Combining Human Induced Pluripotent Stem Cells Derived Cardiac monolayers, CRISPR-based Genome Editing, and Optical Mapping of Rotors to Study the Short QT Syndrome

Introduction:
The congenital Short QT Syndrome (SQTS) is a familial arrhythmogenic syndrome caused by abnormal ion channel function leading to action-potential duration (APD) shortening, life-threatening arrhythmias and sudden cardiac death.

Aims:
1) To establish a patient-specific hiPSC 2D cardiac monolayer disease model of the congenital SQTS. 2) To establish an isogenic control line using gene-editing technology. 3) To characterize the electrical properties of the generated patient-specific hiPSC cardiac monolayers at baseline and following drug interventions.

Results:
Patient/disease-specific hiPSC lines were established from a healthy individual and a symptomatic SQTS patient carrying missense mutation (N588K) in the hERG gene, leading to gain of function in the rapidly activating delayed rectifier potassium current (I_Kr). A corrected-SQTS-hiPSC line was established using CRISPR-Cas9 system. All hiPSC lines were directly differentiated into hiPSC-derived cardiomyocytes (hiPSC-CMs). Optical mapping studies revealed that the SQTS-hiPSC-CM monolayers recapitulated the disease phenotype presenting abbreviated mean APD, impaired APD-rate adaptation, yet a similar conduction velocity compared to healthy-control and corrected-SQTS-hiPSC-CM monolayers. Furthermore, applying an arrhythmia induction protocol on SQT-hiPSC-CM monolayers resulted in higher rates of sustained functional reentries, higher frequency and lower diameter rotors (resulting in higher rotor stability), compared to both isogenic and healthy controls. 1 µM quinidine normalized mean APD, and prevented sustained reentry inducibility in SQT-hiPSC-CM monolayers. Finally, application of pentamidine, antimicrobial medication inhibiting hERG channel trafficking, succeeded prolonging mean APD in SQT-hiPSC-CM monolayers.

Conclusions:
A novel hiPSC-based 2D model of the SQTS was established. Using gene-editing technology allowed establishing isogenic control and rescuing the diseased phenotype. This model allowed recapitulating the disease phenotype (shortened APD, impaired rate adaptation, and sustained functional reentry inducibility). The response to quinidine demonstrated the potential of the model for drug screening. Our study on SQTS-hiPSC 2D model suggests pentamidine as a potential novel drug for SQTS treatment.