DPI on O₂⁻ production by the mitochondrial fraction (*P < 0.05). (C) The level of H₂O₂ in LV blocks from WT and c-Nox4^−/− mouse hearts was evaluated with the Amplex Red method. (D) LV myocardial sections obtained from WT and c-Nox4^−/− mice were subjected to immunostaining with anti–8-OHdG antibody. (Scale bars, 50 μm.) (E) MDA content was measured in heart homogenates from WT and c-Nox4^−/− mice. In A, B, C, and E, bar graphs indicate mean ± SEM obtained from three to five experiments.

Mitochondrial function is preserved in c-Nox4^−/− mice during PO. The cardiac mitochondrial fraction was prepared from c-Nox4^−/− mice and WT littermates subjected to TAC or sham operation. (A–D) Mitochondrial function was evaluated by the mitochondrial swelling assay (A), ATP production assay (B), quantitative real-time PCR for mitochondrial DNA (C), and aconitase assay (D). (E) Release of cytochrome c into the cytosol was evaluated with immunoblot analyses. In A–D, bar graphs indicate mean ± SEM obtained from three to four experiments.