Functional analysis across model systems implicates ribosomal proteins in growth and proliferation defects associated with hypoplastic left heart syndrome

Abstract

Hypoplastic left heart syndrome (HLHS) is the most lethal congenital heart disease (CHD). The pathogenesis of HLHS is poorly understood and due to the likely oligogenic complexity of the disease, definitive HLHS-causing genes have not yet been identified. Postulating that impaired cardiomyocyte proliferation as a likely important contributing mechanism to HLHS pathogenesis, and we conducted a genome-wide siRNA screen to identify genes affecting proliferation of human iPSC-derived cardiomyocytes (hPSC-CMs). This yielded ribosomal protein (RP) genes as the most prominent class of effectors of CM proliferation. In parallel, whole genome sequencing and rare variant filtering of a cohort of 25 HLHS proband-parent trios with poor clinical outcome revealed enrichment of rare variants of RP genes. In addition, in another familial CHD case of HLHS proband we identified a rare, predicted-damaging promoter variant affecting RPS15A that was shared between the proband and a distant relative with CHD. Functional testing with an integrated multi-model system approach reinforced the idea that RP genes are major regulators of cardiac growth and proliferation, thus potentially contributing to hypoplastic phenotype observed in HLHS patients. Cardiac knockdown (KD) of RP genes with promoter or coding variants (RPS15A, RPS17, RPL26L1, RPL39, RPS15) reduced proliferation in generic hPSC-CMs and caused malformed hearts, heart-loss or even lethality in Drosophila. In zebrafish, diminished rps15a function caused reduced CM numbers, heart looping defects, or weakened contractility, while reduced rps17 or rpl39 function caused reduced ventricular size or systolic atrial dysfunction, respectively. Importantly, genetic interactions between RPS15A and core cardiac transcription factors TBX5 in CMs, Drosocross, pannier and tinman in flies, and tbx5 and nkx2-7 (nkx2-5 paralog) in fish, support a specific role for RP genes in heart development. Furthermore, RPS15A KD-induced heart/CM proliferation defects were significantly attenuated by p53 KD in both hPSC-CMs and zebrafish, and by Hippo activation (YAP/yorkie overexpression) in developing fly hearts. Based on these findings, we conclude that RP genes play critical roles in cardiogenesis and constitute an emerging novel class of gene candidates likely involved in HLHS and other CHDs.

Multiplatform modeling of atrial fibrillation identifies phospholamban as a central regulator of cardiac rhythm

“…To address some of these modeling requirements, we have assembled ( Schuttler et al, 2020 ) a new phenotypic platform that enables rapid evaluation of the effects of gene function on the regulation of APD and rhythm in model systems, providing (1) a human and atrial context (ACM model), and (2) an intact, functionally mature and aging heart (fly model). Because both ACM and fly systems have genome-wide screening capacity ( Neely et al, 2010 ; Nielsen et al, 2022 preprint), they unlock the exploratory power of functional genomics that is needed for the unbiased identification of novel genes and pathways controlling cardiac rhythm beyond those identified in GWAS. A remarkable feature of the ACM platform is the ability to assess APD and rhythm parameters at single-cell resolution, which enables (1) the development of co-culture conditions mimicking aspects of known AF-associated risk factors, such as fibrosis, and (2) the quantification of the heterogeneity in cellular responses to environmental perturbations.…”

Multiplatform Modeling of Atrial Fibrillation Identifies Phospholamban as Central Regulator of Cardiac Rhythm

“…To address some of these modeling requirements, we have assembled (Schuttler et al, 2020) a new phenotypic platform that enables rapid evaluation of gene function on the regulation of APD and rhythm in model systems providing 1) a human and atrial context (ACM model), and 2) an intact, functionally mature and aging heart (fly model). Because both ACM and fly systems have genome-wide screening capacity (Neely et al, 2010;Nielsen et al, 2022), they unlock the exploratory power of functional genomics that is needed for the unbiased identification of novel genes and pathways controlling cardiac rhythm beyond those identified in GWAS studies. A remarkable feature of the ACM platform is the ability to assess APD and rhythm parameters with single cell resolution, which enables 1) the development of co-culture conditions mimicking aspects of known AF-associated risk factors such as fibrosis and 2) the quantification of the heterogeneity in cellular responses to environmental perturbations.…”