A PROTEOMICS APPROACH TO IDENTIFY NEW CARDIAC INTERCALATED DISC PROTEINS

2130 Background: Human atrial fibrillation (AF) is hypothesized to be driven by localized sources that are possible targets for radiofrequency ablation (RFA). However, accurate identification of AF drivers in the 3D atrial structure may require simultaneous epi/endocardial (Epi/Endo) mapping. Methods: Coronary perfused human right atria (RA, n ¼ 6, 35–72 years) were optically mapped simultaneously by 3 high-resolution CMOS cameras (2 aligned Epi/Endo views, 3.3  3.3 cm2, and 1 entire Endo view, 9  9 cm2) with near-infrared dye. Sustained AF was induced by burst pacing during 10– 100 mM pinacidil (n ¼ 6). The AF driver was defined in the area of highest dominant frequency as a stable repetitive source of fibrillatory conduction. Results: Dual-sided intramural mapping revealed the common mechanism of sustained AF (430 minutes) in all 6 RA was a single, spatially and temporally stable, leading reentrant driver in the pectinate muscle region. Reentrant circuits (11.7 ⫾ 5  3.1 ⫾ 2  5.0 ⫾ 3 mm3, 93 ⫾ 44 ms cycle length [CL]) had 2 limbs and intramural pivot points or “U turns” in conduction, creating stable patterns seen from sub-Endo but not sub-Epi. Endo RFA through pivot points consecutively organized (þ10% CL, n ¼ 5) and/or terminated AF (n ¼ 11). Attempts to reinduce AF resulted in slower AF drivers in different locations (0–2 per RA) or macroreentrant AT around the ablation lesion that was terminated by further ablation (n ¼ 3). Conclusions: Our integrative mapping study demonstrates, for the first time, that stable intramural reentry may drive AF in the human heart. Furthermore, this study represents a proof of concept that targeted ablation of localized drivers may successfully treat AF.

Heart Rhythm, Vol 11, No 11, November 2014 anesthetized rabbits and in 8 cAVB dogs with dofetilide as a positive control. Results: DHE had an IC50 of 250 ⫾ 26 nM for IKr and dose dependently (0.01–1 μM) prolonged APD in cAVB cells, whereas APD prolongation after the highest dose of 10 μM was less severe. Early afterdepolarizations (EADs) were seen in 14%, 67%, 100%, and 67% of cells after 0.01, 0.1,1, and 10 μM DHE, respectively. Dofetilide (1 μM) increased APD and induced EADs in 60% of cells. In rabbits, QT interval increased significantly by 12% ⫾ 10% and 60% ⫾ 26% after 0.05 and 0.5 mg/kg/ 5 min DHE, respectively. The latter dose induced torsades de pointes arrhythmias (TdP) in 2 rabbits. Dofetilide (0.04 mg/kg) increased repolarization beyond the increase seen with either dose of DHE; in contrast, no TdP was induced. In cAVB dogs, 0.33 mg/kg/5 min DHE reached a blood plasma concentration of 1.2 μM, increased QT duration by 48% ⫾ 10% (Po.05*), and induced TdP in 3/4 dogs, which was comparable to dofetilide in the same animals. Surprisingly, a higher dose of 0.5 mg/kg/5 min DHE (blood plasma concentration 2.3 μM) induced a similar increase in QT duration (51 ⫾ 10%*), but this did not induce TdP (0/4), whereas after dofetilide 3 of these 4 animals developed TdP. Conclusions: DHE considerably prolonged repolarization, and proarrhythmic effects were observed in all experimental models used. However, it seems likely that, apart from IKr, DHE affects other ion currents and/or has extracardiac effects that could limit its proarrhythmic potential and explain the self-protecting properties observed at higher concentrations.

A PROTEOMICS APPROACH TO IDENTIFY NEW CARDIAC INTERCALATED DISC PROTEINS T.A.B. van Veen,1 S. Soni,1 H.J.A. Raaijmakers,1 L.M. Raaijmakers,1 M.A. Damen,2 L. van Stuijvenberg,1 M.A. Vos,1 A.J.R. Heck,2 A. Scholten3 1 University Medical Center Utrecht, Utrecht, Netherlands, 2Netherlands Proteomic Center, Utrecht, Netherlands, 3Utrecht Institute for Pharmaceutical Sciences, Utrecht, Netherlands.

DEHYDROEVODIAMINE, ISOLATED FROM THE DRIED UNRIPE FRUIT OF EVODIA RUTAECARPA PRESENT IN THE TRADITIONAL CHINESE MEDICINE WU CHU YU, HAS PROARRHYTHMIC EFFECTS IN VITRO AND IN VIVO THAT DISAPPEAR AT HIGHER CONCENTRATIONS M.A. Vos,1 R. Varkevisser,1 I. Baburin,2 A. Schramm,3 M. van der Heyden,1 M. Houtman,1 M. Jonsson,1 H. Takanari,1 J. Beekman,1 M. Hamburger,3 S. Hering2 1 UMC Utrecht, Utrecht, Netherlands, 2University of Vienna, Vienna, Austria, 3University of Basel, Basel, Switzerland. Background: Dehydroevodiamine (DHE) is a major phytochemical in fruits of Evodia rutaecarpa, a traditional Chinese herb known to have cardiovascular effects. These fruits are freely available and are contained in various products that are commercialized via the Internet. We aimed to determine whether DHE prolongs cardiac repolarization and induces proarrhythmia in suitable model systems. Methods: For in vitro action potential duration (APD) measurements, freshly isolated left ventricular cardiomyocytes obtained from dogs with chronic atrioventricular block (cAVB) were used. The electrophysiologic effects and proarrhythmic potential of DHE in vivo were evaluated in 8

Background: Orchestration of every heartbeat is highly dependent on efficient functioning of the cardiac intercalated disc. The intercalated disc is composed of a complex protein network that enables electrical continuity and chemical communication between individual cardiomyocytes. It also ensures mechanical integration of the individual cardiomyocytes into the syncytium of the cardiac muscle. Many studies have recently shed light on increasingly prevalent cardiac diseases involving dysfunction of the intercalated disc. Insufficient knowledge of its composition makes it difficult to study these disease mechanisms in more detail; therefore, we aimed to identify the intercalated disc proteome. Methods and Results: Here, for the first time using a combination of enrichment of membrane proteins, in-depth proteomics analysis, and bioinformatics, we discovered a defined set of new putative intercalated disc proteins in rat ventricular tissue. An enriched membrane fraction was prepared using differential centrifugation steps. We quantitatively evaluated 3455 proteins for their enrichment in the enriched membrane fraction with respect to their levels in the counterpart soluble fraction. An intuitive data filtering approach was used to generate a final set of 97 high potential intercalated disc proteins. These included several well-known markers (eg, Cx43 and N-cadherin) but also many interesting novel candidates. In the first approach to confirm their presence in the intercalated disc, we selected 4 candidates: Flotillin-2 (flot2), Nexilin (nexn), Popeye-domain-containingprotein 2 (popdc2), and thioredoxin-related-transmembrane-protein 2 (tmx2). Using immunocytochemistry we were able to confirm their complete colocalization with N-cadherin in the intercalated discs of human, dog, and rat heart cryosections and isolated ventricular cardiomyocytes. In tissue derived from patients who suffered from dilated cardiomyopathy or arrhythmogenic cardiomyopathy, flotillin-2 appeared to be present in an increased amount in the intercalated disc.

2131 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 IKACH 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. Noujaim1 1 Tufts Medical Center, Boston, MA, 2University of Michigan, Ann Arbor, MI, 3Tufts 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-IKACh); (2) ca-IKACh is due to interaction between Kir3.1 (a molecular correlate of IKACh) and protein kinase C epsilon (PKCε); and (3) oxidative stress (OS) is increased. However, the structural and molecular bases of ca-IKACh 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-IKACh 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 μM H2O2 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 phosphorylation at S185, and IKACh 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 IKACh increase. In mouse atrial myocytes, OS shortened APD90 from 98 ⫾ 13 ms (n ¼ 6) to 37.9 ⫾ 5.3 ms (n ¼ 13, Po.01). Tertiapin Q, a selective IKACh blocker, significantly prolonged APD90 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 phosphorylated in vitro by recombinant activated PKCε. Conclusions: Arrhythmogenic ca-IKACh 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. Gourdie1 1 Virginia Polytechnic Institute, Roanoke, VA, 2 California Polytechnic State University, San Luis Obispo, CA, 3University 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 (INa) 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 INa 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: INa blockade alone decreased CV isotropically but preferentially decreased CV-T during edema or Gj uncoupling. This is consistent with impairment of ephaptic coupling due to INa 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. Fedorov1 1 The Ohio State University, Columbus, OH z2The 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, 949494 mm3 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 pacinginduced 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 (11.73.15.0 mm3). 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-