Zeng, Xin-Xin I.

Hypoplastic left heart syndrome (HLHS) is the most lethal congenital heart disease (CHD) whose genetic basis remains elusive, likely due to oligogenic complexity. To identify regulators of cardiomyocyte (CM) proliferation relevant to HLHS, we performed a genome-wide siRNA screen in human iPSC-derived CMs, revealing ribosomal protein (RP) genes as the most prominent effectors of CM proliferation. Whole-genome sequencing of 25 HLHS proband–parent trios similarly showed enrichment of rare RP gene variants, including a damaging RPS15A promoter variant shared in a familial CHD case. Cross-species functional analyses demonstrated that perturbation of RP genes impairs cardiac growth: knockdown of RPS15A, RPS17, RPL26L1, RPL39, or RPS15 reduced CM proliferation, caused cardiac malformations in Drosophila , and produced hypoplastic or dysfunctional hearts in zebrafish. Genetic interactions between RP genes and key cardiac transcription factors (TBX5 and NKX2–7) further support their developmental role. Importantly, p53 suppression or Hippo activation partially rescued RP deficiency phenotypes. Together, these findings implicate RP genes as critical regulators of cardiogenesis and candidate contributors to HLHS.

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 affecting RPS15A that 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 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 of the atrium, 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 novel critical roles in cardiogenesis and constitute an emerging class of gene candidates likely involved in HLHS.

Hypoplastic left heart syndrome (HLHS) is the most lethal congenital heart disease (CHD) whose genetic basis remains elusive, likely due to oligogenic complexity. To identify regulators of cardiomyocyte (CM) proliferation relevant to HLHS, we performed a genome-wide siRNA screen in human iPSC-derived CMs, revealing ribosomal protein (RP) genes as the most prominent effectors of CM proliferation. Whole-genome sequencing of 25 HLHS proband–parent trios similarly showed enrichment of rare RP gene variants, including a damaging RPS15A promoter variant shared in a familial CHD case. Cross-species functional analyses demonstrated that perturbation of RP genes impairs cardiac growth: knockdown of RPS15A, RPS17, RPL26L1, RPL39, or RPS15 reduced CM proliferation, caused cardiac malformations in Drosophila , and produced hypoplastic or dysfunctional hearts in zebrafish. Genetic interactions between RP genes and key cardiac transcription factors (TBX5 and NKX2–7) further support their developmental role. Importantly, p53 suppression or Hippo activation partially rescued RP deficiency phenotypes. Together, these findings implicate RP genes as critical regulators of cardiogenesis and candidate contributors to HLHS.

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 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 affecting RPS15A that 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 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 of the atrium, 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 novel critical roles in cardiogenesis and constitute an emerging class of gene candidates likely involved in HLHS and other CHDs.

Congenital heart diseases (CHDs), including hypoplastic left heart syndrome (HLHS), are genetically complex and poorly understood. Here, a multidisciplinary platform was established to functionally evaluate novel CHD gene candidates, based on whole-genome and iPSC RNA sequencing of a HLHS family-trio. Filtering for rare variants and altered expression in proband iPSCs prioritized 10 candidates. siRNA/RNAi-mediated knockdown in healthy human iPSC-derived cardiomyocytes (hiPSC-CM) and in developing Drosophila and zebrafish hearts revealed that LDL receptor-related protein LRP2 is required for cardiomyocyte proliferation and differentiation. Consistent with hypoplastic heart defects, compared to parents the proband’s iPSC-CMs exhibited reduced proliferation. Interestingly, rare, predicted-damaging LRP2 variants were enriched in a HLHS cohort; however, understanding their contribution to HLHS requires further investigation. Collectively, we have established a multi-species high-throughput platform to rapidly evaluate candidate genes and their interactions during heart development, which are crucial first steps toward deciphering oligogenic underpinnings of CHDs, including hypoplastic left hearts.