Abstract Body: Introduction: Hypoplastic right heart syndrome (HRHS) is a rare congenital heart defect characterized by the underdevelopment and malformation of right-sided cardiac structures. Due to the intrinsic complexity and oligogenic nature of HRHS pathogenesis, suitable animal models that faithfully recapitulate right ventricular hypoplasia remain limited. Patient iPSC-derived cardiac organoids (cardioids) serve as a promising platform to examine the molecular mechanisms underlying HRHS.
Hypothesis: We hypothesize that three-dimensional patient-specific iPSC-derived cardioids can recapitulate cardiac differentiation defects observed in HRHS patients.
Approaches: Cardioids were generated from iPSC lines derived from three HRHS probands and sex-matched unaffected family controls using a protocol that recapitulates key stages of cardiac development. Cardioids were collected at day (D)3, D5, D7, and D14, representing early patterning, late patterning, cardiac specification, and early maturation stages. Samples were subjected to single-nucleus RNA-seq and single-cell ATAC-seq. Differential gene expression, pathway enrichment, trajectory inference, and gene regulatory network analysis were performed to characterize molecular changes during cardioid development.
Results: Cardiac mesoderm (D3), early cardiac progenitor (D5), and cycling cardiac progenitor (D7) populations were identified during cardioid development. In these populations, pathways related to muscle cell differentiation, cardiac chamber formation, and ventricular development were downregulated in HRHS cardioids. Integration of trajectory inference and gene regulatory network analysis identified key transcription factors underlying disrupted cardiac differentiation. Regulatory relationships were inferred by integrating multiomics data to connect transcription factors with enhancer elements and downstream target genes. FOXC2 repressing regulatory activity showed the largest decrease at D3, which indicates early disruption during mesoderm specification. Meanwhile, MEF2C repressing regulatory activity declined from D3 to D5 and may contribute to downstream specification defects. During cardiac specification and early maturation (D7–D14), HRHS cardioids failed to adequately upregulate NKX2-5, HAND1, HAND2, and SRF regulatory activity.
Conclusions: Together, these findings indicate that early disruption of transcription factor-mediated regulatory networks drive defective cardiac lineage specification in HRHS.