Effects and Mechanisms of Pitx2c Modulation for Atrial Electrophysiological and Structural Remodelings in Heart Failure

The genome-wide association studies (GWAS) have identified genetic variation from large cohorts and provided the potential molecular mechanisms for atrial fibrillation (AF). Through GWAS, three AF susceptibility loci (KCNN3 on 1q21, 4q25 near PITX2, and ZFHX3 on 16q22) were identified. Gene variants on chromosome 4q25 adjacent to Pitx2c, is the most significantly associated with the occurrence of AF. Pitx2c is mainly expressed in left atrium (LA) and pulmonary veins, and proposed to control atrial structure and electrophysiology, which plays a critical role in the genesis of AF. Pitx2 promoter has a high content of CpG dinucleotides. Hypermethylation of Pitx2 promoter will down-regulate Pitx2 and plays an important role in cancer actions. Similarly, Pitx2c promoter has abundant CpG islands, which can be hypermethylated to regulate the expressions of Pitx2c. Our previous studies have shown that heart failure (HF) can modulate cardiac methylaton. HF significantly increased the occurrences of AF. Therefore, HF may increase Pitx2c promoter methylation and reduce Pitx2c expression with regulations on atrial electrophysiology. Through HF animal experiments, we have found that HF reduced atrial Pitx2c expressions with hypermethlation on Pitx2c promoter. However, the mechanism underlying the methylation effects of HF are not clear. Angiotensin II, oxidative stress or inflammation may play a role in the pathophysiology of hypermethylation in HF or AF. Calcium dysregulation increases atrial arrhythmogenesis and plays a critical role in the pathophysiology of AF and HF. MicroRNAs (MiRNAs) has several electrophysiological and calcium regulations effects. Nevertheless, it is not clear whether modulation of Pitx2c expressions can regulate atrial electrophysiology and miRNAs. Moreover, through modulation of Pitx2c expression may provide a way to regulate the pathophysiology of HF and AF. Therefore, the purposes of this study in the first year are to identify the effects and mechanisms of HF on atrial methylation. In the second year, we will study the effects of Pitx2c on atrial electrophysiology and calcium handling. In the third year, we will evaluate whether overexpression of Pitx2c can modulate miRNAs and attenuate the genesis of HF and atrial arrhythmogenesis. Methods: In the first year experiment, pyrosequencing, real-time PCR and Western blot will be used to study the promoter methylation status, RNA and protein levels of Pitx2c, Kir 2.1, Nav1.5 or angiotensin II receptor and oxidative stress-related proteins in LA from control and isoproterenol-induced HF rats or in HL-1 cells treated with angiotensin II (1❍M), tumor necrosis factor-α (50 ng/ml) or H2O2 (200 ❍M) with or without methylation inhibitor (DAC, 0.1 ❍M). In the second year, whole-cell patch clamp techniques and confocal microscopy with fluorescence detection will be used to study the electrical activity, ionic currents and calcium homeostasis in Pitx2c overexpressed or RNA silenced and control HL-1 cells. Real-time PCR or Western blot will be used to evaluate the differences on calcium regulation of SERCA2a, ryanodine receptor, sodium/calcium exchanger, calmoduline kinase II and phopholamban (total & phosphorylated phospholamban) in these cells. In the third year, Pitx2c overexpressed HL-1 cells will be analyzed for the miRNA expression profile by miRNA microarray and compare the miRNA expression association with AF or HF by real-time PCR. We will study whether overexpression of Pitx2c can attenuate the rapid pacing-induced atrial arrhythmogenesis (10 Hz, for 6h) by patch clamp, calcium image in HL-1 cells. Through injecting Pitx2c encoding plasmid directly into LA in HF rats, we will study the fibrosis, electrophysiology and conduction properties, by histology, conventional micro-electrodes, high density mapping and Western blot (ionic channel and calcium regulating proteins) in HF and Pitx2c-treated LA. Preliminary results: The HF atrium exhibited increased Pitx2c promoter methylation with increased DNA methyltransferase 1 and decreased Pitx2c proteins compared to the normal atrium. We found that Kir2.1 protein increased in Pitx2c overexpressed HL-1 cells.