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

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 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 a familial CHD case we identified a rare, predicted-damaging promoter variant affectingRPS15Athat was shared between the HLHS 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 the 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 inDrosophila. In zebrafish, diminishedrps15afunction caused reduced CM numbers, heart looping defects, or weakened contractility, while reducedrps17orrpl39function caused reduced ventricular size or systolic atrial dysfunction of the atrium, respectively. Importantly, genetic interactions betweenRPS15Aand core cardiac transcription factorsTBX5in CMs,Drosocross, pannierandtinmanin flies, andtbx5andnkx2-7(nkx2-5paralog) in fish, support a specific role for RP genes in heart development. Furthermore,RPS15AKD-induced heart/CM proliferation defects were significantly attenuated byp53KD in both hPSC- CMs and zebrafish, and by Hippo activation (YAP/yorkieoverexpression) in developing fly hearts. Based on these findings, we conclude that RP genes play novel critical roles in cardiogenesis and constitute an emerging class of gene candidates likely involved in HLHS and other CHDs.