Abstract Body: Background: RPL5 encodes a core component of the 60S ribosomal subunit required for ribosome biogenesis and translational control. Germline RPL5 variants cause Diamond–Blackfan anemia (DBA), a congenital marrow-failure syndrome frequently associated with congenital heart disease (CHD). Individuals with loss-of-function RPL5 variants have an increased incidence of septal and conotruncal defects, but the cardiomyocyte-intrinsic effects of RPL5 deficiency in humans remain poorly defined.
Methods: Genetic data from the Pediatric Cardiac Genomics Consortium and the Boston Children’s Hospital DBA cohort were queried for RPL5 variants. A human RPL5 knockout (KO) induced pluripotent stem cell (iPSC) line was generated using CRISPR/Cas9. Wild-type (WT) and RPL5-KO iPSCs were differentiated into iPSC-derived cardiomyocytes (iPSC-CMs). Cardiomyocytes were harvested at day 30 for single-nucleus RNA sequencing, with downstream analyses performed using a Seurat-based workflow.
Results: Five individuals with loss-of-function RPL5 variants were identified; all had CHD, and two had DBA. Single-nucleus transcriptomic profiling identified two major clusters demonstrating genotype-dependent differences in cellular composition. A WT-enriched cluster characterized by activation of cardiac and circulatory system development, muscle tissue development and contraction, and growth factor–mediated signaling comprised a larger fraction of WT cells compared with RPL5-KO cells (59.8% vs. 22.9%). In contrast, RPL5-KO cells were enriched in a cluster marked by coordinated downregulation of cardiac and vascular functional pathways, including muscle contraction, circulatory system processes, and regulation of transport and secretion (WT 19.5% vs. RPL5-KO 57.1%), alongside relative enrichment of early developmental, neurodevelopmental, and morphogenetic transcriptional programs. These findings indicate disruption of cardiomyocyte developmental progression with transcriptional arrest at earlier lineage stages rather than lineage conversion.
Conclusions: Loss of RPL5 impairs human cardiomyocyte development by disrupting progression through cardiac lineage states and attenuating activation of gene programs required for functional maturation. This human RPL5 knockout model provides mechanistic insight into how ribosomal dysfunction perturbs cardiac developmental trajectories and establishes a platform to study ribosome biology in human cardiogenesis and congenital heart disease.