Abstract
Atrial fibrillation (AF) is the most common cardiac arrhythmia in the human population, with an estimated incidence of 1–2% in young adults but increasing to more than 10% in 80+ years patients. Pituitary Homeobox 2, Paired Like Homeodomain 2 (PITX2c) loss-of-function in mice revealed that this homeodomain (HD)-containing transcription factor plays a pivotal role in atrial electrophysiology and calcium homeostasis and point to PITX2 as a candidate gene for AF. To address this issue, we recruited 31 AF patients for genetic analyses of both the known risk alleles and PITX2c open reading frame (ORF) re-sequencing. We found two-point mutations in the homedomain of PITX2 and three other variants in the 5’untranslated region. A 65 years old male patient without 4q25 risk variants but with recurrent AF displayed two distinct HD-mutations, NM_000325.5:c.309G>C (Gln103His) and NM_000325.5:c.370G>A (Glu124Lys), which both resulted in a change within a highly conserved amino acid position. To address the functional impact of the PITX2 HD mutations, we generated plasmid constructs with mutated version of each nucleotide variant (MD4 and MD5, respectively) as well as a dominant negative control construct in which the PITX2 HD was lacking (DN). Functional analyses demonstrated PITX2c MD4 and PITX2c MD5 decreased Nppa-luciferase transactivation by 50% and 40%, respectively, similar to the PITX2c DN (50%), while Shox2 promoter repression was also impaired. Co-transactivation with other cardiac-enriched co-factors, such as Gata4 and Nkx2.5, was similarly impaired, further supporting the pivotal role of these mutations for correct PITX2c function. Furthermore, when expressed in HL1 cardiomyocyte cultures, the PITX2 mutants impaired endogenous expression of calcium regulatory proteins and induced alterations in sarcoplasmic reticulum (SR) calcium accumulation. This favored alternating and irregular calcium transient amplitudes, causing deterioration of the beat-to-beat stability upon elevation of the stimulation frequency. Overall this data demonstrate that these novel PITX2c HD-mutations might be causative of atrial fibrillation in the carrier.
Highlights
Atrial fibrillation (AF) is the most common cardiac arrhythmia in the human population, with an estimated incidence of 1–2% in young adults but increasing to more than 10%in 80+ years patients [1]
The 4q25 risk variant rs13143308 has recently been linked to defective calcium homeostasis [14], pointing to PITX2 as a candidate gene for AF
We have conducted a genetic screening on the PITX2 open reading frame (ORF) (NM_000325_5) in 31 patients diagnosed with AF according to the ESC Guidelines for treatment of AF referred to the Cardiology Unit of the Regional Hospital “Ciudad de Jaen”
Summary
Atrial fibrillation (AF) is the most common cardiac arrhythmia in the human population, with an estimated incidence of 1–2% in young adults but increasing to more than 10%in 80+ years patients [1]. Atrial fibrillation (AF) is the most common cardiac arrhythmia in the human population, with an estimated incidence of 1–2% in young adults but increasing to more than 10%. Thanks to genome wide association studies (GWAS) an increasing number of single nucleotide polymorphisms (SNPs) have been associated with risk of AF [2,3,4,5] with some of the most significant are located in 4q25 locus. Experimental evidence in distinct laboratories, including ours, have demonstrated that PITX2c loss of function predisposes to atrial arrhythmogenesis [9,10,11,12] and provokes disturbances in the calcium homeostasis [13]. The 4q25 risk variant rs13143308 has recently been linked to defective calcium homeostasis [14], pointing to PITX2 as a candidate gene for AF.
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