Background: Atrial fibrillation (AF) is the most common heart rhythm disorder in adults and a major cause of stroke. Unfortunately, current treatments of AF are suboptimal as they are not targeted to the molecular mechanisms underlying AF. Using a highly novel gene therapy approach in a canine, rapid atrial pacing model of AF, we demonstrate that NADPH oxidase 2 (NOX2) generated oxidative injury, by causing upregulation of a constitutively active form of acetylcholine-dependent K+ current (IKACh) - called IKH - is an important mechanism underlying not only the genesis but also the perpetuation of electrical remodeling in the intact, fibrillating atrium.
Methods: To understand the mechanism by which oxidative injury promotes the genesis and/or maintenance of AF, we performed targeted injection of NOX2 shRNA (followed by electroporation to facilitate gene delivery) in atria of normal dogs followed by rapid atrial pacing. We used in-vivo high density electrical mapping, isolation of atrial myocytes, whole-cell patch clamping, in-vitro tachypacing of atrial myocytes, lucigenin chemiluminescence assay, immunoblotting, real-time PCR, immunohistochemistry and Masson's trichrome staining.
Results: First, we demonstrate that generation of oxidative injury in atrial myocytes is a frequency-dependent process, with rapid pacing in canine atrial myocytes inducing oxidative injury through induction of NOX2 and generation of mitochondrial reactive oxygen species. We show that oxidative injury likely contributes to electrical remodeling in AF by upregulating IKH by a mechanism involving frequency-dependent activation of protein kinase C epsilon (PKCε). The time to onset of non-sustained AF increased by more than 5-fold in NOX2 shRNA treated dogs. Furthermore, animals treated with NOX2 shRNA did not develop sustained AF for up to 12 weeks. The electrophysiological mechanism underlying AF prevention was prolongation of atrial effective refractory periods, at least in part due to attenuation of IKH. Attenuated membrane translocation of PKCε appeared to be a likely molecular mechanism underlying this beneficial electrophysiological remodeling.
Conclusions: NOX2 oxidative injury: a) underlies onset as well as maintenance of electrical remodeling in AF, and b) can be successfully prevented with a novel, gene-based approach. Future optimization of this approach may lead to a novel, mechanism-guided therapy for AF.