Inducibility, but not stability, of atrial fibrillation is increased by NOX2 overexpression in mice

Abstract

Aims: Gp91-containing NADPH oxidases (NOX2) are a significant source of myocardial superoxide production. An increase in NOX2 activity accompanies atrial fibrillation (AF) induction and electrical remodelling in animal models and predicts incident AF in humans; however, a direct causal role for NOX2 in AF has not been demonstrated. Accordingly, we investigated whether myocardial NOX2 overexpression in mice (NOX2-Tg) is sufficient to generate a favourable substrate for AF and further assessed the effects of atorvastatin, an inhibitor of NOX2, on atrial superoxide production and AF susceptibility.

Methods and results: NOX2-Tg mice showed a 2- to 2.5-fold higher atrial protein content of NOX2 compared with wild-type (WT) controls, which was associated with a significant (twofold) increase in NADPH-stimulated superoxide production (2-hydroxyethidium by HPLC) in left and right atrial tissue homogenates (P = 0.004 and P = 0.019, respectively). AF susceptibility assessed in vivo by transoesophageal atrial burst stimulation was modestly increased in NOX2-Tg compared with WT (probability of AF induction: 88% vs. 69%, respectively; P = 0.037), in the absence of significant alterations in AF duration, surface ECG parameters, and LV mass or function. Mechanistic studies did not support a role for NOX2 in promoting electrical or structural remodelling, as high-resolution optical mapping of atrial tissues showed no differences in action potential duration and conduction velocity between genotypes. In addition, we did not observe any genotype difference in markers of fibrosis and inflammation, including atrial collagen content and Col1a1, Il-1β, Il-6, and Mcp-1 mRNA. Similarly, NOX2 overexpression did not have consistent effects on RyR2 Ca2+ leak nor did it affect PKA or CaMKII-mediated RyR2 phosphorylation. Finally, treatment with atorvastatin significantly inhibited atrial superoxide production in NOX2-Tg but had no effect on AF induction in either genotype.

Conclusion: Together, these data indicate that while atrial NOX2 overexpression may contribute to atrial arrhythmogenesis, NOX2-derived superoxide production does not affect the electrical and structural properties of the atrial myocardium.

Keywords: Arrhythmia (mechanisms); Atorvastatin; Atrial fibrillation; NADPH oxidases; Oxidant stress.

Graphical abstract

Figure 1

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.

Figure 2

NOX2-Tg mice are more prone to pacing-induced AF. (A) Surface ECG recordings illustrate episodes of atrial tachyarrhythmias resembling AF, characterized by absent P-waves and increased R-R interval variance, with recovery of sinus rhythm (SR). (B) Percentages of NOX2-Tg and WT mice with inducible AF episodes lasting at least 2 s. Note higher incidence of pacing-induced AF (≥2 s) in NOX2-Tg mice (P = 0.037; n = 58 WT and n = 56 NOX2-Tg mice). (C) The probability of AF induction was not different between genotypes (P = 0.83; n = 40 WT and n = 49 NOX2-Tg mice). Maximum AF duration (D) and cumulative AF duration (E) tended to be longer in NOX2-Tg mice but was not statistically significant (P > 0.05; n = 40 WT and n = 49 NOX2-Tg mice). Categorical data are shown as percentages and P-values were calculated using the Fisher’s exact test. Continuous data are shown as individual data points with medians and IQRs and P-values were calculated using the Mann–Whitney U test.

Figure 3

Atrial action potentials, conduction velocity, and diastolic Ca²⁺ leak in WT and NOX2-Tg atria following exposure to ANG-II. (A) Representative RA and LA optical action potentials recorded from WT or NOX2-Tg atria incubated with ANG-II. Summarized data for APD30, APD50, and APD80 in the RA (B) and LA (C) show no significant difference between WT and NOX2-Tg mice. Statistical significance between groups was determined for each APD using unpaired Student’s t-test. n = 11 and 9–10 for WT and NOX2-Tg atria, respectively. (D) Representative activation maps of a single spontaneous wave depolarization show a similar pattern of impulse conduction in WT and NOX2-Tg atria. Each isochrone line represents 1 ms. Summarized CV data show no significant difference in mean (E; n = 12 for WT and n = 10 for NOX2-Tg) or maximum (F; for RA, n = 12 for WT and n = 10 for NOX2-Tg; for LA, n = 9 for WT and n = 12 for NOX2-Tg) CV in the RA or LA between genotypes. Statistical significance between groups was determined for each parameter using unpaired Student’s t-test. (G) Diastolic Ca²⁺ leak was present in a greater proportion of NOX2-Tg RA myocytes (58%, 23/40 cells) compared with WT controls (33%, 11/33 cells) (P = 0.06, Fisher’s exact). The SR Ca²⁺ content (H) and the RyR2 leak—SR load ratio (I) was similar between NOX2-Tg (n = 23 cells from 9 mice) and WT RA myocytes (n = 11 cells from 9 mice). (J) The proportion of cells with diastolic Ca²⁺ leak was not different between NOX2-Tg and WT LA myocytes (P = 0.63, Fisher’s exact). (K) SR Ca²⁺ content was significantly greater in NOX2-Tg LA myocytes (n = 38 cells from 11 mice; 0.785, 25th–75th percentile: 0.56–1.02) compared with WT (n = 32 cells from 8 mice; 0.64, 25th–75th percentile: 0.52–0.81; P = 0.04), which meant that the RyR2 leak—SR load ratio (L) was reduced in NOX2-Tg LA myocytes (F365/380: 1.93, 25th–75th percentile: 0.64–3.78 for NOX2-Tg LA vs. 3.94, 25th–75th percentile: 2.3–4.7 for WT LA, P = 0.022, Mann–Whitney U test). Graphs show individual data points with means and SDs or medians and IQRs, as appropriate.

Figure 4

Atorvastatin prevents the increase in NADPH-stimulated atrial superoxide production secondary to NOX2 overexpression. (A) Representative HPLC chromatograms of superoxide production (2-OHE peak) in LA homogenates from WT and NOX2-Tg mice given ATV or placebo diet are shown. Blue, NADPH-stimulated superoxide production; Red, Tiron-inhibited trace. (B) NADPH-stimulated superoxide production is higher in RA homogenates of placebo-treated NOX2-Tg mice (P = 0.003 vs. WT-placebo). This difference is abolished by ATV treatment (P = 0.9). For placebo, n = 16 for WT and n = 11 for NOX2-Tg; and for ATV, n = 12 for WT and n = 9 for NOX2-Tg. (C) The increase in NADPH-stimulated superoxide production in LA homogenates of placebo-treated NOX2-Tg mice (n = 8) compared with WT controls (n = 11, P = 0.006) is prevented by ATV treatment, with no differences between ATV-treated groups (P = 0.84). For placebo, n = 11 for WT and n = 8 for NOX2-Tg; and for ATV, n = 12 for WT and n = 9 for NOX2-Tg. Two-way ANOVA with Tukey post hoc analysis was used to determine statistical significance of placebo and superoxide production with ATV between groups. Graphs show individual data points with medians and IQRs. For these experiments, two atria were pooled for each biological replicate.

Figure 5

Pacing-induced AF susceptibility in placebo and atorvastatin-treated WT and NOX2-Tg mice. (A) Percentages of NOX2-Tg and WT mice with inducible AF episodes lasting at least 2 s (n numbers as indicated). (B) The probability of AF (≥2 s) induction per mouse was not different between genotypes (P = 0.83) and there was no effect of ATV (P = 0.89). (C) Maximum AF durations tended to be longer in placebo-fed NOX2-Tg mice (117 s, 25th–75th percentile: 40–381 vs. 68 s, 25th–75th percentile: 32–433 s for placebo-fed WT) and shorter with ATV treatment (P = 0.07; WT, 43 s, 25th–75th percentile: 25–242 s vs. NOX2-Tg, 65 s, 25th–75th percentile: 22–261 s). (D) Cumulative AF duration was significantly lower in mice who received ATV (P = 0.038 vs. placebo), independent of genotype. AF probability and durations were quantified from mice that developed AF lasting at least 2 s (for placebo, n = 19 WT and n = 25 NOX2-Tg mice; for ATV, n = 22 WT and n = 27 NOX2-Tg mice). Categorical data are shown as percentages and P-values were calculated using the Fisher’s exact test. Continuous data are shown as individual data points with means and SDs or medians and IQRs and P-values were calculated by two-way ANOVA with Tukey post hoc analysis. To achieve normality, non-parametric data were log-transformed before statistical analysis.