Verma, R. S.; Zhang, I.; Chen, X.; Pierce, M. S.; Kumar, S. R.; McCain, M. L.

Background: Hypoplastic Left Heart Syndrome (HLHS) is a severe congenital heart defect characterized by underdevelopment of left-sided cardiac structures, requiring staged surgical palliation that establishes the right ventricle as the single ventricle maintaining systemic circulation. After surgery, patients remain highly susceptible to heart failure, possibly due to the increased hemodynamic load imposed on the single surviving ventricle. Because the etiology of HLHS remains poorly understood, elucidating the mechanisms that drive ventricular dysfunction requires model systems that capture both patient-specific genetics and the dynamic mechanical environment of the ventricle. Thus, our objective was to compare how induced pluripotent stem cell (iPSC)-derived cardiomyocytes (iPSC-CMs) from patients with HLHS differ from control in static culture and in response to cyclic stretch. Methods: Control iPSCs from two unaffected donors and iPSCs from four HLHS patients were differentiated into iPSC-CMs, engineered into aligned tissues, and cultured statically or exposed to cyclic 20% uniaxial stretch at 1 Hz for 48h. Sarcomere organization was quantified in static tissues using images of immunostained -actinin. Calcium transients at 1 Hz pacing frequency in static and stretched tissues were evaluated using Fluo-4 calcium indicator dye. Bulk RNA sequencing was performed to evaluate stretch-induced transcriptomic changes in control and HLHS iPSC-CMs. Results: In static culture, HLHS iPSC-CMs had modest defects in sarcomere organization and calcium transient morphology compared to control. In response to cyclic stretch, control iPSC-CMs exhibited calcium transients with quicker rise time, a response observed in only one of four lines of HLHS iPSC-CMs. Transcriptomic analysis revealed that all four lines of HLHS iPSC-CMs failed to upregulate calcium handling and sarcomere-associated genes in response to stretch, unlike control iPSC-CMs. Stretch also induced HLHS iPSC-CMs to exhibit pronounced activation of inflammatory signaling pathways, a response not observed in control iPSC-CMs. Conclusions: iPSC-CMs from HLHS patients demonstrate aberrant responses to cyclic stretch, including blunted adaptation of calcium transients and heightened inflammatory signaling. These findings implicate defective mechanoadaptation as a potential contributor to single ventricle failure and identify potential candidate biomarkers and new opportunities for therapeutic interventions for HLHS patients.