ARRESTIN-BIASED M3 DESIGNER RECEPTOR: A NOVEL THERAPEUTIC TOOL IN THE UPREGULATION OF HERG CHANNEL ACTIVITY

Abstracts

VF for up to 4 times in each heart. Spatial organization of VF was subsequently evaluated on phase maps constructed based on data from optical recordings. VF became significantly organized, resembling monomorphic or polymorphic VT in all (5 of 5) Ranolazine-treated-hearts compared to non treated hearts. Phase map analysis of VF revealed a significant organization of VF in Ranolazine treated heart. The mean number of calcium wave fronts were not significantly different at time 0 (before treatment) between the 2 groups (2.6
0.1 and 2.13
0.16 in ranolazine-treated and untreated control hearts, respectively; p>0.05). In Ranolazine-treated hearts, the number of Ca2+ wave fronts were significantly reduced to 2.14
0.33 at 4 min of VF, while in control hearts, it increased significantly to 2.76
0.19 (p¼0.007 for interaction). Following initial VF and defibrillation, calcium transient duration 50 (CaTD50) (p¼0.04), CaTD80 (p¼0.01) and calcium alternans (p¼0.03) were significantly reduced in Ranolazine-treated hearts, whereas there was no significant difference in spontaneous diastolic calcium elevation and APD50 and APD80 between the two groups After defibrillation of initial VF, successful re-fibrillation was inducible only in 37.5% (6 of 16) of attempts in Ranolazine-treated hearts compared to 80% (16 of 20) in control hearts (p¼ 0.009). CONCLUSION: Treatment with Ranolazine during VF improves organization of electrical activities during long duration VF, mitigates dysregulation in calcium dynamics and reduces susceptibility to subsequent re-fibrillation. Ranolazine as a therapeutic strategy needs to be tested in cardiac arrest models. CIHR 363 ARRESTIN-BIASED M3 DESIGNER RECEPTOR: A NOVEL THERAPEUTIC TOOL IN THE UPREGULATION OF HERG CHANNEL ACTIVITY M Sangoi, S Lamothe, S Zhang Kingston, Ontario BACKGROUND:

The human ether-a-go-go-related gene (hERG) encodes the pore-forming subunit of the voltagegated K+ channel (Kv11.1), which passes the rapidly activating delayed rectifier K+ current (IKr). Unique gating kinetics of the hERG channel establishes the important role of IKr in setting the cardiomyocyte action potential duration (APD) and maintenance of cardiac repolarization. A reduction in IKr due to loss-of-function mutations or drug interactions may cause long QT syndrome (LQTS), a cardiac electrical disorder characterized by a prolonged QT interval on an ECG. Individuals with inherited or acquired LQTS are predisposed to ventricular arrhythmias (Torsade de pointes) and sudden cardiac death. Spontaneous loss-of-function hERG mutations reduce IKr and cause inherited type 2 LQTS. Furthermore, many medications or reductions in extracellular K+ concentration (hypokalemia) have been shown to cause acquired LQTS. Since a reduction in hERG expression and

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IKr cause LQTS, methods to enhance or restore hERG channel activity are recognized as appropriate treatment strategies. In particular, we discovered that M3 receptor activation increases hERG expression and IKr through a Gq proteindependent PKC pathway. Similar to other G protein-coupled receptors (GPCRs), M3 receptors recruit -arrestins in response to ligand binding to mediate G protein-independent signaling pathways. Although these findings establish a basis for M3mediated regulation of hERG activity, there is little therapeutic potential due to complex G protein signaling and heterologous M3 expression. However, the development of an M3 designer receptor (rM3Darr) has allowed for exclusive -arrestin signaling in response to an otherwise pharmacologically inert ligand, clozapine-N-oxide (CNO). These designer receptors may prove to be of therapeutic benefit for upregulating hERG channel activity through -arrestin-dependent signaling. METHODOLOGY: Human embryonic kidney cells stably expressing hERG protein (hERG-HEK cells) were transiently transfected with empty pcDNA3 or rM3Darr plasmids, and cultured for 24 hours in the absence (control) or presence of CNO. Western blot analysis was performed to assess hERG expression in the groups. Whole-cell patch clamp recordings were conducted to evaluate hERG channel current. RESULTS: Western blot analysis revealed rM3Darr-expressing cells treated with CNO to have significantly increased hERG expression compared to control groups. Furthermore, these cells displayed significantly greater hERG current compared to controls. CONCLUSIONS: These findings suggest that rM3Darr activation via CNO increases hERG expression and current through a -arrestin-dependent pathway. Furthermore, these arrestin-biased M3 designer receptors may prove to be a valuable therapeutic tool in restoring IKr in LQTS individuals. 364 LONG DURATION VF RESULTANT PHOSPHORYLATION RYANODINE RECEPTOR-2 IS MAINLY MEDIATED BY PKA MECHANISM MA Azam, G Kichigina, N Zamiri, N Jackson, M Kusha, S Massé, J Asta, PF Lai, K Nanthakumar Toronto, Ontario INTRODUCTION:

The calcium release channel ryanodine receptor-2 (RyR2) is considered to become dysfunctional during long duration ventricular fibrillation (LDVF) leading to dysregulation of calcium transient during VF. Phosphorylation of RyR2, mediated by calcium calmodulin-dependent protein kinase II (CaMKII) and protein kinase A (PKA), is a major control mechanism of the channel activity. The objective of the present study was to determine the effects of LDVF on RyR2 phosphorylation. METHODS AND RESULTS: Hearts from normal, healthy NZW rabbits were Langendorff-perfused and subjected to simulated