Abstract Body: Background:
Hypoplastic left heart syndrome (HLHS) is a severe congenital heart defect (CHD) marked by the underdevelopment of the left heart. Despite the prevalent theories of genetic and hemodynamic perturbations being the potential causes, molecular mechanisms underlying the HLHS pathogenesis remain obscure. Recently, we reported a human induced pluripotent stem cell (hiPSC)-based 3D bioprinted embryonic heart tube (eHT) model that shows recapitulation of robust cardiac function and cardiogenic cellular activities upon hemodynamic flow initiation.

Methods:
To delineate the individual and synergistic effects of genetic and hemodynamic perturbations in HLHS, we generated wildtype (WT) vs. HLHS cardiomyocytes (CMs) and endocardial cells (ECs) from hiPSCs via WNT and BMP10 signaling modulation. 2D monocultures and 3D eHT perfusion cocultures were conducted and assessed for HLHS-associated gene expression, contractile function, and cellular structure and interactions using qPCR, video-based contractility analysis, single cell RNA-sequencing, and immunohistochemistry. Computational simulation was also performed to characterize the hemodynamic-contractile coupling under varying contractility and flow conditions. Further, integrated perfusion-electrical conditioning was applied chronically to WT vs. HLHS eHTs, modeling HLHS phenotype induction in response to developmental cardiac loading and electrophysiological changes.

Results:
Transcriptomic and immunohistochemistry analyses in 2D monocultures revealed intrinsic proliferation defects in CMs and novel endocardial defects in ECs derived from HLHS hiPSCs. In 3D, both WT and HLHS eHTs demonstrated long-term viability and cardiac function under flow perfusion, while the additional electrical conditioning resulted in the enhancement of contractile adaptation over time. Preliminary data from flow perfusion also suggested an effect of hemodynamic perturbations on developmental EC gene expression.

Conclusion:
This study highlights the causal and exacerbatory interactions of intrinsic genetics and extrinsic hemodynamic perturbations in HLHS pathogenesis in a 3D bioprinted eHT model, suggesting novel therapeutic targets.