Maturing hiPSC-cardiomyocytes in cardiac microtissues for accurate preclinical in vitro modelling of long-QT syndrome type 1
Authors: Campostrini G , Ward Van-Oostwaard D , Verkerk A , Mummery C , Bellin M 
Published: May 2024

Abstract

Funding Acknowledgements
Type of funding sources: Foundation. Main funding source(s): reNEW - Novo Nordisk Foundation Center for Stem Cell Medicine

Background
Long-QT syndrome type 1 (LQT1), a cardiac arrhythmia often leading to sudden cardiac death, is due to mutations in KCNQ1 gene. Human iPSC-derived cardiomyocytes (hiPSC-CMs) have been widely used to model LQT1, promoting the concept of their use in preclinical studies. However, KCNQ1 locus is subjected to developmentally regulated genomic imprinting in the heart: in fetal cardiomyocytes the paternal allele is repressed, while both alleles become expressed after birth. Since hiPSC-CMs are typically immature fetal-like cells, residual imprinting may affect evaluation of KCNQ1 mutations in this system.

Purpose
This study wants to address the imprinting status of KCNQ1 in hiPSC-CMs and propose maturing hiPSC-CMs as a solution for reliable LQT1 modelling.

Methods
We derived hiPSC-CMs from a LQT1 patient carrying a heterozygous KCNQ1 mutation on the paternal allele and compared their electrical properties with the isogenic corrected hiPSC-CMs by patch clamp. We analysed KCNQ1 expression by ddPCR and DNA methylation. We used three-dimensional cardiac microtissues (MTs) composed by hiPSC-CMs, hiPSC-derived cardiac fibroblasts and endothelial cells to induce cardiomyocte maturation.

Results
LQT1 and isogenic corrected hiPSC-CMs showed no difference in action potential duration (APD). We hypothesized that residual KCNQ1 imprinting in hiPSC-CMs due to their immaturity was masking the phenotype. When we analyzed different hiPSC-CM lines, we found in all an unbalanced KCNQ1 allelic expression, with paternal allele accounting for 5-15% of the total. We then investigated whether maturing hiPSC-CMs could remove KCNQ1 imprinting. We included hiPSC-CMs in MTs, as this system previously showed to promote functional maturation and upregulation of KCNQ1. Here, we observed a substantial increased expression of the paternal allele and a reduction in the expression of KCNQ1OT1, the long non-coding RNA inducing paternal allele repression. LQT1 hiPSC-CMs dissociated from MTs showed prolonged APD compared to the corrected line. We also demonstrated that this was due to the reduction of the ionic current mediated by KCNQ1 (IKs) in LQT1 hiPSC-CMs compared to the corrected controls, thus revealing the mutation causative effect.

Conclusions
Our findings show that residual KCNQ1 imprinting in immature hiPSC-CMs can lead to underestimate (or overestimate) functional effects of pathological variants based on the parental allele that carries the mutation. This is overcome by maturation of hiPSC-CMs in tri-cellular cardiac microtissue which promotes KCNQ1 bi-allelic expression and allow dissecting mutation mechanisms. This study brings attention to the epigenetic regulation of hiPSC models and demonstrates the utility of using matured hiPSC-CMs as in vitro preclinical model for cardiac arrhythmias.