Abstract Body:
Introduction: Hypoplastic left heart syndrome (HLHS) is a rare congenital defect marked by the underdevelopment of the left-sided structures in the heart. Currently, available animal models mimicking left ventricular hypoplasia are constrained because they often fail to reproduce the full spectrum of structural and hemodynamic abnormalities. In this study, we aim to employ a self-organizing cardioid model derived from patient-specific induced pluripotent stem cells to investigate the molecular and cellular mechanisms that underlie ventricular malformation in HLHS.
Approaches: We generated cardioids from induced pluripotent stem cell (iPSC) lines derived from three pairs of HLHS probands and their sex-matched unaffected family controls. We measured the overall size and recorded the onset of cavity formation in cardioids from both HLHS patients and healthy controls between day 2 and day 8. Cardioids collected at days 2, 5, 8, 10, 14, and 20 were subjected to bulk RNA sequencing. Additionally, spatial profiling of cryo-sectioned and embedded day 20 cardioids was performed using Visium spatial transcriptomics.
Results: Cardioids derived from HLHS patients displayed a smaller overall size accompanied by a slower growth rate compared to healthy controls. HLHS patient-specific cardioids show delayed formation of chamber-like cavity than gender-matched family controls. Time-course transcriptomic profiling of cardioids confirms efficient cardiomyocyte differentiation, as demonstrated by abundant expression of cardiac lineage marker genes. Bulk RNA sequencing of day 2 cardioids reveals significant downregulation of NFATC4, a gene essential for cardiomyocyte proliferation, and FHL1, which is typically upregulated in patients with hypertrophic cardiomyopathy. Further analysis of day 10 cardioids shows notable downregulation in pathways associated with cardiac muscle contraction, cardiac chamber development, and ventricular cardiac muscle tissue development. Spatial transcriptomics performed on day 20 cardioids, along with supporting immunofluorescence data, demonstrate the presence of myocardial and endocardial layers in both HLHS patients and healthy controls. Enrichment analysis of biological pathways indicates that myocardial layer in HLHS-derived cardioids is less proliferative compared to healthy controls.
Conclusions: HLHS patient-specific cardioids provide a unique in vitro model to dissect the cellular etiologies of HLHS in a 3D context.