Conclusion: The presented proteomics dataset of intercalated disc proteins is a valuable resource for future research into this important molecular intersection of the heart. STRUCTURAL AND MOLECULAR BASES FOR THE CONSTITUTIVE ACTIVITY OF I KACH IN CHRONIC ATRIAL FIBRILLATION G. Bassil, 1 M. Haburcak, 1 Y. Takemoto, 2 D. Slough, 3 R. Karas, 1 J. Jalife, 2 Y. Lin, 3 S. Noujaim 1 1 Tufts Medical Center, Boston, MA, 2 University of Michigan, Ann Arbor, MI, 3 Tufts University, Medford, MA. Background: A hallmark of remodeling in chronic atrial fibrillation (AF) is action potential duration (APD) shortening. It has been suggested that in chronic AF: (1) the acetylcholine-sensitive inward rectifier potassium current is constitutively active (ca-I KACh ); (2) ca-I KACh is due to interaction between Kir3.1 (a molecular correlate of I KACh ) and protein kinase C epsilon (PKCε); and (3) oxidative stress (OS) is increased. However, the structural and molecular bases of ca-I KACh and its possible role in APD shortening and AF maintenance remain poorly understood. We hypothesized that OS phosphorylates Kir3.1 at residue S185 via PKCε, leading to ca-I KACh and contributing to APD shortening and AF perpetuation. Methods and Results: We tested our hypothesis using molecular, electrophysiologic, and numerical approaches. We induced OS with 100 μMH 2 O 2 for 1 hour. In HEK cells transfected with Kir3.1/4, OS increased reactive oxygen species as assayed by dihydroethidium fluorescence. In addition, PKCε translocated to the membrane, Kir3.1 showed phosphoryla- tion at S185, and I KACh increased from –16.1  5.1 pA/pF (n ¼ 3) to –34.2  3.8 pA/pF (n ¼ 6, Po.01). PKCε silencing prevented OS-induced phosphorylation of Kir3.1 and I KACh increase. In mouse atrial myocytes, OS shortened APD 90 from 98  13 ms (n ¼ 6) to 37.9  5.3 ms (n ¼ 13, Po.01). Tertiapin Q, a selective I KACh blocker, significantly prolonged APD 90 in stressed but not unstressed myocytes. In sheep atria with tachypacing-induced chronic AF, phospho-Kir3.1 increased 2.45-fold in chronic AF (n ¼ 4) vs sham (n ¼ 4, Po.05). Optical maps showed that tertiapin Q progressively reduced chronic AF dominant frequency until sinus rhythm resumed. Molecular dynamics simulations using the chimeric Kir3.1 crystal structure suggested that the region containing S185 was a switch that initiated conformational changes resulting in channel opening upon phosphorylation. Finally, purified chimeric Kir3.1 was phosphory- lated in vitro by recombinant activated PKCε. Conclusions: Arrhythmogenic ca-I KACh is due in part to OS-mediated PKCε activation and Kir3.1 phosphorylation at residue S185. These results shed new light on the complex ionic mechanisms of AF and may help in the development of new anti-AF agents. ANISOTROPIC CONDUCTION SLOWING DURING SODIUM CHANNEL BLOCKADE: A ROLE FOR EPHAPTIC COUPLING? R. Veeraraghavan, 1 J. Lin, 2 J.P. Keener, 3 S. Poelzing, 1 R. Gourdie 1 1 Virginia Polytechnic Institute, Roanoke, VA, 2 California Polytechnic State University, San Luis Obispo, CA, 3 University of Utah, Salt Lake City, UT. Background: We previously demonstrated that conduction slows during edema, possibly due to weakened ephaptic coupling. We hypothesized that sodium current (I Na ) inhibition during intercellular uncoupling would unmask anisotropic conduction dependence on ephaptic coupling. Methods: Conduction velocity (CV) and anisotropy (AR) were quantified by optical mapping in Langendorff-perfused guinea pig ventricles (n ¼ 5 all groups). Intermembrane distance was quantified by electron microscopy. Edema was induced with mannitol (26.1 g/L), gap junctions (Gj) were uncoupled by carbenoxolone (Cbx, 25 mM), and I Na was inhibited by flecainide (0.5 mM). Differences are reported with Po.05. Results: Edema increased intermembrane distance within the perinexus (18.5  1.1 nm vs 10.2  1.3 nm) but not at nonperinexal sites within the intercalated disc (14.5  0.7 nm vs 12.7  0.7 nm). The perinexus is a microdomain with elevated connexin43 and sodium channel levels located around gap junctions. During control, longitudinal CV (CV-L) and transverse CV (CV-T) were 52  1 cm/s and 21  1 cm/s, respectively; AR was 2.5  0.1. Flecainide alone uniformly decreased CV from control without changing AR. Mannitol (edema) preferentially decreased CV-T and increased AR to 3.0  0.1 relative to control. Importantly, flecainide þ mannitol preferentially decreased CV-T, increased AR to 3.3  0.2, and increased spontaneous arrhythmias (7/9 vs 4/11) relative to mannitol alone. Cbx preferentially decreased CV-T and increased AR to 2.9  0.1 relative to control. Importantly, flecainide þ cbx decreased CV-T preferentially, raised AR to 3.3  0.2, and increased spontaneous arrhythmias (7/9 vs 3/ 12) relative to Cbx alone. Interestingly, only a computer model including ephaptic coupling and colocalization of sodium channels with Gj could recapitulate these results. Conclusions: I Na blockade alone decreased CV isotropically but preferen- tially decreased CV-T during edema or Gj uncoupling. This is consistent with impairment of ephaptic coupling due to I Na blockade under these conditions and suggests that cellular localization of Nav1.5 with Gjs is a key determinant of anisotropic cardiac conduction. INTEGRATION OF GADOLINIUM-ENHANCED MRI AND SIMULTANEOUS EPICARDIAL–ENDOCARDIAL OPTICAL MAPPING REVEALS MICROANATOMIC SUBSTRATES ANCHORING REENTRANT DRIVERS DURING SUSTAINED ATRIAL FIBRILLATION IN HUMAN HEART T.A. Csepe, 1 J. Zhao, 2 B.J. Hansen, 1 N. Li, 1 L. Jayne, 1 B. Moore, 1 P. Lim, 2 A. Bratasz, 1 K.A. Powell, 1 O. Simonetti, 1 R.S.D. Higgins, 1 A. Kilic, 1 P.J. Mohler, 1 P.M.L. Janssen, 1 R. Weiss, 1 J.D. Hummel, 1 V.V. Fedorov 1 1 The Ohio State University, Columbus, OH z 2 The University of Auckland, Auckland, New Zealand. Background: The complex 3D microstructure of diseased human atria could provide substrates for reentry that can drive atrial fibrillation (AF). However, current clinical approaches cannot capture the precise structure of these AF driver substrates. Methods: Ex vivo, 6-day gadolinium-enhanced MRI (GE MRI, 949494 mm 3 resolution) was used, with histologic validation, to resolve the underlying 3D structure and fibrosis distribution in regions of AF drivers in right atria (RA) from explanted human hearts (n ¼ 4, 34–65 years old) with structural cardiac remodeling. Epi–endocardial optical mapping (3 CMOS cameras) was used to identify the drivers during sustained pacing- induced AF. Results: 3D GE MRI analysis of epi–endocardially mapped RA (n ¼ 4) revealed that stable reentrant AF drivers were anchored on microanatomic tracks (11.73.15.0 mm 3 ). These 3D microanatomic tracks consisted of 2 limbs: a “central” pectinate muscle (PM) (2.0  0.5mm thick) and the subepicardial wall, neighboring PM, and/or atrial vestibule. The limbs were connected by small intramural bundles (0.16–0.8 mm thick) where the reentrant driver repetitively made a “U turn” (Figure). Perimysial fibrosis (152  49 mm) insulated central PMs and intramural bundles from surrounding atria and may stabilize reentrant AF drivers. Conclusions: Our ex vivo integrative structural/functional study of diseased human RA revealed that microanatomic tracks for reentrant AF drivers are insulated by fibrosis and consist of a central PM connected to small intramural bundles. Our study suggests that GE MRI-defined microana- 2131