Majid, Q. A. | Search bioRxiv

bioRxiv2026

Patient-Derived hiPSC-Cardiomyocytes and Engineered Heart Tissues Reveal Distinct Functional Phenotypes in Inherited Cardiomyopathies

Background Hypertrophic and dilated cardiomyopathies (HCM and DCM) are the most common inherited cardiomyopathies. However, genotype-specific molecular and functional cardiomyocyte phenotypes and responses to neurohormonal stimulation remain incompletely understood. Here, we investigated whether patient-derived HCM and DCM cardiomyocytes exhibit distinct baseline phenotypes or differential responses to hypertrophic stimulation and pharmacological treatment. Methods Three human-induced pluripotent stem cell (hiPSC) lines were used: a control line, an HCM patient-derived line carrying a MYBPC3 mutation, and a DCM patient-derived line carrying an LMNA mutation. The cells were differentiated into hiPSC-cardiomyocytes, which were exposed to endothelin-1 and the GATA4-targeted compound 3i-1262, followed by transcriptional and protein expression analyses. In addition, engineered heart tissues (EHTs) were generated and cultured for 40 days, with endothelin-1 and 3i-1262 treatment applied during the final 20 days. Lastly, {beta}-adrenergic stimulation with isoprenaline was performed. EHT contractile function was quantified using MUSCLEMOTION. Results Patient-derived hiPSC-cardiomyocytes exhibited genotype-dependent responses to endothelin-1 at the transcriptional and protein levels. DCM-cardiomyocytes failed to maintain structural integrity in the EHTs, resulting in tissue fracture or cessation of beating. Functional analyses demonstrated distinct baseline contractile properties between control and cardiomyopathy EHTs, as well as differential responses to endothelin-1 and isoprenaline. Conclusions Patient-derived hiPSC-cardiomyocytes exhibit genotype-specific molecular and functional phenotypes. EHTs generated from HCM hiPSC-derived cardiomyocytes showed a progressive decline in apparent force, whereas DCM EHTs fractured over time, suggesting mutation-associated phenotypes in 3D cardiac tissue models. These findings highlight the utility of hiPSC-based cardiac models for investigating molecular and functional disease mechanisms and pharmacological responses in inherited cardiomyopathies. Keywords: hypertrophic cardiomyopathy, dilated cardiomyopathy, engineered heart tissues, hiPSC-derived cardiomyocytes, GATA4

pharmacology and toxicology↗

bioRxiv2025

The novel SCN5A-P1891A mutation is associated with left ventricular hypertrabeculation and links Nav1.5 to cardiomyocyte proliferation and disrupted 3D cardiac tissue formation

BackgroundLeft ventricular hypertrabeculation (LVHT) is a heterogenous cardiac condition with a complex and poorly understood aetiology. We comprehensively characterised the effect of a novel P1891A mutation in the SCN5A gene, which encodes the voltage-gated sodium channel Nav1.5, identified in a Finnish family diagnosed with LVHT. MethodsWe generated SCN5A-P1891A mutation-carrying human induced pluripotent stem cell-derived cardiomyocytes (P1891A-hiPSC-CMs) and performed electrophysiological assessments, including patch-clamp studies, and fluorescent calcium imaging, to determine the mutations effect on hiPSC-CM electrophysiology. We also evaluated the impact of the mutation on the proliferative capacity in response to mitogenic stimuli and on the hypertrophic response following cyclic mechanical stretch or endothelin-1 treatment. Further, we assessed the effect on contractile parameters in three-dimensional (3D) contractile hydrogels (engineered heart tissues, EHTs) and conducted advanced proteomics to understand the consequences of the mutation on Nav1.5 protein-protein interactions. ResultsThe SCN5A-P1891A mutation reduced the sodium current densities and increased both the sodium window current and arrhythmogenicity; however, action potential parameters were unaffected. Advanced proteomics characterised, for the first time, the complete Nav1.5 interactome and revealed that the SCN5A-P1891A mutation negated interactions with fibroblast growth factor 12 (FGF12) and FGF13, that are known to modulate sodium channel activity. Baseline proliferation was unchanged, although aged P1891A-hiPSC-CMs demonstrated enhanced proliferative capacity following mitogenic stimulation. Further, P1891A-hiPSC-CMs exhibited a heightened stress response upon mechanical stretch, resulting in the upregulation of heart failure-associated genes. Strikingly, EHTs derived from P1891A-hiPSC-CMs yielded disparate phenotypes. Whilst the majority condensed only partially and failed to beat synchronously, a small subset condensed fully yet exhibited weak contractile properties, alongside age-associated functional decline. In contrast, EHTs derived from healthy control hiPSC-CMs consistently condensed fully and demonstrated a positive correlation between post-fabrication age and contractile properties ConclusionsOur study presents a unique aetiology of LVHT and reveals a novel association between SCN5A mutations and enhanced human cardiomyocyte proliferation. Further, the inability of P1891A-hiPSC-CMs to consistently form fully condensed 3D cardiac tissues may be linked to their abnormal response to mechanical stretch and provides a powerful 3D model for future mechanistic research and drug development studies to better understand and treat LVHT.

molecular biology↗