Background: Hypoplastic left heart syndrome (HLHS) is a severe congenital heart defect characterised by underdevelopment of left-sided cardiac structures. While genetic predisposition contributes to HLHS, the relevance of environmental stressors is increasingly recognised, yet the cellular mechanisms linking genetic susceptibility to environmental vulnerability remain unclear. We aimed to identify molecular and functional differences between cardiomyocytes derived from HLHS patients and healthy controls to uncover potential susceptibilities contributing to the HLHS phenotype.

Methods: Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from HLHS patients and healthy controls were used to examine intrinsic cellular differences. Single-cell RNA sequencing compared baseline transcriptional profiles. Functional assays assessed responses to endothelin-1 (ET-1)-induced stress, cyclic mechanical stretch, and basal or mitogen-stimulated proliferation. These approaches were used to identify intrinsic functional impairments and altered stress responses in HLHS cardiomyocytes.

Results: Single-cell transcriptomics revealed downregulation of gene networks associated with cardiac stress responses, metabolic resilience, and rhythm regulation in HLHS cardiomyocytes. Regulon analysis revealed broad reductions in transcription factor activity across key cardiac regulatory networks. Functionally, HLHS cardiomyocytes showed heightened vulnerability to ET-1, with exaggerated proBNP induction compared with controls. No significant differences were observed following cyclic mechanical stretch. Basal proliferation varied across HLHS lines, while mitogen-induced proliferation remained comparable to controls.

Conclusions: These findings support a model in which intrinsic molecular and functional vulnerabilities in HLHS cardiomyocytes might reduce resilience to developmental stressors. Such gene-environment interactions may contribute to HLHS pathogenesis, underscoring the interplay between genetic predisposition and environmental influences in congenital heart disease.

© 2026. The Author(s).