Through genome-wide association studies, 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. Studies have shown that epigenetic mechanisms significantly participate in AF pathogenesis. Increasing evidence shows that histone deacetylase (HDAC)-mediated histone and non-histone protein modifications play roles in the genesis of AF. Moreover, Hilton and Kioussi demonstrated that Pitx2c can recruit HDAC to regulate gene expression during embryonic development. Besides, our previous studies showed HDAC inhibitors attenuate cardiac fibrosis and possess anti-AF potential through multiple target modifications. MicroRNAs (miRNAs) has electrophysiological and calcium regulations effects. Nevertheless, it is not clear whether modulation of Pitx2c expressions can regulate atrial histone modification and microRNAs. Moreover, through modulation of Pitx2c expression may provide a way to regulate the pathophysiology of AF. Therefore, the purposes of this study in the first year are to investigate the epigenetic effect of Pitx2c and to study whether overexpression of Pitx2c can attenuate the genesis of HF and atrial arrhythmogenesis. In the second year, we will evaluate the Pitx2c effects on co-culture of fibroblasts and cardiomyocytes for cardiac electrophysiology and calcium handling in atrial cardiomyocytes. Methods: In the first year experiment, Pitx2c overexpressed HL-1 cells will be analyzed for HDAC activity and compare the miRNA expression associated with AF by miRNA microarray and confirmed by real-time PCR. We will study whether overexpression of Pitx2c can attenuate the rapid pacing-induced atrial arrhythmogenesis (5 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-overexpressed LA. In the second year, we will co-culture HL-1 cells with atrial fibroblasts with or without Pitx2c overexpression into different groups as following: HL-1 cells/atrial fibroblasts (normal HL-1 cells and fibroblasts), HL-1 cells/ atrial fibroblasts-Pitx2c (normal HL-1 cells, Pitx2c-overexpressed fibroblasts), or HL-1 cells-Pitx2c/ atrial fibroblasts-Pitx2c (Pitx2c overexpressed in both HL-1 cells and fibroblasts) with and without tachy-pacing (5Hz). 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 HL-1 cardiomyocytes. Western blot will be used to evaluate the differences on calcium regulation proteins (SERCA2a, ryanodine receptor, sodium/calcium exchanger, calmoduline kinase II and total & phosphorylated phospholamban) in these cells. Preliminary results: Pitx2c can regulate migration ability of atrial fibroblasts. The Pitx2c overexpressed cells decreased cell migration but increased cell mobility in Pitx2c knockdown cells. Moreover, the connexin 40 (Cx40) and Cx45 of gap junction proteins were also decreased in Pitx2c knockdown cells. These finding suggest that Pitx2c play a pivotal role in anti-atrial fibrosis and regulation Pitx2c may change atrial electrophysiology and AF occurrence through modulating fibroblast activity.
|Effective start/end date||8/1/16 → 7/31/17|
- Atrial fibrillation
- Histone deacetylase