Electrophysiological and Molecular Insights into the Cardiac Regulations of ZFHX3 Gene---Implication in the Pathophysiology of Atrial Fibrillation

Atrial fibrillation (AF) is the most commonly sustained arrhythmia, which can induce heart failure and increase mortality. A growing number of studies have shown that GWAS (genome-wide association studies) has potential to understanding the molecular mechanisms of pathogenesis. GWAS studies have been successfully identify three loci for AF: PITX2、ZFHX3 and KCNN3. It has been shown that PITX2 and KCNN3 associated with AF and heart failure, but the role of ZFHX3 in AF is not clear. For the first time, we have successfully knock-down the ZFHX3 level in HL-1 cells by RNA interference technology and found that sarcoplasmic reticulum Ca2+-ATPase (SERCA2a) was down-regulated in ZFHX3-siRNA cell. Since SERCA2a is essential in the calcium homeostasis and contributes to the pathophysiology of AF, our findings suggest that ZFHX3 may play a critical role in the atrial electrophysiology and genesis of AF. Nevertheless, it is not clear how ZFHX3 can regulate atrial electrophysiology and knowledge about the underling mechanisms was limited. In this study, we will investigate that role of ZFHX3 on atrial electrophysiology and calcium regulations to clarify the role of ZFHX3 in AF. In the first year experiment, we will evaluate the functions of ZFHX3 and analysis of his regulatory targets in HL-1 cells. In the second year experiment, we will study how the ZFHX3 can modulate the atrial electrophysiology and calcium homeostasis. In the third year experiment, we will study whether ZFHX3 regulate miRNA expression profile by miRNA microarray and precipitating factors for AF genesis will also investigate miRNA expressions by ZFHX3 regulated to cause AF. Methods: In the first year, whole-cell clamp techniques will be used to study the HL-1 cells electrical activity and ionic currents (L type calcium current, sodium current, transient outward current, ultra repaid and delayed rectifier potassium current, inward rectifier potassium currents) over-expression, siRNA and control ZFHX3 HL1 cells. Confocal microscopy will be used to measure the intracellular calcium transient, store and Ca2+distributed. In the second year, real-time PCR or western blot will be used to evaluate the differences on calcium regulation SERCA2a, ryanodine receptor blocker, sodium/calcium exchanger, calmoduline kinase, phopholamban, phosphorylated phospholamben) and ionic currents in these cells. Moreover, we used Chromatin immunoprecipitation (ChIP)/ ChIP-seq to detect the direct or indirect effects of SERCA2 regulation from ZFHX3. Over-expression and RNAi against the candidates from ChIP-seq were performed in HL-1 cells and compared the whole-cell clamp techniques to validate these findings. In the third year, over-expression and siRNA against ZFHX3 of will analysis the miRNA expression profile by miRNA microarray and compare the miRNA expression association with AF (miRNA-150, 155, 21, 133, 30, 328, 29, 1, 26, 590 etc.) by real-time PCR. Moreover, we will use angiotensin II (50 nM), tumor necrosis factor-α (50 ng/ml) and H2O2 (200 M) in over-expression, siRNA and control ZFHX3 HL-1 and compare the miRNA expression profile, western blot association with these precipitating factors. We will use patch clamp, calcium image and high density mapping to study these cells electrophysiological characteristics and conduction properties.