Mechanisms of Apelin in Electrophysiological Characteristics and Calcium Dynamics in Left Atrium and Pulmonary Vein Cardiomyocytes Underlying the Pathophysiology of Atrial Fibrillation

Apelin is a novel peptide that acts through the APJ receptor, sharing similarities with the angiotensin II–angiotensin II type 1 receptor pathway. It is a peripheral vasodilator, powerful inotrope and may affect central fluid homeostasis. Animal and human studies suggest that it may play a role in the pathogenesis of heart failure and atrial fibrillation (AF) by modulating the harmful effects of angiotensin II. Apelin is reduced in patients with heart failure and AF and up-regulated following favourable left ventricular remodelling. AF is the most common cardiac arrhythmia and decreased apelin has been found to be a risk factor of AF. However, the pathophysiological mechanisms underlying the higher incidence of AF in patients with reduced apelin level were not clear. Pulmonary veins (PVs) are known to be important pathological role in the genesis of AF. PVs have been shown to contain cardiomyocytes with a high arrhythmogenic activity. However, knowledge about the effects of apelin on the electrophysiological characteristics of PV was limited. Moreover, abnormal calcium regulation has been suggested to play an important role in the genesis of AF and PV arrhythmogenesis. Enhanced Na+/Ca2+ exchanger (NCX), dysfunction of ryanodine receptor (RyR), spontaneous nonpropagating Ca2+ releases (Ca2+ spark) have been reported to have a role in the pathophysiology of AF. It is possible that apelin may have effects on calcium homeostasis to regulate atrial electrophysiology and PV arrhythmogenesis. Therefore, the purposes of this study in the first year are to investigate the effects of apelin on the electrophysiological characteristics in the PVs and atrial cardiomyocytes. In the second year experiment, we will investigate the RyR function, calcium regulation, calcium spark and expressions of calcium regulation proteins in PV and atrial cardiomyocytes from control and apelin-treated cardiomyocytes. In the third study, we will approach the potential upstream and downstream signal transduction that apelin exerts its distinct effects on cardiomyocytes. Moreover, we will study the arrhythmogenic drugs, including isoproterenol and angiotensin II, which may induce AF through mitogen-activated protein kinases pathway, to validate the putative role of apelin in the therapy for AF. Methods: The first year experiment- Single cardiomyocytes are isolated from rabbit PVs and atrum through perfusion of Tyrode solution containing digestive enzymes. Cardiomyocytes will be incubated with (apelin group) and without (control group) apelin (0.1, 1, 10 nM) for 4~6 hours. Whole-cell clamp techniques will be used to study the APs and electrical activity, L-type calcium current (ICa-L), transient inward currents, NCX currents, transient outward currents (Ito), and delayed (IK) and inward rectified outward potassium (IK1) current between control and experimental PV and atrial cardiomyocytes. Second year experiment: Isolated single rabbit PV and atrial cardiomyocytes will be incubated with (apelin group) and without (control group) apelin (0.1, 1, 10 nM) for 4~6 hours. Confocal microscopy will be used to measure the intracellular calcium ([Ca2+]i) transient, [Ca2+]i store, and Ca2+ sparks with fluorescence between control and apelin group PV and atrial cardiomyocytes. Western blot and RT-PCR will be used to detect and measure the expression of proteins and RNA in ICa-L, RyR, NCX, and SERCA2a, in PV and atrial cardiomyocytes from control and apelin groups. Third year experiment: Isolated single cardiomyocytes will be incubated with and without incubation of apelin (0.1, 1, 10 nM) for 4~6 hours Whole-cell clamp techniques will be used to measure the APs and ionic currents (ICa-L, transient inward currents, NCX, Ito, IK and IK1) before and after isoproterenol (10 nM) or angiotensin II (100 nM) in control and apelin groups PV and atrial cardiomyocytes. Western blot and PCR will be used to detect and measure the expression of proteins of phospho-p38/ERK and p38/ERK in PV and atrial cardiomyocytes from control and apelin groups. We also will use the HL-1 cells with and without hypoxia (1% O2, 2hours) and study the RNA expression of apelin and apelin receptor by RT-PCR, the binding of HIF within the apelin promoter by ChIP assay, and promoter reporter gene Assay Preliminary result: Apelin (1 nM) significantly decrease the PV spontaneous activity and ICa-L, which confirm our speculation that apelin have direct electrical effects.