Abstract 2275: Nox4 is a Major Source of Superoxide Production in the Mouse Heart
NAD(P)H oxidases (Noxs) produce superoxide and play an important role in cardiovascular pathophysiology. The Nox4 isoform is expressed in cardiac myocytes. In order to elucidate the function of endogenous Nox4, we have generated cardiac-specific Nox4 −/− (c-Nox4−/−) mice. Cardiac specific deletion of Nox4 was achieved using αMHC-Cre. Neither early mortality nor obvious cardiac abnormality was observed. Nox4 expression in the heart was markedly inhibited in c-Nox4 −/− mice, whereas Nox4 expression in other organs, such as the kidneys, in c-Nox4 −/− mice was similar to that in wild-type (WT) mice. There was no compensatory upregulation of other members of the Nox family in the heart. Expression of p22phox, a membrane-integrated protein that forms a heterodimer with Nox proteins, was reduced, possibly due to destabilization caused by the depletion of Nox4 (48.3% of WT). SOD-inhibitable (SI)-superoxide production in whole heart homogenates, measured by a chemiluminescence method, was significantly less in c-Nox4 −/− mice (485 ± 95 RLU, n = 3) than in WT mice (1332 ± 266 RLU, n = 3; p < 0.001). SI-superoxide production by the mitochondrial fraction (768 ± 163 vs. 2781 ± 548 RLU, n = 3; p <0.01) and the microsomal fraction (464 ± 17 vs. 682 ± 49 RLU, n = 3; p < 0.05) was also less in c-Nox4 −/− mice than in WT mice. Dihydroethidium staining of the heart sections showed significantly less fluorescence in c-Nox4 −/− mice (64.0 ± 4.2 % of WT; p < 0.005) than in WT mice. These results suggest that Nox4 plays an important role in mediating superoxide production in the heart, especially in the mitochondria and microsomes. LV weight/body weight (3.3 vs 3.4 mg/g, NS) and echocardiographically determined cardiac function, such as ejection fraction (73.2 vs 70.5%, NS), in c-Nox4 −/− mice were similar to those in WT mice at 3 months of age. In summary, c- Nox4 −/− mice exhibited significantly reduced levels of superoxide production in the heart, suggesting that Nox4 is a major producer of superoxide in the heart at baseline. Despite significant reduction in superoxide production, c-Nox4 −/− mice exhibited an apparently normal cardiac phenotype, suggesting that Nox4 is dispensable for physiological growth and maintenance of LV function in the postnatal heart at baseline.