Abstract 20885: A Novel Mechanism for Cardiac Abnormal Automaticity: Orai-Linked Calcium Signaling
Objective: The plasma membrane Orai proteins permit cell calcium entry in response to calcium store depletion. We address whether the heart contains these voltage-independent calcium channels & whether their activation affects heart electromechanical (EM) stability.
Methods: Western analysis & immunofluorescence were used to validate that rat left atria (LA) & ventricle contain Orais. Isolated LA & left ventricular anterior papillary muscles (PM) were superfused and their mechanical function was recorded with a Grass polygraph; action potentials were recorded using 3µm glass microelectrodes. 2-amino-ethoxydiphenyl borate (2APB) interrogated how Orais affect cardiac EM stability as 10–30µM 2APB activates Orai calcium entry (J Biol Chem 283:17662 (2008)).
Data: LA & PM myocytes contain Orai1 & Orai3. Untreated intact LA & PM require pacing to produce action potentials & contractions. With superfusate 2APB <10µM, unpaced LA & PM are quiescent. 10–25µM 2APB, however, provokes calcium replete unpaced LA & PM to spontaneously contract at sustained maximum rates of 230±8 & 282±13 contractions/min (c/m) at 30°C. At 37°C, LA produces 549±13 spontaneous c/m. Right atrial normal automaticity is 179±5 & 359±9 c/m at 30 & 37°C. Untreated LA has resting potentials of 79±5mV vs. 82±3mV for 2APB-treated, spontaneously contracting LA. This tachycardia is a novel form of abnormal automaticity as exposing unpaced LA or PM to 25µM 2APB produces spontaneous EM activity 242±12 or 117±29sec after adding 2APB. The Orai inhibitor SKF-96365 prevents & reverses 2APB automaticity with IC50s of 10±2 and 6.2±2µM in LA & PM. Treating spontaneously contracting LA with 800nM ryanodine for 10min decreases their mechanical function 90% but does not affect spontaneous contraction rates (218±18c/m), the production of spontaneous action potentials or their properties. Pretreating LA & PM with the calmodulin inhibitor fluphenazine-N2-chloroethane suppresses Orai abnormal automaticity. Pretreating with 20µM KN93, a calmodulin kinase inhibitor does not.
Conclusions: An Orai channel activator induces abnormal automaticity which an Orai inhibitor suppresses. Thus these voltage-independent calcium channels are an unrecognized mechanism to provoke abnormal automaticity.
- Triggering mechanisms
- Ventricular tachycardia
- Ventricular arrhythmia
- Atrial arrhythmias
- Supraventricular tachycardia
- © 2010 by American Heart Association, Inc.