Biomechanical stress unmasks a fibroblast-dependent hypercontractile-disarray phenotype in MYBPC3 truncation HCM.

Hypertrophic cardiomyopathy (HCM) is commonly caused by pathogenic variants in the sarcomere, such as truncations in myosin-binding protein C (MYBPC3), yet clinical severity and adverse outcomes correlate poorly with genotype alone and are strongly influenced by fibrosis. We developed an engineered human micro-heart tissue (μHT) combining iPSC-derived cardiomyocytes (CM) with defined primary cardiac fibroblast (cFB) fractions and mechanically modulated afterload. This design allowed us to test how stromal context and pro-fibrotic signaling shape HCM pathogenesis in micro-engineered heart tissues made from iPSCs harboring an HCM-linked frameshift variant in MYBPC3 (MYBPC3fs). Under low afterload, genotype-associated differences in total active force were limited at 0% and 5% added cFB but became apparent at 25% added cFB. However, high afterload markedly amplified MYBPC3fs μHT hypercontractility, with tissues generating greater total active force at both 5% and 25% added cFB. We also observed afterload- and cFB-dependent remodeling of Ca2+ handling, indicating that excitation-contraction coupling in MYBPC3fs tissues is modulated by combinatorial actions of mechanical stress and the stromal environment. Despite heightened contractile output, the MYBPC3fs μHT exhibited blunted improvements in Z-disc alignment with increasing cFB content, whereas cardiomyocytes within the isogenic control μHT showed progressive structural ordering as cFB density increased. Finally, the MYBPC3fs μHT exhibited a greater response to TGF-β, leading to exaggerated increases in tissue-level resting tension and α-smooth muscle actin expression. Our findings provide important insights into the pathophysiologic pathways leading to adverse outcomes in clinical HCM and a foundation for future studies of cardiomyocyte-fibroblast interactions in HCM.