Atrial NOX2 expression and superoxide production in WT and NOX2-Tg hearts. (A) Quantitative RT-PCR detection of the human NOX2 mRNA transcript confirms the presence of the transgene in the LA and RA of NOX2-Tg, but not WT, hearts (n = 8/group). (B) Expression of mouse Nox2 mRNA is significantly higher in the RA (P = 0.0034 vs. LA-WT and P < 0.0001 vs. LA-NOX2-Tg; one-way ANOVA), with no differences between genotypes. (C) Western blot shows detection of a 58 kDa NOX2 protein in atrial tissue homogenates from WT and NOX2-Tg mice. NOX2 protein content is significantly higher in NOX2-Tg hearts in both RA (P = 0.015 vs. WT, Mann–Whitney U test, n = 6 for WT and n = 5 for NOX2-Tg) and LA (P = 0.0025 vs. WT; Mann–Whitney U test; n = 7 for WT and n = 5 for NOX2-Tg) tissue homogenates. Two atria were pooled for each biological replicate. (D) Representative HPLC chromatograms showing the formation of 2-hydroxyethidium (2-OHE), the superoxide-derived oxidation product of DHE, and ethidium (E), the non-specific two-electron oxidation product, in LA homogenates. The tiron-inhibitable fraction of 2-OHE (i.e. the difference in the area under the 2-OHE peak between NADPH and tiron) was taken as a measure of the NADPH-stimulated superoxide production and found to be significantly higher in NOX2-Tg RA (P = 0.019 vs. WT, Mann–Whitney U test, n = 8 for WT and n = 5 for NOX2-Tg) and LA (P = 0.004 vs. WT, Mann–Whitney U test, n = 6/genotype). Graphs show individual data points with means and SDs or medians and IQRs, as appropriate.