Molecular and Cellular Mechanism of Adipocyte Modulatory Effects on Pulmonary Vein and Atrium

Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia, which was enhanced in obesity and metabolic syndrome. Pericardial adipose tissues are highly associated with the genesis of AF. Local interactions between pericardial adipose tissue and adjacent myocardium may induce myocardial electrical or anatomical remodeling and give rise to AF occurrence. Abnormal calcium regulations 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) play a critical role in the pathophysiology of AF. Additionally, protein kinase C plays a critical role in the mechanisms of AF. Since adipocytes can produce inflammatory cytokine and adipocytokine, we hypothesized that pericardial adipocytes contain distinctive arrhythmogenicity by releasing adipocytokines, and adipocyte-cardiomyocyte interactions to induce AF though its effects on calcium regulations. Moreover, adiponectin has been shown to prevent the occurrences of AF. However, it is not clear whether adiponectin may modulate the effects of adipocytes on AF. Therefore, the purposes of this study in the first year are to investigate the components and effects of adipocytes on the electrophysiological characteristics and calcium regulations in the PVs and atrial cardiomyocytes, and compare the differences between different adipocytes. In the second year experiment, we will study the effects of fat tissue on conduction properties and electrical characteristics in PV and atrial tissues and the changes in RNA and protein expressions of various calcium regulating proteins. In the third year study, we will investigate potential signal transduction pathways evoked by adipocytes and their paracrine effects on cardiomyocytes, which may be modulated by adiponectin and protein kinase C. In the four year experiment, we will evaluate the changes of micro-RNA and how they regulate the effects of adiopocytes on cardiomyocytes. Methods: The first year experiment- Single cardiomyocytes will be isolated from rabbit PVs and atrium through perfusion of Tyrode solution containing digestive enzymes and adiopocytes will be isolated from pericardial fat and abdominal fat. Cardiomyocytes will be incubated with (fat group) and without (control group) adipocytes 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. Confocal microscopy will be used to measure the intracellular calcium ([Ca2+]i) transient, [Ca2+]i store, and Ca2+ sparks. We also will measure the components of adipocytokines through adipokine array. The second year experiment– Through MED64 Multi-electrode system and conventional microelectrodes to measure the conduction properties and electrophysiological characteristics between PV and LA tissue preparations cocultured with pericardial fats for 4~6 hours. Western blot and RT-PCR will be used to detect the changes in the expression of proteins and RNA levels of ICa-L, RyR, NCX, phospholamban (total or phopholated) and SERCA2a between PV and LA tissue preparations in contact with pericardial fats for 4~6 hours and compared with controls. Third year experiment: In co-culture of HL-1cells and 3T3-L1 adipocytes, whole-cell clamp techniques and confocal microscopy will be used to measure the APs and ionic currents, and Ca2+ i homeostasis in HL-1cardiomyocytes. Western blot and PCR will be used to detect the expression of protein kinase C, angiotensin II receptor, phospho forms of P38 MAPK, ERK1/2, NFATc4, TNF-, TGF- and CTGF in HL-1 cardiomyocytes with and without the presences of protein kinase C alpha (PKC) inhibitor (Go6976) or exogenous administration of adiponectin. The Fourth year experiment: In HL-1cells with and without co-cultured with 3T3-L1 adipocytes, the miRNA expression profile will be analyzed by miRNA microarray and verified by real-time PCR. Furthermore, the intracellular calcium ([Ca2+]i) transient, [Ca2+]i store, and Ca2+ sparks, ICa-L, NCX, and calcium regulation proteins will be evaluated for mir functional assay to study the potential modulations. Preliminary result: Adipocytes significantly prolonged the AP duration, depolarized the resting membrane potentials in atrial myocytes with increased genesis of delayed afterdepolarization and enhancement of ICa-L, which confirm our speculation that adipocytes have electrical effects on atrium.