NADPH Oxidase 4 Induces Cardiac Arrhythmic Phenotype in Zebrafish

Oxidative stress has been implicated in cardiac arrhythmia, although a causal relationship remains undefined. We have recently demonstrated a marked upregulation of NADPH oxidase isoform 4 (NOX4) in patients with atrial fibrillation (AF), which is accompanied by overproduction of reactive oxygen species (ROS). In the present study we investigated impact on cardiac phenotype of NOX4 overexpression in zebrafish. One cell stage embryos were injected of NOX4 RNA prior to video recording of GFP-labeled (myl7:GFP zebrafish line) beating heart in real time at 24-31 hpf (hrs post fertilization). Intriguingly, NOX4 embryos developed cardiac arrhythmia that is characterized by irregular heartbeats. When quantitatively analyzed by an established LQ-1 program, the NOX4 embryos displayed much more variable beat-to-beat intervals (mean of SDs of beat-to-beat intervals was 0.027 sec/beat in control embryos, vs. 0.038 sec/beat in NOX4 embryos). Both the phenotype and the increased ROS in NOX4 embryos were attenuated by NOX4 morpholino (MO) co-injection, treatments of the embryos with PEG-SOD, or NOX4 inhibitors Fulvene-5, 6-dimethylamino-Fulvene and Proton Sponge Blue. Injection of NOX4-P437H mutant RNA had no effect on cardiac phenotype or ROS production. In addition, CaMKII phosphorylation was increased in NOX4 embryos but diminished by PEG-SOD, while its inhibitor KN93 or AIP abolished the arrhythmic phenotype. Taken together, our data for the first time uncover a novel pathway that underlies development of cardiac arrhythmia, namely NOX4 activation, subsequent NOX4-specific, NADPH-driven ROS production, and redox-sensitive CaMKII activation. These findings may ultimately lead to novel therapeutics targeting cardiac arrhythmia.