Pitx2c Modulations on Atrial Fibroblast Activity, Electrical Characteristic, and Myocyte-Fibroblast Interaction: Implications in the Genesis of Atrial Fibrillation

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. Genetic variants on chromosome 4q25 adjacent to PITX2 are the most significantly associated with the occurrence of AF. Pitx2c can regulate atrial structure and electrophysiology. In addition, angiotensin II (the key factor of AF or heart failure, HF) can dysregulate Pitx2c and Kir2.1 through promoter hypermethylation. Therefore, Pitx2c plays an important role in the cardiac electrophysiology and arrhythmogenesis. However, the arrhythmogenetic effect of Pitx2c is not fully elucidated. Atrial fibrosis significantly contributes AF structural and electrical remodeling and may underlie atrial arrhytmogenesis in HF. For the first time, we found that Pitx2c can regulate atrial fibroblast activity. However, it is not clear whether modulation of Pitx2c expressions in atrial fibroblast can regulate atrial electrophysiology through regulations on fibroblast activity, cellular (myocytes and fibroblasts) interactions, or extracellular matrix. Moreover, HF may modulate atrial fibroblast activity through modulation of Pitx2c expressions via angiotensin II activation. Therefore, the purposes of this study will investigate the effects of Pitx2c modulation on atrial fibroblast activity, electrical characteristics, and cell interactions. In the first year study, we will identify the effects and mechanisms of Pitx2c on atrial fibroblast electrophysiological characteristics and fibroblast activities. In the second year, we will study whether overexpression of Pitx2c can attenuate the genesis of HF and atrial arrhythmogenesis. In the third 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, Patch clamp, cell proliferation, migration, and collagen production assay will be used to evaluate effects of Pitx2c in atrial fibroblast transfected with Pitx2c encoding plasmid or vector. We will treat angiotensin II (100 nM) to control and Pitx2c-overexpressed fibroblast to evaluate the role of Pitx2c on angiotensin II-induced fibrosis activity. Immunofluorescence and RNA and Western blot will be used to study the myofibroblast marker and RNA and proteins levels of ionic currents in control and Pitx2c overexpressed fibroblasts. In the second year, through injecting Pitx2c encoding plasmid directly into LA of 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 RA, LA , and PVs from control HF and Pitx2c-overexpressed HF Rats. In the third year, we will co-culture HL-1 cardiomyocytes with atrial fibroblast with or without Pitx2c overexpression into different groups as following: HL-1 cell/atrial fibroblast (normal HL-1 and fibroblast), HL-1 cell/atrial fibroblast-Pitx2c (normal HL-1, Pitx2c-overexpressed fibroblast), or HL-1 cell-Pitx2c/atrial fibroblast-Pitx2c (Pitx2c overexpressed HL-1 and fibroblast) 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 II5 and total & phosphorylated phospholamban) in these cells. Preliminary results: The Pitx2c overexpressed atrial fibroblast decreased cell proliferation and migration ability. These finding suggest that Pitx2c play a role in atrial fibrosis and regulation Pitx2c may change atrial electrophysiology and AF occurrence through modulating fibroblast activity.