Subtype-specific promoter-driven action potential imaging for precise disease modelling and drug testing in hiPSC-derived cardiomyocytes

Aims Cardiomyocytes (CMs) generated from human induced pluripotent stem cells (hiPSCs) are increasingly used in disease modelling and drug evaluation. However, they are typically a heterogeneous mix of ventricular-, atrial-, and nodal-like cells based on action potentials (APs) and gene expression. This heterogeneity and the paucity of methods for highthroughput functional phenotyping hinder the full exploitation of their potential. We aimed at developing a method for rapid, sequential, and subtype-specific phenotyping of hiPSC-CMs with respect to AP morphology and single-cell arrhythmias. Methods and results We used cardiac lineage-specific promoters to drive the expression of a voltage-sensitive fluorescent protein (VSFP-CR) in hiPSC-CMs, enabling subtype-specific optical AP recordings. In a patient-specific hiPSC model of long-QT syndrome type 1, AP prolongation and frequent early afterdepolarizations were evident in mutant ventricular- and atrial like, but not in nodal-like hiPSC-CMs compared with their isogenic controls, consistent with the selective expression of the disease-causing gene. Furthermore, we demonstrate the feasibility of sequentially probing a cell over several days to investigate genetic rescue of the disease phenotype and to discern CM subtype- specific drug effects. Conclusion By combining a genetically encoded membrane voltage sensor with promoters that drive its expression in the major subtypes of hiPSC-CMs, we developed a convenient system for disease modelling and drug evaluation in the relevant cell type, which has the potential to advance the emerging utility of hiPSCs in cardiovascular medicine.