Abstract 

Aims

Electrical remodelling in human persistent atrial fibrillation (AF) is believed to result from rapid electrical activation of the atria, but underlying genetic causes may contribute. Indeed, common gene variants in an enhancer region close to PITX2 are strongly associated with AF, but the mechanism behind this association remain unknown. This study evaluated consequences of PITX2 deletion in human induced pluripotent stem cell derived atrial cardiomyocytes (hiPSC-aCM).

Methods and Results

CRISPR/Cas9 was used to delete PITX2 (PITX2^-/^-) in a healthy human iPSC line which served as isogenic control. HiPSC-aCM were differentiated with retinoic acid (RA) and cultured in atrial engineered heart tissue (aEHT). Force and action potential were measured in aEHTs. Single hiPSC-aCM were isolated from aEHT for ion current measurements. PITX2^-/^- aEHT beat slightly slower than isogenic control without irregularity. Force was lower in PITX2^-/^- than in isogenic control (0.053±0.015 vs. 0.131±0.017 mN, n=28/3 vs. n=28/4, PITX2^-/^- vs. isogenic control; p<0.0001), accompanied by lower expression of CACNA1C and lower L-type Ca²⁺ current density. Early repolarisation was weaker (APD₂₀; 45.5±13.2 vs. 8.6±5.3 ms, n=18/3 vs. n=12/4, PITX2^-/^- vs. isogenic control; p<0.0001 and maximum diastolic potential was more negative (-78.3±3.1 vs. -69.7±0.6 mV, n=18/3 vs. n=12/4, PITX2^-/^- vs. isogenic control; p=0.001), despite normal inward rectifier currents (both I_K1 and I_K,ACh) and carbachol-induced shortening of APD.

Conclusion

Complete PITX2 deficiency in hiPSC-aCM recapitulates key findings of electrical remodelling of AF in the absence of fast beating, indicating that at least some of these abnormalities could be primary consequences of lower PITX2 levels.

Keywords

Atrial fibrillation, remodelling, PITX2, hiPSC-CM, engineered heart tissue, calcium currents