Transcriptomic and functional analysis of induced pluripotent stem cell-derived cardiomyocytes from tetralogy of fallot patient with and without DiGeorge syndrome

Abstract

Tetralogy of Fallot (TOF) is the most common cyanotic congenital heart disease. Most of the TOF cases are of unknown origin while DiGeorge syndrome (DG), a microdeletion on chromosome 22q11.2, accounts for most of the cases with identifiable genetic causes. Technological advancement and better clinical management have significantly improved the survival rate and increased the longevity of patients with TOF. Yet, the incidence rate of late complications, including pulmonary regurgitation, arrhythmia and right ventricular dysfunction, is high in older patients with surgically corrected TOF. The assessment of cardiac function in TOF is confounded by the secondary compensation to the structural defects or the surgery. The central objective of this thesis is to assess the functional parameters and the transcriptome of cardiomyocytes from TOF patients, in the absence of structural defects or secondary compensation, with the use of a human-induced pluripotent stem cell (hiPSC) model. Two groups of TOF patients were recruited, one with DG (TOF-DG) and one without DG (TOF-ND). HiPSC lines were established by reprogramming the CD34+ peripheral mononuclear cells from the patients and healthy control. High purity of cardiomyocytes could be generated from all the hiPSC lines (hiPSC-CMs). hiPSC-CMs were fabricated in a cardiac anisotropic sheet (CAS) for the assessment of electrophysiological properties and arrhythmogenicity. Significant alterations in the kinetics of the action potential and calcium handling were found in some of the TOF-DG-CAS and TOF-ND-CAS. A significantly increased risk of arrhythmia was identified in one TOF-DG-CAS. Transcriptomic differences between Control-, TOF-DG- and TOF-ND-CAS were interrogated by single-cell RNA sequencing (scRNA-seq). Different disease groups showed group-specific transcriptomic signatures. Genes related to contractile function were upregulated and downregulated in TOF-ND-CAS and TOF-DG-CAS, respectively. scRNA-seq further unveiled the downregulation of cardiac genes and upregulation of neural genes in a subgroup of hiPSC-CMs in TOF-DG-CAS. In addition to the group-specific transcriptome, CHCHD2, implicated in Parkinson’s Disease, was found to be downregulated in both TOF-DG and TOF-ND groups. Another scRNA-seq experiment was performed on the progenitors during in vitro cardiac differentiation. Through the scRNA-seq of cardiac progenitors, the impairment in cardiac gene expression was evident in the subset of TOF-DG as early as Day 7 post differentiation. To conclude, this study is the first to demonstrate impairments in the differentiation and cardiac functions of hiPSC-CMs derived from TOF-DG patients in the absence of structural anomalies and secondary compensations. On the other hand, such impairments were not found in hiPSC-CMs derived from TOF-ND patients. Yet, the downregulation of CHCHD2 implies the risk of neurodegenerative disease in all the TOF patients studied here, with or without DG. These provide a new perspective on the clinical management of TOF patients and new directions for future studies on TOF.