Sequential Defects in Cardiac Lineage Commitment and Maturation Cause Hypoplastic Left Heart Syndrome

Abstract

Background: Complex molecular programs in specific cell lineages govern human heart development. Hypoplastic left heart syndrome (HLHS) is the most common and severe manifestation within the spectrum of left ventricular outflow tract obstruction defects occurring in association with ventricular hypoplasia. The pathogenesis of HLHS is unknown, but hemodynamic disturbances are assumed to play a prominent role. Methods: To identify perturbations in gene programs controlling ventricular muscle lineage development in HLHS, we performed whole-exome sequencing of 87 HLHS parent–offspring trios, nuclear transcriptomics of cardiomyocytes from ventricles of 4 patients with HLHS and 15 controls at different stages of heart development, single cell RNA sequencing, and 3D modeling in induced pluripotent stem cells from 3 patients with HLHS and 3 controls. Results: Gene set enrichment and protein network analyses of damaging de novo mutations and dysregulated genes from ventricles of patients with HLHS suggested alterations in specific gene programs and cellular processes critical during fetal ventricular cardiogenesis, including cell cycle and cardiomyocyte maturation. Single-cell and 3D modeling with induced pluripotent stem cells demonstrated intrinsic defects in the cell cycle/unfolded protein response/autophagy hub resulting in disrupted differentiation of early cardiac progenitor lineages leading to defective cardiomyocyte subtype differentiation/maturation in HLHS. Premature cell cycle exit of ventricular cardiomyocytes from patients with HLHS prevented normal tissue responses to developmental signals for growth, leading to multinucleation/polyploidy, accumulation of DNA damage, and exacerbated apoptosis, all potential drivers of left ventricular hypoplasia in absence of hemodynamic cues. Conclusions: Our results highlight that despite genetic heterogeneity in HLHS, many mutations converge on sequential cellular processes primarily driving cardiac myogenesis, suggesting novel therapeutic approaches.

Cell cycle defects underlie childhood-onset cardiomyopathy associated with Noonan syndrome

“…The healthy iPSC lines were reprogrammed with the CytoTune-iPS 2.0 Sendai Reprogramming kit (Invitrogen A16517) using the peripheral blood mononuclear cells (PBMCs) of male (#1: Human Pluripotent Stem Cell Registry ID MRIi003-A) and female (#2: ID MRIi001-A) volunteers, as previously described ( Krane et al., 2021 ; Moretti et al., 2020 ). PTPN11 N308S/+ iPSCs (Human Pluripotent Stem Cell Registry ID MRIi025-A) were reprogrammed with the same protocol from the PBMCs of a male Noonan syndrome patient carrying a heterozygous 923A→G mutation in the PTPN11 gene leading to a p.N308S substitution in the protein tyrosine phosphatase domain of the SHP-2 protein.…”

Genomic and transcriptomic data analyses highlight KPNB1 and MYL4 as novel risk genes for congenital heart disease

“…Lower expression levels of MYL4 and KPNB1 were seen in the scRNAseq data of three of four hiPS-CM cell lines derived from LVOTO patients as compared to heathy controls. Recent studies have indicated that despite the genetic heterogeneity, similar abnormal phenotypes and gene expression profiles in LVOTO patient derived hiPS-CMs can be observed, indicating for example diminished capacity for cardiomyocyte differentiation, more immature transcriptomic profile, and reduced expression of cardiac transcription factors (Jiang et al, 2014;Kobayashi et al, 2014;Krane et al, 2021;Theis et al, 2015;Yang et al, 2017). The lower expression levels of MYL4 and KPNB1 in patient-derived cells may, thus, indicate a role of these genes in the development of CHD.…”

Polyploidy as a Fundamental Phenomenon in Evolution, Development, Adaptation and Diseases

“…In the proliferating cells, physiological stress associated with genome instability can promote polyploidy via the premature cell cycle and disrupted cell differentiation [ 94 ]. This phenomenon was described in cardiomyocytes from the hypoplastic left ventricle in the human and neonatal mice [ 94 , 102 ]. It was also observed in cardiomyocytes and hepatocytes of neonatal rats that survived severe inflammatory stress [ 7 , 9 , 75 ], and in drosophila epithelial cells involved in wound healing [ 1 , 103 ].…”