The precise mechanisms by which oxidative stress (OS) causes atrial fibrillation (AF) are not known. Since AF frequently originates in the posterior left atrium (PLA), we hypothesized that OS, via calmodulin-dependent protein kinase II (CaMKII) signaling, creates a fertile substrate in the PLA for triggered activity and reentry. In a canine heart failure (HF) model, OS generation and oxidized-CaMKII-induced (Ox-CaMKII-induced) RyR2 and Na v 1.5 signaling were increased preferentially in the PLA (compared with left atrial appendage). Triggered Ca 2+ waves (TCWs) in HF PLA myocytes were particularly sensitive to acute ROS inhibition. Computational modeling confirmed a direct relationship between OS/CaMKII signaling and TCW generation. CaMKII phosphorylated Na v 1.5 (CaMKII-p-Na v 1.5 [S571]) was located preferentially at the intercalated disc (ID), being nearly absent at the lateral membrane. Furthermore, a decrease in ankyrin-G (AnkG) in HF led to patchy dropout of CaMKII-p-Na v 1.5 at the ID, causing its distribution to become spatially heterogeneous; this corresponded to preferential slowing and inhomogeneity of conduction noted in the HF PLA. Computational modeling illustrated how conduction slowing (e.g., due to increase in CaMKII-p-Na v 1.5) interacts with fibrosis to cause reentry in the PLA. We conclude that OS via CaMKII leads to substrate for triggered activity and reentry in HF PLA by mechanisms independent of but complementary to fibrosis. insight.jci.org https://doi.org/10.1172/jci.insight.120728 R E S E A R C H A R T I C L Epropensity for spontaneous calcium (Ca 2+ ) release (in the form of Ca 2+ waves) in atrial myocytes of patients with chronic AF (16). Our work indicates that atrial myocytes in the setting of HF have increased susceptibility to the development of Ca 2+ waves during rapid atrial pacing (i.e., triggered Ca 2+ waves; TCWs) (17). Ca 2+ waves are known to create conditions in both the ventricle and atrium for the genesis of afterdepolarizations, which can lead to triggered activity and/or dispersion of repolarization (18,19). Another major mechanism thought to underlie the development of a vulnerable AF substrate in HF is slow and inhomogeneous conduction in the intact atrium, which creates conditions for reentry (20)(21)(22). While several studies suggest that increased fibrosis in the HF atrium is a major cause of this inhomogeneous conduction (21,23), it is also known that the HF atrium undergoes significant electrical remodeling (24), with alterations in the I Na having been implicated in some studies in creation of the AF disease state (25). Our specific hypotheses for this study were, therefore, as follows: (a) OS generation -and downstream oxidation of key signaling proteins -is preferentially increased in the PLA/PVs in the HF atrium; (b) the increased vulnerability of HF atrial myocytes to TCW generation is at least partially mediated by OS, with OS creating substrate for TCWs by increasing CaMKII-p-RyR2 (S2814) in the HF PLA; and (c) OS, via Ox-CaMKII, increases the level of C...
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 ( I KACh ), called I KH ; 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 I KACh by a mechanism involving frequency-dependent activation of PKC ε (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 I KACh . Attenuated membrane translocation of PKC ε appeared 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.
Background: We have identified a novel form of abnormal Ca 2+ wave activity in normal and failing dog atrial myocytes which occurs during the action potential (AP) and is absent during diastole. The goal of this study was to determine if triggered Ca 2+ waves affect cellular electrophysiological properties. Methods: Simultaneous recordings of intracellular Ca 2+ and APs allowed measurements of maximum diastolic potential and AP duration during triggered calcium waves (TCWs) in isolated dog atrial myocytes. Computer simulations then explored electrophysiological behavior arising from TCWs at the tissue scale. Results: At 3.3 to 5 Hz, TCWs occurred during the AP and often outlasted several AP cycles. Maximum diastolic potential was reduced, and AP duration was significantly prolonged during TCWs. All electrophysiological responses to TCWs were abolished by SEA0400 and ORM10103, indicating that Na-Ca exchange current caused depolarization. The time constant of recovery from inactivation of Ca 2+ current was 40 to 70 ms in atrial myocytes (depending on holding potential) so this current could be responsible for AP activation during depolarization induced by TCWs. Modeling studies demonstrated that the characteristic properties of TCWs are potentially arrhythmogenic by promoting both conduction block and reentry arising from the depolarization induced by TCWs. Conclusions: Triggered Ca 2+ waves activate inward NCX and dramatically reduce atrial maximum diastolic potential and prolong AP duration, establishing the substrate for reentry which could contribute to the initiation and maintenance of atrial arrhythmias.
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