Table 3.

Key studies determining atrial PITX2 expression in patients and in experimental models and systems (upper part); key studies linking 4q25 rs13143308T allele to atrial cardiomyocyte function of AF patients (lower part).

Study first author surname and year	Pubmed ID	Details and quality of evidence			Summary of key findings
		Species	Model system	Key inclusion & exclusion criteria	Relevant outcome(s) to atrial remodeling	Key findings and important biases	Conclusion(s)
Key studies determining atrial PITX2 expression in patients and in experimental models and systems
Kahr et al., 2011	92	Human	Right and left atrial appendages (RAA, LAA)	Patients in sinus rhythm undergoing heart surgery for CABG or valve replacement		Protein levels of PITX2 two-fold higher in LA vs RA	Systematic differences between LA and RA gene expression exist and support a potential role of PITX2 in shaping LA
Donate Puertas et al., 2017	55	Human	LAA and pulmonary veins-LA junction	Patients with or without AF undergoing heart surgery for valve replacement	Increased LA surface in AF is inversely correlated with PITX2 mRNA levels	-mRNA levels of both PITX2 and PITX2c about two-fold lower in LA of AF vs SR patients -PITX2 promoter is hypermethylated in AF vs SR patients -It is possible that besides AF co-morbidities also affect PITX2 mRNA -PITX2 may not be changed at the protein level in human AF	AF is associated with epigenetic LA changes including PITX2 promoter hypermethylation
Kao et al., 2013	93	Rat Mouse	Rat LA Mouse atrial HL-1 cells	Rats with isoprenaline-induced heart failure (HF) vs control animals	HF and Ang-II decrease atrial PITX2c protein levels	-PITX2c promoter is hypermethylated in LA of HF rats -Ang-II causes PITX2c promoter hypermethylation, along with a decrease in PITX2c and Kir2.1 protein levels	Heart failure and Ang-II promote atrial PITX2c promoter hypermethylation
Scridon et al., 2015	94	Rat	Rat LA	Young (14 weeks-old), adult (24 weeks-old) and aged (48 weeks-old) spontaneously hypertensive rats (SHRs)	Hypertension decreases atrial PITX2c mRNA levels	-mRNA levels of PITX2 were lower in LA of SHR vs age-matched control rats -Protein levels of PITX2 were not assessed	In SHRs PITX2 down-regulation is an age-dependent process that starts before the occurrence of atrial arrhythmias
Torrado et al., 2015	95	Pig Mouse	Pig LA Mouse atrial HL-1 cells	Pigs with AF induced by rapid atrial pacing	AF itself decreases LA PITX2 (and TBX5) protein levels	-Rapid atrial pacing reduces the protein levels (by 70%), but not the mRNA levels of PITX2c (and TBX5) in LA of pigs, along with an increase in miR-21 -Overexpression of miR-21 in HL-1 cells causes downregulation of PITX2c protein levels	Rapid atrial pacing mimicking AF decreases atrial PITX2c and TBX5 protein levels by upregulating miR-21
Key studies assessing the consequences of genetic manipulation of PITX2 expression levels for atrial function and AF susceptibility
Wang et al., 2010	96	Mouse	Whole hearts and LA	Mice with global PITX2 deficiency (PITX2null ± mice)	PITX2 deficiency creates an atrial arrhythmogenic substrate	-PITX2null ± mice have a higher susceptibility to inducible atrial arrhythmias -PITX2c is a suppressor of sinoatrial node-specific gene expression in LA: PITX2c deficiency enhances the mRNA levels of HCN4, NPPA, KCNQ1, SHOX2, and TBX3	PITX2 prevents susceptibility to atrial arrhythmias by inhibiting left-sided pacemaker specification
Chinchilla et al., 2011	97	Human Mouse	Human RAA and LAA Mouse RA and LA Mouse atrial HL-1 cells	Patients with or without AF undergoing heart surgery for valve replacement Conditional atrial-specific PITX2 deficient mice (NppaCre + PITX2−/−)	Atrial-specific PITX2 deficiency causes atrial electrical and structural changes	-mRNA levels of PITX2c strongly decreased (80–90%) in both LA and RA of AF vs SR patients -PITX2c may not be changed at the protein level in human AF -On ECG P wave was missing in 85% of NppaCre + PITX2−/− mice, pointing to AF presence; Sinoatrial node function normal -NppaCre + PITX2−/− show atrial and ventricular enlargement and increased ventricular, but not atrial fibrosis; mRNA of Col1a1 and Col3a1 increased in the ventricle but reduced in RA and LA of mutant mice -Multicellular AP recordings: more depolarized RMP with smaller AP amplitude, but no change in Vmax or APD in LA only of NppaCre + PITX2−/−, along with mRNA reduction of SCN5A, SCN1B, KCNJ2, KCNJ12, and KCNJ4. Kir2.1 and Nav1.5 protein levels reduced in mutant mice, along with miR-1 increases. -Overexpression of PITX2c in HL-1 cells decreases miR-1, whereas miR-1 overexpression in HL-1 cells decreases GJA1 and KCNJ2, but not SCN5A and SCN1B transcripts -Ventricular dysfunction is a confounder of the atrial phenotype of NppaCre + PITX2−/−	PITX2 could be an upstream transcriptional regulator of atrial function left-sided pacemaker specification
Kirchhof et al., 2011	98	Human Mouse	Human RAA and LAA Mouse RA and LA	Patients in sinus rhythm with or without AF undergoing heart surgery for CABG or valve replacement PITX2c deficient mice (PITX2c+/−)	PITX2c deficiency causes atrial electrical disturbances (action potential shortening) but no structural changes	-mRNA levels of PITX2c about 100-fold higher in human LA vs RA -PITX2c mRNA levels do not correlate to rs2200733 AF risk allele on chromosome 4q25 -PITX2c ± mice show action potential shortening and a higher susceptibility to inducible AF; Sinoatrial node function normal -On optical mapping, no difference in conduction time and activation patterns between mouse groups -Cardiac dimensions, structure and contractile function preserved in PITX2c ± mice (echocardiography); no evidence of fibrosis -PITX2c ± have normal ventricular function	Reduction of atrial PITX2c expression promotes AF inducibility by causing action potential shortening and is associated with complex changes in gene expression in the atria
Tao et al., 2014	99	Mouse	Whole atria	Conditional PITX2 deficient mice (PITX2 CKO)	Conditional PITX2 deficiency causes abnormal cardiac conduction, sinoatrial node dysfunction, and alterations in cardiomyocyte ultrastructure	-PITX2 CKO mice have sinoatrial node dysfunction and abnormal cardiac conduction -PITX2 CKO mice show disrupted intercalated discs and swollen and vacuolated mitochondria in LA cardiomyocytes -PITX2 CKO exhibit alterations in ion channel and calcium handling genes, and in genes that stabilize the intercalated discs	PITX2 directly regulates ion transport, calcium handling and intercalated dics genes
Wang et al., 2014	58	Mouse	Whole atria	Different genetically modified mouse lines	PITX2 deficiency upregulates miR-17-92 and miR-106b-25 and upregulation of these miRs promote pacing-induced AF in mice	-PITX2 positively regulates both miR-17-92 and miR-106b-25 expression -Mice with cardiac-specific miR-17-92 or miR-106b-25 both show higher susceptibility to inducible AF	PITX2 suppresses expression of miR-17-92 and miR-106b-25 and inhibits predisposition to AF
Nadadur et al., 2016	100	Mouse	Whole LA Isolated atrial myocytes	TBX5 and PITX2 deficient and double mutant mouse lines	PITX2 deficiency causes atrial APD abbreviation, but does not induce cellular triggered activity	-Atrial myocytes of PITX2 ± mice show shortened APD90 and APD50 and an increased AP amplitude, but no cellular triggered activity -PITX2 ± mice have a higher susceptibility to pacing-induced AF -Cross-breeding of PITX2 ± with TBX5 ± mice rescues the proarrhythmic phenotype (slowed atrial conduction, Ca2+ dependent triggered activity, APD50 and APD90 prolongation) of the latter	The TBX5 deficiency associated susceptibility to inducible AF was rescued by PITX2 haploinsufficiency in mice
Syeda et al., 2016	101	Human Mouse	Human LAA Mouse whole hearts Mouse LA Mouse LA myocytes	Patients undergoing bilateral thoracoscopic AF ablation PITX2c deficient mice (PITX2c+/−)	Pitx2 deficiency causes APD shortening and a more depolarized RMP	-PITX2 mRNA levels vary substantially in LAA of AF patients requiring rhythm control therapy with ablation -PITX2 mRNA levels do not correlate to rs2200733, rs6838973 or rs14448818 AF risk alleles on chromosome 4q25 -Flecainide suppresses atrial arrhythmias more efficiently in PITX2c ± mice -RMP is more depolarized in PITX2c+/−, along with decreased protein levels of TASK-2 (but not Kv1.6 or Nav1.5) -IK1 similar in PITX2c ± and WT mice -Conduction velocity similar in PITX2c ± and WT mice -LA optical APD is shorter in PITX2c ± mice	PITX2 deficiency results in more depolarized RMP, which causes greater sodium channel inactivation, thereby potentiating the antiarrhythmic effects of flecainide
Kao et al., 2019	102	Human	Cultured human atrial fibroblasts	PITX2c knockdown in human atrial fibroblasts	PITX2c deficiency increases activity of atrial fibroblasts, promoting their transition to collagen-secreting myofibroblasts	-PITX2c KO increase fibroblast migration, with no change in proliferation; CaMKII inhibition normalizes migration -PITX2c KO enhances Ca2+ influx and increases protein levels of CaMKII-P, αSMA, and MMP2, but not of Col1 or MMP9 -Effect of culture-driven transdifferentiation is a bias	PITX2c deficiency increases human atrial fibroblast activity potentially promoting atrial fibrosis
Collins et al., 2019	103	Zebra fish	Whole heart	PITX2c deficient zebra fish (PITX2c ± and PITX2c−/−)	PITX2c deficiency causes atrial conduction defects and alterations in cardiomyocyte metabolism and ultrastructure	-PITX2c deficiency causes atrial conduction defects, atrial enlargement and fibrosis -PTX2c deficiency induces sarcomere disassembly, alters mitochondrial morphology and cardiac metabolism, and increases ROS production, all of which precede the onset of cardiac arrhythmias -Incidence and severity of PITX2c deficiency related arrhythmias are reduced by antioxidant treatment with the ROS scavenger N-acetyl-cysteine, pointing to oxidative stress as a driver of the arrhythmia -Ventricular function is normal in PITX2c deficient zebra fish	PITX2c deficiency in zebra fish induces sarcomere and metabolic defects that precede the development of atrial conduction disturbances and arrhythmias
Holmes et al., 2021	104	Human Mouse	Human IPSCs HEK293 cells Mouse LA	HEK293 cells expressing Scn5a + Scn1b Human IPSC-derived myocytes PITX2c deficient mice (PITX2c+/−)	PITX2c deficiency causes a more positive RMP	-PITX2c ± mice have more positive RMP -Dronedarone causes stronger action potential prolongation in PITX2c+/− -Human IPSC-derived myocytes are not atrial-like	The more depolarized RMP due to PITX2c deficiency increases the efficacy of dronedarone to prolong atrial action potential
Tarifa et al., 2023	105	Mouse	Pooled LA and RA myocytes	Atrial-specific PITX2 deficient mice (NppaCre + PITX2+/−; NppaCre + PITX2−/−)	Atrial-specific PITX2 deficiency causes atrial electrical and Ca2+ handling alterations that may promote atrial ectopy	-PITX2 deficiency increases cell capacitance of RA and LA myocytes, pointing to cell hypertrophy -PITX2 deficiency causes a reduction in ICa,L in RA and LA -PITX2 deficiency increases frequency of Ca2+ sparks and waves and of transient inward current ITI in LA and RA myocytes -PITX2 deficiency enhances caffeine releasable SR Ca2+ load in RA and LA -PITX2 deficiency promotes DADs and triggered action potentials in RA myocytes	Atrial-specific PITX2 deficiency reduces ICa,L that may cause re-entry promoting action potential shortening and induces cellular Ca2+ handling abnormalities that may cause atrial ectopy and triggered activity
Kim et al., 2023	106	Mouse	Atrial myocytes	PITX2 deficient mice (PITX2+/−)	PITX2 deficiency causes Ca2+-dependent cellular triggered activity	-PITX2−/− mice show higher susceptibility to inducible AF, which is suppressed by the selective RyR2 inhibitor ent-verticulide -Normal sinoatrial node function -Atrial myocytes from PITX2 ± mice show increased frequency, amplitude and mass of Ca2+ sparks, along with more spontaneous Ca2+ waves -No changes in diastolic Ca2+, SR Ca2+ load or CaT decay in PITX2 ± mice -Ent-verticulide suppresses spontaneous Ca2+ waves in PITX2−/−	The higher susceptibility to AF of PITX2 ± mice is suppressed by selective RyR2 inhibition suggesting that ectopic (triggered) activity is one potential arrhythmogenic mechanism of PITX2 deficiency
Schulz et al., 2023	107	Human	Humans IPSCs	CRISPER/Cas9-mediated PITX2 deletion in healthy human atrial-like IPSCs Atrial engineered heart tissue	Atrial-specific PITX2 deficiency causes atrial electrical changes	-PITX2−/− reduces cell capacitance -PITX2 deficiency causes a reduction in Ito and ICa,L -Lack of Pitx2 decreases force of contraction and slows force relaxation, with beating rate being slightly lower in PITX2−/− -RMP more negative in PITX2−/−, but not due to a higher IK1 -APD20 is longer, APD90 is shorter; AP amplitude is higher with faster Vmax in PITX2−/− -PITX2−/− reduces mRNA of Cav.1.2, SERCA2a, RyR2, NCX1; no change in KCNJ2, KCNJ4, KCNJ12 -Atrial-like IPSCs differ from native atrial myocytes and there is no LA myocyte specification	PITX2 knockout induces key findings of electrical (shorter triangular AP with decreased Ito and ICa,L) and contractile (reduced force of contraction) remodeling typical of persistent AF
Perez-Hernandez et al., 2016	108	Human Mouse	Human RAA Mouse atrial HL-1 cells	Patients in sinus rhythm with or without AF undergoing heart surgery for CABG or valve replacement Mouse atrial HL-1 cells	Increased PITX2c is associated with reduced ICa,L and increased IKs, both potentially abbreviating action potential duration	-PITX2c mRNA is increased in RA myocytes from AF patients -The higher PITX2c mRNA in RA correlates with reduced ICa,L and increased IKs in RA cardiomyocytes of AF patients -Overexpression of PITX2 in HL-1 cells reduces ICa,L and increases IKs by direct modulation of KCNQ1 promoter activity -These findings might not apply to human LA cardiomyocytes of AF patients	Human chronic AF is associated with increased PITX2c expression and a reduction of ICa,L and an increase in IKs in RA cardiomyocytes that may contribute to re-entry promoting AP shortening
Key study linking 4q25 rs13143308T allele to atrial cardiomyocyte function of AF patients
Herraiz-Martinez et al., 2019	109	Human	Human RAA	Patients in sinus rhythm with or without AF undergoing heart surgery for CABG or valve replacement	The risk variant rs13143308T associates with DAD-dependent triggered activity, but not with ICa,L function	-Rs13143308T increases SR Ca2+ load and frequency of Ca2+ sparks, transient inward current ITI, and DADs in RA myocytes -Rs13143308T does not associate with ICa,L changes -Ser2808-RyR2 increased, Ser2014-RyR2 unchanged -Protein levels of SERCA2a increased, those of NCX1 and CSQ unaltered -Putative association of the risk allele with atrial PITX2 expression levels not assessed	The 4q25 variant rs13143308T increase the risk for AF by causing ectopic (triggered) activity