Abstract 17814: Nadph Oxidase 4 Induces Cardiac Arrhythmia in Zebrafish
Accumulating evidence has indicated cardiac arrhythmias, particularly the most common form atrial fibrillation (AF), are associated with increased oxidative stress and structural remolding of the heart. We recently found that AF patients have elevated hydrogen peroxide production that is more than 3-fold of non-AF transplant patients, and this is correlated with an upregulation of cardiac NADPH oxidase 4 (NOX4) expression (Frontiers in Oxidant Physiology 2012 in press). The present study investigated whether overexpression of NOX4 in a zebrafish model induces cardiac arrhythmia. Full-length human NOX4 RNA (300 pg) was injected to one-cell stage embryos of cardiac specific GFP transgenic zebrafish. Interestingly, 52% of the NOX4 embryos developed arrhythmia at 24-30 hours postfertilization (hpf), while most of the control embryos had normal heart beats at the same stage (10% incidence). Quantification of the heart rates was carried out by video typing and software analysis. We discovered that the standard deviations of heart beats in NOX4 embryos were significantly bigger than those of control embryos (0.038±0.0020 vs. 0.027±0.0026 second/beat, n=11 and 17, all data as Mean±SEM, p=0.0028). The NOX4 embryos also had increased superoxide production (5.3±0.69 vs. 10.9±1.5 μM/min/mg, n=12 and 9, p<0.01). Overexpression of mutant NOX4 (proline 437 substituted with histidine led to inactivation) however failed to induce arrhythmia (n=15) and superoxide accumulation (n=5). The incidence rates of arrhythmogenesis in NOX4 embryos can be attenuated by NOX4 antisense morpholino co-injection, PEG-SOD treatment, or NOX inhibitor NSC23766 to 20%, 13% and 19%. All of these treatments abolished NOX4-induced superoxide production (5.9±1.1 (n=3), 4.7±0.54 (n=4) and 6.9±0.91 (n=5) μM/min/mg). At 24 hpf, the Calmodulin-dependent Protein Kinase II (CaMKII) phosphorylation (Thr286) was upregulated in NOX4 embryos (n=4). In summary, our data identified a novel phenotype of cardiac arrhythmia in NOX4 over-expressed zebrafish. This may represent the first direct evidence oxidative stress, specifically derived from NOX4 activation, plays a causal role in arrhythmia development.
- Heart rate/Heart rate variability
- Atrial fibrillation
- Reactive oxygen intermediates
- Oxidative stress
- Super oxide
- © 2012 by American Heart Association, Inc.