Human induced pluripotent stem cell platform for assessment of cardiac electrophysiological and contractile function in heterotaxy syndrome

Abstract

Heterotaxy syndrome is a set of congenital disorders identified by abnormal arrangement of thoraco-abdominal organs across the left-right axis of the body, and the long-term outcomes of heterotaxy syndrome improved due to advanced surgical procedures and enhanced management of cardiac complications. While the clinical outcomes of both right isomerism (RI) and left isomerism (LI) are well-documented, the cellular contribution on contractile dysfunction and cardiac arrhythmogenicity remained unknown. To address this gap, heterotaxy patient-specific induced pluripotent stem cells (iPSC) and their derived cardiomyocytes (CM) were successfully generated in this dissertation for subsequent functional assessments. The central hypothesis of this dissertation is that in the absence of structural abnormality and secondary remodelling, intrinsic cellular defects of heterotaxy patient-derived cardiomyocytes contribute to malfunctional contractility and altered electrophysiological properties leading to emergence of cardiac arrhythmias. In this dissertation, using three-dimensional tissue constructs, contractile force generated by RI-iPSC-CMs was shown to be significantly reduced, and the contractile and relaxation time were increased. This alteration in contractility is associated with reduced calcium transient and reduced expression of contractile proteins as shown with single cell RNA sequencing. Illustrated by pathway analysis, worsening of contractile function in RI-iPSC-CMs is likely attributed to reduced sarcomerogenesis and weakened myocardial filaments sliding. Moreover, electrophysiological features including action potential duration (APD) and effective refractory period (ERP) were altered in RI-iPSC-CMs, and potassium channels were dysfunctional, which are associated with increased risk of arrhythmogenicity. Effective refractory period was reduced due to altered potassium current and caused patient-specific cardiomyocytes to be more readily available for re-excitation and increased the occurrence of reentrant events. Whereas in LI-iPSC-CMs, although calcium transient was shortened due to increased expression of ryanodine receptors as shown by differential gene expression, it did not translate into altered contractile force elicited. This discordance between calcium handling and contractility may be caused by compensatory responses including increased expression of titin, troponin I and MyCB-P. As a result, an increase in contractile kinetic was observed. Furthermore, increased risk of arrhythmogenicity was also observed in LI-iPSC-CMs. While both sodium and potassium channels remained intact, electrophysiological properties including APD, ERP and conduction velocity (CV) were reduced, which may be due to changes in cell-cell interaction such as dysregulated gap junctions. In addition to gap junctions, as seen with pathway analysis of LI-iPSC-CMs single cell RNA sequencing data, various collagenases were upregulated and implicated the possibility of fibrosis in these cells, which may also contribute to the reduced CV, leading to increased risk of developing arrhythmias. However, further investigation is required to validate these speculations. To conclude, these findings suggested that contractile force generated was reduced in RI-iPSC-CMs, and contractile kinetic was altered in both RI-iPSC-CMs and LI-iPSC-CMs. While stemmed from different changes in electrophysiological features, both RI-iPSC-CMs and LI-iPSC-CMs were affected by increased risk of arrhythmias. These cellular defects in heterotaxy patient-derived cardiomyocytes provide new perspectives on current and future clinical management for improving long-term healthcare of patients with heterotaxy syndrome.