Suppression of autonomic-mediated triggered firing in pulmonary veins, 24 hrs post coronary artery occlusion

Poster 6 P6-3 INHIBITION OF HETEROMERIC KIR2.X CHANNELS BY ALPHA1A ADRENERGIC RECEPTORS: CRUCIAL ROLE OF PROTEIN KINASE C DEPENDENT REGULATION OF KIR2.3 SUBUNITS Claudia Kiesecker, MS, Edgar Zitron, Daniel Scherer, Sonja Lueck, Ramona Bloehs, Eberhard P. Scholz, Sven Kathofer, MD, Dierk Thomas, MD, Volker A. Kreye, MD, Johann Kiehn, MD, Wolfgang Scho¨ls, MD, Hugo A. Katus, MD and Christoph A. Karle, MD. Medical University Hospital Heidelberg, Heidelberg, Germany and University of Heidelberg, Heidelberg, Germany. Introduction: The cardiac inwardly rectifying potassium current IK1 stabilises the resting membrane potential of cardiomyocytes. Activation of adrenergic alpha-1a receptors leads to an inhibition of IK1 currents. In native cells the protein kinase C pathway is crucial in this regulation. IK1 is probably generated by heteromeric assembly of Kir2.1, Kir2.2 and Kir2.3 channels. Thus, in this study we focused on the PKC-dependent regulation of Kir2-heteromers by alpha-1a adrenergic receptors. Methods: Human adrenergic alpha-1a receptors and Kir2.x channels were co-expressed in Xenopus oocytes and experiments were performed using two-microelectrode voltage-clamp. Results: First, regulation of heteromeric Kir2-channels by protein kinase C (PKC) was studied. Activation of PKC by PMA (1 ␮M) induced a currrent reduction in all heteromeric channels. The degree of inhibition, however, was different in Kir2.1/Kir2.2 (-24%), Kir2.1/Kir2.3 (-10%) and Kir2.2/ Kir2.3 (-88%) channels. Then, adrenergic alpha-1a receptors were coexpressed with the heteromeric channels. Activation of the receptors with phenylephrine (10 ␮M) lead to an inhibition of currents in Kir2.1/Kir2.3 and in Kir2.2/Kir2.3 heteromers, but not in Kir2.1/Kir2.2 heteromers. This inhibitory regulation could be suppressed by coapplication of PKC inhibitor chelerythrine (10 ␮M). In order to elucidate the contribution of subunit-dependent regulation to this effect, we also studied homomeric channels. After coexpression with adrenergic alpha-1a receptors, the inhibitory regulation could be observed in homomeric Kir2.2 and Kir2.3 channels, but not in Kir2.1 channels. Coapplication of chelerythrine suppressed the effect in Kir2.3 channels, but not in Kir2.2 channels. Conclusion: Inhibition of IK1 by adrenergic alpha-1a receptors in native cells is mainly mediated by PKC-dependent pathways. This regulation can be reproduced in heteromeric Kir2.x channels. On the molecular level, the effect depends on a PKC regulation of Kir2.3 channel subunits. In contrast, Kir2.1 and Kir2.2 are not affected by receptor-induced PKC activation. P6-4 MOLECULAR, CELLULAR, AND FUNCTIONAL ANALYSIS OF A NOVEL LONG QT TYPE 2 DELETION Blake Anson D. Anson, PhD, Jianfang Lian, MD, PhD, Changzong Cui, MD, Xiaolin Xue, MD, Li Zhang, MD, Cheng Huang, PhD, Aiqun Ma, MD, G. Michael Vincent, MD and Craig T. January, MD, PhD. UW-Madison, Madison, WI, Xi’an Jiaotong University, Xi’an, China and LDS Hospital, Salt Lake City, UT. KCNH2 ( HERG) encodes the ␣-subunit of the rapidly activating delayed rectifier K⫹ channel that mediates a principle cardiac repolarizing ionic current, IKr. Long QT Syndrome Type 2 (LQT2) is caused by genetic variations in KCNH2, the overwhelming majority of which are single nucleotide missense mutations thought to decrease IKr through any of four mechanistic classifications comprising alterated; transcription and/or translation, protein processing and channel trafficking, biophysics, or ionic selectivity/permeability (classes 1 through 4, respectively). Additionally, dominant-negative interactions with normal protein may exacerbate mutational effects. The functional effects of deletions are less well understood. KCNH2-L539fs/18 is a LQT2-linked 19 base pair deletion that generates a prematurely truncated protein. We tested the hypothesis that this deletion is a class 2 (altered trafficking) mutation with dominant negative effects. Methods: KCNH2-L539fs/18 was incorporated into cDNA expression

S295 vectors and transiently introduced into human embryonic kidney (HEK293) cells alone or in the presence of wild-type (WT) KCNH2 channels followed by biochemical, immunocytochemical, and electrophysiological analysis. Results: Transient transfection of KCNH2-L539fs/18 cDNA into HEK293 cells produced protein (n⫽4 Western blots) that trafficked to the plasma membrane (n⫽ 6 cells) but did not produce measurable current (n⫽10 cells). Furthermore, transient transfection of KCNH2-L539fs/18 cDNA into HEK293 cells stably expressing KCNH2-WT channels did not alter WT current levels (82.8⫾16.0 pA/pF versus 81.3⫾11.4 pA/pF, n ⫽ 5 cells each), steady-state activation (V1/2 ⫽ -11.3⫾2.3mV versus -10.7⫾2.8mV, n⫽ 4 cells each), or inactivation and deactivation kinetics (n⫽4 - 6 cells). Discussion: KCNH2-L539fs/18 is not a functional channel, but it does traffick to the plasma membrane. Thus this deletion is not a class 2 mutation. Furthemore, unlike some missense mutations, KCNH2L539fs/18 protein does not exhibit dominant negative effects on WT channels, thus providing a mechanism whereby some KCNH2 deletions may be less detrimental than missense mutations. P6-5

WITHDRAWN

P6-6 SUPPRESSION OF AUTONOMIC-MEDIATED TRIGGERED FIRING IN PULMONARY VEINS, 24 HRS POST CORONARY ARTERY OCCLUSION Eugene Patterson, PhD, Xichun Yu, MD, Shijun Huang, MD, Marion Garrett, BSc, David C. Kem, MD and *Benjamin J. Scherlag, PhD. OUHSC and DVAMC, Oklahoma City, OK. Early afterdepolarizations (EADs) are observed in superfused canine pulmonary vein sleeves following the administration of norepinephrine (NE) (3.2 x 10-8 M). Triggering of rapid sustained arrhythmias is observed with tachycardia-pause pacing interventions following further shortening of the abbreviated pulmonary vein action potential with the addition of acetylcholine (10-7M)(N⫽18 of 23). Triggered arrhythmias (782 ⫾ 158 bpm) are also observed during autonomic nerve stimulation (300 msec duration trains of 0.1 msec duration stimuli at 100 Hz) (N⫽18 of 23), associated with both action potential shortening (atropine-suppressible) and EAD formation (both atenolol and ryanodine suppressible). In superfused pulmonary veins removed from dogs, 24 hrs following anterior descending coronary artery ligation, autonomic nerve stimulation at multiple sites in each preparation produced triggered arrhythmia in only 1 of 12 preparations (p⫽0.002). The failure to produce triggered arrhythmia was reversed by administration of 3.2 x 10-7M norepinephrine in 2 of 5 preparations. Tachycardia-pause pacing produced similar EAD formation in pulmonary vein preparations from both control and infarct dogs despite the failure to further enhance EAD formation and induce sustained triggered arrhythmia in the presence of NE and acetylcholine (N⫽ 0 of 12)(p⫽0.0001). A 29-fold shift in the EC50 for norepinephrine-induced EAD formation was observed for preparations from control and infarct dogs (1.1 x 10-9M to 3.2 x 10-8M, respectively; p⬍0.02) associated with a 32 ⫾ 8% increase in ␤-adrenergic receptor kinase (␤-ARK). Conclusion: The data demonstrate a decreased sensitivity of superfused canine pulmonary veins for triggered arrhythmia formation produced by local autonomic nerve stimulation and combined norepinephrine-acetylcholine administration, 24 hrs post coronary artery ligation, The absence of EAD formation and triggered arrhythmia were associated with a decreased response to the ␤-adrenergic receptor agonist NE, resulting from ␤-ARKmediated desensitization of ␤-adrenergic receptors. P6-7 MULTIPLE PATTERNS OF AH WENCKEBACH BLOCK RESULTING FROM LONGITUDINAL DISSOCIATION AND REENTRY WITHIN THE SUPERFUSED RABBIT AV JUNCTION Eugene Patterson, PhD. OUHSC and DVA Medical Center, Oklahoma City, OK.