Abstract 19452: Prrx1 Interacts with and Inhibits Pitx2 Function
Background: Genome-wide association studies have identified 9 genomic loci associated with atrial fibrillation (AF) two of which are located at chromosomes 4q25 and 1q24 near the genes PITX2 and PRRX1 respectively. Pitx2 is crucial for the heart development and its loss leads to the inducible AF in mice. Since Prrx1 is a transcriptional co-activator we hypothesized that it may interact with and modulate Pitx2 function.
Methods: We assessed the expression pattern of Pitx2 and Prrx1 in mouse atria by qPCR. Using an in vitro overexpression system we tested if Prrx1 and Pitx2 are present in a complex via co-immunoprecipitation studies (co-IP). We then made serial deletion constructs to determine the site of Prrx1 interaction with Pitx2. We assessed functional consequences of this interaction by siRNA knock-down followed by qPCR and luciferase reporter assays on the Prrx1, ANF, and HCN4 promoters.
Results: Pitx2 and Prrx1 had a similar expression pattern in adult mouse atria with a 3.4±0.85 fold overexpression for Pixt2 and 1.7±0.55 fold for Prrx1 in LA, compared to RA (p<0.05). Our co-IP data show that Prrx1 and Pitx2 are present in a complex with binding site located at the homeodomain of Pitx2 (aa72-92, aa119-139, and aa131-151 for Pitx2a, Pitx2b and Pitx2c respectively). SiRNA knockdown studies in atrial cell line showed that knockdown of Pitx2 reduced Prrx1 expression by 71±29% (p<0.05) and Pitx2 knockdown reduced Prrx1 expression by 56±21% (p<0.05). Prrx1 but not Pitx2 knockdown caused 68±29% and 65±28% reduction in HCN4 and ANF mRNA levels respectively. All Pitx2 isoforms activated Prrx1 promoter by 4.3±2.7 fold in luciferase reporter assay, and this effect was abolished by addition of Prrx1 (p<0.05). Similar results were seen with ANF promoter: Pitx2 increased promoter activity by 2.2±0.2 fold, and addition of Prrx1 brought it back to baseline (p<0.05). In contrast, neither Prrx1, nor Pitx2 had significant effect on HCN4 promoter activity.
Conclusions: Our data show that in vitro Prrx1 and Pitx2 are present in a complex. Further, we demonstrate that Prrx1/Pitx2 interaction has functional consequences that alter expression of some atria-specific genes. Thus, the Prrx1/Pitx2 interaction may be an interesting regulatory mechanism in the susceptibility to the AF.
- © 2012 by American Heart Association, Inc.