Whole Genome siRNA Screen identified Ribosomal proteins as agonists of cardiomyocyte proliferation.

(A) High-throughput iPSC-derived CM proliferation screen overview. (B) Screen result showing normalized % EdU+ CMs (X-axis) and relative total number of CMs (Y-axis) upon knockdown of genome-wide siRNAs (18,055 siRNAs). siRNAs for RPL and RPS genes highlighted in red. (C) Representative immunofluorescence images of proliferation (EdU incorporation, green, CM marker ACTN2, red) of induced hPSC-CMs upon TP53 and RPS14 knockdown. Insets: nuclei (DAPI) (D) Gene ontology enrichment analysis for WGS hits (BP, biological process; FDR-corrected analysis using gprofiler2). (E) Overview of hits corresponding to top 4 non-redundant BP categories.

Ribosomal gene variants identified in HLHS.

(A) Gene prioritization scheme of 25 poor-outcome proband-parent trios. (B) Testing 292 HLHS candidate genes from all poor-outcome families in CMs identified RPs as major regulators of hPSC-CM proliferation (Normalized fraction of ACTN2+/EdU+ cells). (C) Drosophila cardiac phenotypes induced by loss of RP genes affected in HLHS patients with poor outcome. Heart is visualized by RFP expression specifically in cardiomyocytes (R94C02::tdTom). Knockdown is achieved by sustained Gal4/UAS activity using Hand4.2-Gal4. (D) Wild-type zebrafish larva, rps17 and rps28 CRISPR mutants and rpl39 morphants at 72 hpf. rpl39 morphants, injected with 1 ng MO in lateral view, show mild edema. (E) Systolic surface area (SA) upon rps17 and rps28 CRISPR, and diastolic SA after rpl39 MO in the atrium and ventricle of zebrafish hearts.

Characterization of RPS15A from 75H HLHS index family.

(A) (B) (C) Prioritized candidate genes from the 75H family and relative hPSC-CM proliferation capacity upon KD. (D) Representative immunofluorescence images of proliferation (EdU incorporation, green; CM marker ACTN2, red) of induced hPSC-CMs upon siRPS15A knockdown. (E) Heart-specific KD of RpS15Aa in Drosophila adult hearts cause loss of heart tissue and is fully penetrant. (F) Representative immunofluorescence images of control and RpS15Aa-RNAi shows partial heart loss (Myosin heavy chain, Mhc, red; heart tissue-reporter, green). (G) CRISPR-mediated loss of rps15a in F0 larval zebrafish hearts causes decrease in CM number. (H) Representative immunofluorescence of hearts and whole-mount images of control and rps15a-CRISPR F0 larval hearts (CM nuclei reporter, red).

Loss of ribosomal gene function in CMs invokes TP53-stress response.

(A) RNA sequencing of hPSC-CMs following siRNA-treatment for RPL39 and RPS15A shows both, convergent and divergent transcriptomic response. (B) GO term analysis of differentially expressed genes following RP KD shows TP53-mediated response, including upregulation of CDKN1A. (C, D) CDKN1A is highly upregulated in CMs following siRPS15A treatment. (E, F) Reduced CM proliferation upon RPS15A KD is mediated by CDKN1A/TP53 and can be rescued upon their co-KD. (G, H) Larval zebrafish CM number is reduced by morpholino-treatment for rps15a and can be attenuated by P53 morpholino co-injection. Control and morphant (MO) hearts of 72 hpf zebrafish larva stained with Tg(myl7:EGFP) and Tg(myl7:H2A-mCherry). Note that smaller heart with aberrant looping by rps15a MO is partially reversed by p53 co-KD. Student’s t-test, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Rescue of RpS15Aa KD-mediated heart tube loss in Drosophila by YAP/yorkie overexpression depending on its co-factor TEAD/scalloped.

(A) Representative images of RFP expressing fly hearts. RpS15Aa KD-mediated heart tube loss can be partially rescued by overexpression of yorkie (RpS15Aa RNAi + yorkie OE) . The rescue by yki OE depends on its co-factor sd. Flies were raised at 25 °C. (B) Quantification of events presented as a percentage of flies exhibiting whole heart tube versus partial heart loss (defined as 25-75% heart tube length compared to wildtype) or no heart tube. Statistics: Fisher’s exact test, *p<0.05. (C) Proposed signaling cascade underlying cardiac growth, proliferation and differentiation impairment following ribosomal stress (adapted from Baker et al., 2019).

RPS15A genetically interacts with cardiac transcription factors.

(A) The majority of cardiac transcription factors do not impact CM proliferation, except for e.g., HES4 and HOPX. (B) TBX5 genetically interacts with RPS15A and RPL39. siTBX5 does not impact CM proliferation at 0.5, 1, 1.5 or 2nM si-concentration, and neither do siRPS15A and siRPL39 alone at 0.5nM. CM proliferation is reduced in siRP backgrounds with increased titration of siTBX5. Two-way ANOVA for siTBX5 dosage, RP-knockdown and their interaction. (C) Representative fly heart segment (A4) from control flies, heterozygous mutants (tin346/+, pnrVX6/+, DocDf/-, Df(RpS15Aa) +/-) and transheterozygous mutants. tin+/- = tin346/+, pnr+/- = Df(pnr)/+ , Doc+/- = Df(DocA)/+. Note the deformation and myofibrillar disorganization in the transheterozygous mutants. (D) Quantification of adult Drosophila heart defects and genetic interaction. - Statistics: Fisher’s exact test on absolute numbers testing normal vs. severely deformed hearts. *p<0.05, **p<0.005, ***p<0.001.

RP-dependent cardiac-specific regulation of cell proliferation.

(A) Schematic illustrating approach to identify novel RP-dependent and cardiac-specific HLHS-associated gene network controlling CM proliferation. (B) Heatmap showing differential expression (hiPSC-CMs vs hiPSCs) of genes regulating CM proliferation. (C) Histogram showing effect of cardiac-specific and HLHS-associated genes on CM proliferation. (D) Visualization of RP-dependent and cardiac-specific HLHS-associated gene network (GeneMania). HLHS families. (E) Table of HLHS families harboring rare and damaging variant in RP-dependent and cardiac-specific genes. (F) Histogram showing lack of effect of siEWSR1 on OCT4+ hiPSCs. (G) Representative images of OCT4+ cells in siControl and siEWSR1. (H) Histogram showing that siEWSR1 increases the % of CDKN1A+ CMs as compared to siControl. (I) Histogram showing that siEWSR1 concomitantly decreases the % of EDU+ CMs as compared to siControl. (J) Representative images showing immunostaining for CDKN1A (white), EDU (green) and ACTN2 (Red) in siEWSR1 an siControl condition. (K) Pathway reconstruction of cardiac and RP-dependent regulation of CM proliferation by HLHS-associated genes. Chi-square test, *p<0.05, **p<0.005, ***p<0.001

Model for RP-dependent involvement in HLHS-associated phenotypes.

Schematic showing that combined prioritization and unbiased screening led to the identification of a novel HLHS-associated gene network regulating CM proliferation as potential disease-causing mechanism.

Variants in ribosomal genes in HLHS probands.

Abbrevations: MAF – minor allele frequency; TFBS – transcription factor binding site; n.t. – not tested; ASD – atrial septal defect; PLE - Protein-losing enteropathy; RV – right ventricle; RVEF – right ventricular ejection fraction; RVEDP - right ventricular end diastolic pressure

(A,B) Confirmatory siRNA screening for all RP genes confirms critical role for RPs in CM proliferation. (C, D) KD of RPs reduces proliferation in human fibroblasts.

Model organism phenotypes caused by loss of RP genes.

(A) Pedigree of the extended 75H family investigated for shared variants between HLHS proband (black square) and distant cousin with CHD (half-filled circle). (B) (C) Diastolic and (D) and Systolic Surface Area after rps15a CRISPR. Statistics: Mann- Whitney test, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001

rps15a zebrafish larval morphants show heart dysfunction and reduction in cardiomyocyte number.

(A) Lateral view (head to the right) of a wild-type control zebrafish larva (top) and a morphant (bottom) following injection of 2 ng/µl rps15a morpholino (MO). The morphant exhibits near normal body/tail but notable pericardial edema (arrowhead). (B) Control (left) and morphant hearts (right) stained with Tg(myl7:EGFP) and Tg(myl7:H2A-mCherry). Note the smaller heart with aberrant looping in morphant heart. (C) Total CM counts (mCherry+ cells) in control and rps15a morphants shown in (B). (D) Fractional area change (FAC) in control and rps15a morphants. (E-H) Injection of Control MO had little effect on zebrafish cardiac function. There was no significant difference between uninjected and injected controls with respect to (E) Systolic or (F) Diastolic Surface Areas, (G) Fractional Area Change or (H) R-R intervals. Student’s t-test, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Proliferation and heart function is regulated by RPS15A via TP53 in CMs and zebrafish.

(A,B) The number of siRPS15A-treated CMs is strongly reduced, which can be attenuated by co-KD of CDKN1A or TP53. (C,D) Loss of rps15a causes strong reduction in zebrafish heart contractility (measured by Fractional Area Change, C) and heart period (D), which can be rescued by co-KD of p53 in both, zebrafish heart atrium and ventricle.

Genetic interaction between RpS15Aa/RPS15A and cabeza/EWSR1

(A,B) Dose-dependent inhibition of hPSC-CMs proliferation by RPS15A KD. (C) Diastolic Diameter (DD), and Heart Period (HP) of 3-week-old female control and heterozygous RpS15Aa deficient flies with or without additional heterozygous mutation in tinman, pannier or Dorsocross. Statistics: Kruskal-Wallis, *p<0.05, **p<0.005, ***p<0.001, ****p<0.0001. Note, the prolonged HP in transheterozygous mutants for Df(RpS15Aa) and tinman. (D, E) FAC, and HP of the atria and ventricles at 72 hpf in zebrafish embryos injected with 0.5 ng rps15a and/or 1 ng nkx2.7 MOs (E) or 0.5 ng rps15a and/or 0.5 ng tbx5 MOs (F). Note, that FAC and heart periods show synergistic genetic interaction between rps15a and nkx2.7 and tbx5a. Statistics: 2-Way ANOVA, *p<0.05, **p<0.01, ****p<0.0001.(F) Systolic intervals (SI) of 1-week-old adult flies upon heart-specific knockdown of cabeza (caz32990or caz34839 RNAi) using Hand4.2-Gal4 driver with or without additional RpS15Aa heterozygosity (RpS15AaDf/+) and age-matched controls. Automated analysis of SI depicted as median with interquartile range obtained from 5s high-frame-rate videos. Statistics: Kruskal-Wallis test, *p<0.05, **p<0.01, ***p<0.001.