Most LQT-linked KCNH2 mutations are trafficking-defective

Poster 3 maps consisted of 121⫾67 BP. The incidence of fractionated potentials ranged from 19 to 75 [48⫾16]%. The occurrence of fractionated BP within a distance of ⱕ2 cm of the site of earliest activity (’focal area’) was higher compared to the remainder of the atria (71⫾23% versus 31⫾15%, p⬍0.001). Fractionation delays of BP recorded from the ’focal area’ were longer than from the remainder of the atria (BP focal area: P50: 41 ms, [P0: 7- P100: 154 ms], remainder: P50:27 [P0:2-P112 ms], p⬍0.002). Conclusion: Significant differences in fractionation and fractionation delay of BP between the ’focal area’ and the remainder of the atria exist. The results of this study support the hypothesis that a certain degree of cellular uncoupling is required for the development of a FAT thereby providing further insight into the aetiology of FAT. P2-126 DEVELOPMENT OF MANIFEST ACCESSORY PATHWAY CONDUCTION AFTER INITIATION OF DDD PACING IN TWO PATIENTS WITH AV-BLOCK J.-M. Happonen, MD, *Anita Hiippala, MD and Lauri Toivonen, MD. Helsinki University Hospital for Children and Adolescents, Helsinki, Finland and Helsinki University Hospital, Helsinki, Finland. Background: It is common that intermittent accessory pathway (AP) conduction causes ventricular pre-excitation, but it is thought that pre-excitation does not develop in later life. We describe two patients with complete atrioventricular block (AVB), in whom manifest AP conduction appeared during physiologic DDD pacing. Patients: A 4 year-old boy was diagnosed with complete AVB. He was followed for 3 years with serial 24-hour ambulatory ECG recordings, all of which showed complete AVB with narrow complex escape rhythm. At 7 years of age, when his escape rate was 47 bpm, an endocardial DDD pacemaker (PM) was implanted. In follow-up the patient had sinus rhythm at 60 bpm, PR interval of 120 ms and ventricular pre-excitation consistent with a posteroseptal AP. Tested nine years later at PM replacement, there was 1:1 AP conduction up to 165 bpm, AP refractory period was 250 ms, and there was no VA conduction. A 50 year-old male with recurrent syncopal episodes was found to have complete AVB with a wide complex escape rhythm at 35 bpm. When AV conduction recovered temporarily, ECG showed sinus rhythm at 70 bpm and a PR interval of 200 ms, RBBB and LAHB, and Mobitz II type block at exercise. A VVI pacing system was implanted. During follow-up permanent complete AVB was seen in resting ECG and two 24-hour ambulatory ECG recordings. Three years later his PM was upgraded to a DDD system. Next day he had sinus rhythm at 60 bpm, PR interval of 140 ms and ventricular pre-excitation consistent with a posteroseptal AP. VA conduction was blocked at 105 bpm. Ten years later antegrade conduction via AP was still observed. Discussion: Both patients had complete AVB prior to PM implantation or during VVI pacing. After initiation of physiologic pacing antegrade conduction via a posteroseptal AP became apparent. Synchronization of the atrial (and thus AP) activation with late ventricular diastole by physiologic pacing facilitated propagation of the impulse to the ventricles. It seems that modulation of conduction depends on exact timing of the impulse within the cardiac cycle of the receiving myocardial tissue.

POSTER SESSION 3 Thursday, May 5, 2005 Session Time: 2:00 p.m.–5:00 p.m. Presenter Available: 3:45 p.m.– 4:45 p.m. Location: Exhibit Hall P3-1 MOLECULAR CHARACTERIZATION OF VERY EARLY BRADYCARDIC VENTRICULAR ELECTRICAL REMODELING IN RABBITS Fumiaki Suto, MD, PhD, Wei Zhu, MD, Katy-Adair Finlay and Gil J. Gross, MD. Hospital for Sick Children, Toronto, Ontario, Canada.

S175 Background: We previously demonstrated ECG QT interval (QTi) prolongation, spontaneous torsades des pointes, and downregulation of ventricular myocyte rapidly (IKr) and slowly (IKs) activating delayed rectifier currents in rabbits with complete heart block (CHB) paced for 8 days at 140 beats/min (⬃1⁄2 physiologic sinus rate). The aim of the present study was to correlate very early in vivo manifestations of bradycardic remodeling with changes in expression of IKr and IKs as well as their molecular substrates, the K⫹ channels ERG and KvLQT1. Methods: CHB induced by transcatheter radiofrequency AV node ablation was followed by right ventricular endocardial demand pacing at 140 beats/min in New Zealand White rabbits. QTi was measured on CHB days 0 and 2. Beat-by-beat QTi measured for 2 min after abrupt rate change from 140 to 210 beats/min was used to plot dynamic QTi adaptation (QTa) curves fitted by a single exponential decay model yielding a QTa time constant (␶). Ventricular myocytes were enzymatically isolated on day 2 for whole cell patch clamp evaluation of IKr and IKs tail current density. Real time polymerase chain reaction (PCR) was used to semiquantitatively measure ventricular mRNA encoding ERG and KvLQT1. Control tail current and mRNA data were obtained from rabbits undergoing sham AV node ablation. Results: QTi was significantly prolonged by day 2, but QTa was unchanged. IKr and ERG expression was more substantially diminished than that of IKs or KvLQT1 (Table). Conclusions: These observations suggest that IKr/ERG is (i) downregulated sooner than IKs/KvLQT1 in the earliest stages of bradycardic ventricular remodeling, and (ii) associated more directly with steady-state QTi prolongation than with impaired QTi adaptation to abrupt rate changes.

Data are shown as mean ⫾ SD (n ⫽ 6-9) *P ⬍ 0.05 vs day 0 or control; # P ⫽ 0.16 vs. control a.u. ⫽ arbitrary units P3-2 MOST LQT-LINKED KCNH2 MUTATIONS ARE TRAFFICKINGDEFECTIVE Brian P. Delisle, PhD, Corey L. Anderson, BS, Blake D. Anson, PhD, Jennifer A. Kilby, BS, Melissa L. Will, BSc, David J. Tester, BSc, Michael J. Ackerman, MD, PhD, Qiuming Gong, MD, PhD, Zhengfeng Zhou, MD, PhD and Craig T. January, MD, PhD. University of Wisconsin, Madison, WI, Mayo Clinic, Rochester, MN and Oregon Health and Science University, Portland, OR. Over 100 mutations in the KCNH2 or human ether-a-go-go-related gene, which encodes the ␣-subunit peptides of the cardiac rapidly activating delayed rectifier K⫹ channel, have been linked to Long QT syndrome (LQT2). LQT2-linked mutations reduce KCNH2 current (IKCNH2) by altering channel protein trafficking and/or biophysical properties. We tested the hypothesis that the majority of LQT2-linked mutations cause protein trafficking defects. WT and 34 LQT2 missense mutations were heterologously expressed in HEK293 cells. On Western blot analysis, WT KCNH2 channels show two protein bands, a core-glycosylated immature band (⬃135kDa) and a complexly glycosylated mature band (⬃155kDa), whereas trafficking-defective LQT2 mutations are retained in the ER as immature protein. The mature protein band was absent or weakly present in 28 of the 34 LQT2 mutations studied, suggesting they were trafficking defective. Electrophysiological analysis showed that all of the traffickingdefective mutations tested markedly reduced IKCNH2 compared to WT channels. Of the 6 KCNH2 mutations that produced both the immature and mature protein bands, one disrupted gating, one disrupted permeation, and the other 4 expressed IKCNH2 similar to WT. Some trafficking-defective LQT2 mutations can be induced to form the mature protein and increase channel expression in the surface membrane. We tested for this by incu-

S176 bating KCNH2 mutation expressing cells for 24h at reduced temperature (27oC), in the presence of drugs that block IKCNH2 (E4031), or in the presence of the SERCA inhibitor thapsigargin. Of the 28 traffickingdefective mutations, Western blot analysis showed the appearance of the mature band in 16 when cells were incubated at 27oC, 17 when cells were incubated in E4031 (10␮M), and 8 when cells were incubated in thapsigargin (1␮M). Our findings suggest, 1) that LQT2 is a disease dominated by defective protein trafficking, 2) for many trafficking-defective mutations their protein processing can be improved to increase surface membrane expression of functional channels, and 3) not every putative LQT2 mutation has a phenotype that differs from WT channels. P3-3 THE CHANGES OF CX43, METALLOPROTEINASE AND TISSUE INHIBITOT OF METALLOPROTEINASE IN TACHYCARDIAINDUCED CARDIOMYOPATHY INDUCED BY ATRIAL FIBRILLATION IN DOGS Jing-Quan Zhong, MD, PhD, Wei Zhang, MD, PhD, Yan Li, MD, Ming Zhong, MD, PhD and Yun Zhang, MD, PhD. Qilu Hospital, Shandong University, Jinan, China. Objectives: Gap junction remodeling may be involved in initiation and persistence of TIC. Methods: Dogs paced by rapid right atrial pacing at 350⬃ 450 bpm for 8 weeks (n⫽11) were compared with sham dogs (n⫽6). Canine models of TIC were established and each procedure was measured continuously 15 seconds. The content of Cx43 were measured by immunoblotting and confocal immunofluorescence microscopy using isoform-specific antibodies. Left ventricular volume waveforms were obtained by AQ technique and analyzed to compute parameters of systolic and diastolic function. Results: 1.Hemodynamic changes: Compared with control group, after 1 week rapid pacing, TIC group had decreased ⫹dp/dtmax ( p⬍0.05); Left ventricular relaxation time constant was prolonged ( p⬍0.05); left ventricular end-diastolic pressure(LVEDP) was significant increased( p⬍0.05) and -dp/ dtmax was significant decreased ( p⬍0.001) with no statistic diffrences of left ventricular ejection fraction between 1 week, 4 weeks and 8 weeks ( p⬎0.05). These data suggested that canine model of TIC was established successfully. 2.Ultrastructural changes: Remarkable changes in intracellucar membranous ultrastructures such as nucleus, mitochondria and sarcoplasmic reticulum were observed by transmission eletron microscopy. Myocardial fiber disarray was great, which the fibers run in various directions. Mitochondria clustered with waveform. Sarcoplasmic reticulum network density decreased. 3. Compared with the control group, the content of Cx43 in left ventricular in TIC canine was significantly decreased ( p⬍0.001). Conclusions: 1.Ventricular diliation and systolic disfunction occurred after 1-week rapid right atrial pacing. 2.There are structure damages of myofibrills, mitochondria, sarcoplasmic reticulum and nuclear chromatin. 3.The content of Cx43 is decreased significantly and gap junction remodeling occurred during TIC. 4.The formation of the TIC may result from the several mechanisms such as ultrastructural changes, hemodynamic abnormalities and gap junction remodeling. P3-4 STRETCH CHANNEL BLOCKER INHIBITS PULMONARY VEIN ARRHYTHMOGENIC ACTIVITY Shih-Lin Chang, MD, Yi-Jen Chen, MD, PhD, Yao-Chang Chen, ScD, MS, Cheng-I Lin, PhD and Shih-Ann Chen, MD. National Yang-Ming University School of Medicine and Veterans General Hospital-Taipei, Taipei, Taiwan Republic of China, Taipei Medical University, Wan-Fang Hospital, Tapei, Taiwan Republic of China and Institute of Physiology and Department of Biomedical Engineering, National Defense Medical Center, Taipei, Taiwan Republic of China. Background: Atrial fibrillation is frequently associated with dilated pulmonary vein (PV). Stretch has been shown to have cardiac mechanoelectrcial effects and increase PV arrhythmogenic Activity. The purpose of this

Heart Rhythm, Vol 2, No 5, May Supplement 2005 study was to investigate whether stretch channel blocker may inhibit the arrhythmogenic activity of PVs. Methods: Transmembrance action potentials were obtained from rabbit PV tissue specimen with and without stretch tension (300 mg) using the conventional microelectrode recording. Gadolinium and streptomycin were perfused in PVs during stretch, respectively. Results: Gadolinium (0, 1, 3, 10 ␮M) dose-dependently decreased the incidence (100%, 83%, 58%, 33%, P ⬍ 0.05) and beating rates (2.9⫾0.19, 1.9⫾0.92, 1.0⫾0.94, 0.3⫾0.17 Hz, P ⬍ 0.05) of spontaneous activities in 12 PVs with stretch induced spontaneous activities. Gadolinium (0, 1, 3, 10 ␮M) also dose-dependently shortened the 90% of the action potential duration (109⫾13, 117⫾4, 123⫾6, 135⫾9 ms, P ⬍ 0.05) and incidences of delayed afterdepolarization (42%, 17%, 0%, 0%, P ⬍ 0.05) in PV preparations. The other stretch channel blocker, streptomycin (0, 10 and 40 ␮M) dose-dependently decreased the incidence (100%, 71%, 29%, P ⬍ 0.05) and beating rates (2.9⫾0.06, 2.3⫾0.16 , 1.8⫾0.10 Hz, P ⬍ 0.05) of stretch induced spontaneous activities in 7 PVs. However, in the 4 PVs with spontaneous activity during no stretch, gadolinium (0, 1, 3, 10 ␮M) did not have significant effect on the spontaneous activity (1.62⫾0.17, 1.58⫾0.1, 1.54⫾0.08, 1.49⫾0.15 Hz, P ⬎ 0.05). Conclusions: Stretch channel blocker has a potential of antiarrhythmogenic effects on PVs. P3-5 MECHANISMS OF ATRIAL CONTRACTILE DYSFUNCTION IN ATRIAL DILATATION Hans-Ruprecht Neuberger, MD, Maura Greiser, MD, Erik Harks, PhD, Irina Moshkova, PhD, Eva Bodewig, MD, Sunniva De Haan, MD, Maurits A. Allessie, MD, PhD and Ulrich Schotten, MD, PhD. University Hospital HomburgSaar, Homburg, Germany, Maastricht University, Maastricht, Netherlands and University Hospital Aachen, Aachen, Germany. In chronically dilated atria, contractility is impaired. However, the underlying mechanisms are unknown. Methods: In 10 goats the AV-node was ablated and a VVI-pacemaker (45/min) was implanted. After 4 weeks of slow ventricular rhythm, the heart was excised and right atrial muscle bundles were isolated to measure isometric force-frequency-relation, rapid cooling contractures, and transmembrane action potentials. Bundles from 10 goats without AV block served as control. Results: After 4 weeks of AV block the right atrial free wall was enlarged (61⫾2 x 33⫾1mm vs. 49⫾3 x 25⫾1mm, p⬍0.01). In bundles from dilated atria isometric force (FC) was reduced (3.8⫾0.6mN/mm2 [n⫽50] vs. 5.7⫾0.6mN/mm2 in control [n⫽53], p⬍0.05). However, the relative effect of frequency on force was similar in both groups (force at 0.5Hz⫽100%; at 3Hz: 55.8⫾7.6% [n⫽40] vs. 61.6⫾7.3% [n⫽39], p⫽0.58). Contractures after rapid cooling (RCC) were reduced in AV block bundles at 1Hz (4.2⫾0.7mN/mm2 [n⫽11] vs 6.3⫾1.1mN/mm2 [n⫽10], p⫽0.13). This was even more pronounced at higher rates (2.5Hz: 3.4⫾0.5 vs. 12.2⫾3.2mN/mm2, p⬍0.01; see Figure 1). Interestingly, action potential duration at 20% repolarization was increased in AV block (24.5⫾3.9 vs. 13.4⫾1.5ms, p⬍0.05), and the plateau potential of the action potential was similar in both groups (average potential from 10 to 50ms: -13.5⫾2.8 vs. -16.6⫾2.2mV, p⫽0.39).

Conclusions: Despite a prolonged action potential duration, contractility is impaired in muscle bundles from atria dilated due to complete AV block.