TY - JOUR
T1 - Attenuation of Oxidative Injury with Targeted Expression of NADPH Oxidase 2 Short Hairpin RNA Prevents Onset and Maintenance of Electrical Remodeling in the Canine Atrium
T2 - A Novel Gene Therapy Approach to Atrial Fibrillation
AU - Yoo, Shin
AU - Pfenniger, Anna
AU - Hoffman, Jacob
AU - Zhang, Wenwei
AU - Ng, Jason
AU - Burrell, Amy
AU - Johnson, David A.
AU - Gussak, Georg
AU - Waugh, Trent
AU - Bull, Suzanne
AU - Benefield, Brandon
AU - Knight, Bradley P.
AU - Passman, Rod
AU - Wasserstrom, J. Andrew
AU - Aistrup, Gary L.
AU - Arora, Rishi
N1 - Publisher Copyright:
© 2020 Lippincott Williams and Wilkins. All rights reserved.
Copyright:
Copyright 2020 Elsevier B.V., All rights reserved.
PY - 2020/9/29
Y1 - 2020/9/29
N2 - 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 because 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 causes upregulation of a constitutively active form of acetylcholine-dependent K+current (IKACh), called IKH; this is an important mechanism underlying not only the genesis, but also the perpetuation of electric remodeling in the intact, fibrillating atrium. Methods: To understand the mechanism by which oxidative injury promotes the genesis and maintenance of AF, we performed targeted injection of NOX2 short hairpin RNA (followed by electroporation to facilitate gene delivery) in atria of healthy dogs followed by rapid atrial pacing. We used in vivo high-density electric mapping, isolation of atrial myocytes, whole-cell patch clamping, in vitro tachypacing of atrial myocytes, lucigenin chemiluminescence assay, immunoblotting, real-time polymerase chain reaction, immunohistochemistry, and Masson 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 the induction of NOX2 and the generation of mitochondrial reactive oxygen species. We show that oxidative injury likely contributes to electric remodeling in AF by upregulating IKAChby a mechanism involving frequency-dependent activation of PKCϵ(protein kinase C epsilon). The time to onset of nonsustained AF increased by >5-fold in NOX2 short hairpin RNA-treated dogs. Furthermore, animals treated with NOX2 short hairpin RNA 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 attributable to the attenuation of IKACh. Attenuated membrane translocation of PKCϵappeared to be a likely molecular mechanism underlying this beneficial electrophysiological remodeling. Conclusions: NOX2 oxidative injury (1) underlies the onset, and the maintenance of electric remodeling in AF, as well, and (2) 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.
AB - 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 because 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 causes upregulation of a constitutively active form of acetylcholine-dependent K+current (IKACh), called IKH; this is an important mechanism underlying not only the genesis, but also the perpetuation of electric remodeling in the intact, fibrillating atrium. Methods: To understand the mechanism by which oxidative injury promotes the genesis and maintenance of AF, we performed targeted injection of NOX2 short hairpin RNA (followed by electroporation to facilitate gene delivery) in atria of healthy dogs followed by rapid atrial pacing. We used in vivo high-density electric mapping, isolation of atrial myocytes, whole-cell patch clamping, in vitro tachypacing of atrial myocytes, lucigenin chemiluminescence assay, immunoblotting, real-time polymerase chain reaction, immunohistochemistry, and Masson 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 the induction of NOX2 and the generation of mitochondrial reactive oxygen species. We show that oxidative injury likely contributes to electric remodeling in AF by upregulating IKAChby a mechanism involving frequency-dependent activation of PKCϵ(protein kinase C epsilon). The time to onset of nonsustained AF increased by >5-fold in NOX2 short hairpin RNA-treated dogs. Furthermore, animals treated with NOX2 short hairpin RNA 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 attributable to the attenuation of IKACh. Attenuated membrane translocation of PKCϵappeared to be a likely molecular mechanism underlying this beneficial electrophysiological remodeling. Conclusions: NOX2 oxidative injury (1) underlies the onset, and the maintenance of electric remodeling in AF, as well, and (2) 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.
KW - I KH
KW - NADPH oxidase 2
KW - atrial fibrillation
KW - gene therapy
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U2 - 10.1161/CIRCULATIONAHA.119.044127
DO - 10.1161/CIRCULATIONAHA.119.044127
M3 - Article
C2 - 32686471
AN - SCOPUS:85091956958
SP - 1261
EP - 1278
JO - Circulation
JF - Circulation
SN - 0009-7322
ER -