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Mechanical insights into hypertrophic cardiomyopathy (HCM) associated troponin-t variants using human induced pluripotent stem cell derived cardiomyocytes

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) provide a powerful platform for modeling cardiac physiology and disease, yet their inherent immaturity compared to adult cardiomyocytes presents a major challenge. This study investigates the impact of substrate stiffness and micropatterning in promoting hiPSC-CM maturation and their potential for disease modeling. We optimized physiologically relevant micropatterned substrates with controlled stiffness (2–5 kPa and 30–50 kPa) and examined their effects on hiPSC-CM contractility, calcium transient kinetics, sarcomere organization, and metabolic maturation over extended culture periods. Our results demonstrate that hiPSC-CMs cultured on soft micropatterned substrates exhibit the highest contractility and fastest calcium transient kinetics. A proteomic analysis further revealed upregulation of key structural, electrophysiological, and metabolic proteins. Additionally, cardiac remodeling was evident in hiPSC-CMs cultured on stiff patterned substrates. In parallel, we leveraged these engineered substrates to investigate the pathogenicity of hypertrophic cardiomyopathy (HCM)-causing TNNT2 variants under physiological (soft) and fibrotic (stiff) conditions. Functional assays revealed early hypercontractility and prolonged calcium transients in I79N^+/- hiPSC-CMs on soft patterned substrates. While increased stiffness initially mitigated hypercontractility, prolonged culture on stiff substrates exacerbated pathological remodeling, as evidenced by fetal gene reprogramming and maladaptive protein expression changes. Since this model accurately represents the pathogenicity of the I79N^+/- variant, we further assessed the functional mechanisms of two other TNNT2 variants associated with HCM, R278C^+/- and R92Q^+/-. Results demonstrated severe hypercontractility in both I79N^+/- and R92Q^+/- variants, while R278C^+/- exhibited a milder phenotype, aligning with clinical findings. Finally, we evaluated the efficacy of mavacamten in treating HCM caused by variants in TNNT2, which effectively reduced hypercontractility, highlighting its therapeutic potential. Collectively, our findings underscore the critical role of mechanical cues in regulating hiPSC-CM maturation and disease pathogenesis. Engineered micropatterned substrates provide a robust platform for studying mechanotransduction, optimizing cardiac tissue models, and enabling drug screening, paving the way for more physiologically relevant in vitro models of cardiac health and disease.

Keywords. hiPSC-CMs, maturation, hypertrophic cardiomyopathy (HCM), mechanotransduction, mavacamten.