hiPSC-derived models for genetically determined cardiomyopathies and heart regeneration mechanisms

Dilated cardiomyopathy (DCM) is a leading cause of heart failure, sudden death, and cardiac transplantation. Truncating variants in the titin gene (TTNtv) represent the most common genetic cause of familial DCM, yet how these mutations translate into cardiac dysfunction at the cellular and molecular level remains not completely understood. In this work, thanks to a collaborative network endowing the Cardiothoracic Unit at the Cattinara Hospital in Trieste, we established patient-specific induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from four TTNtv carriers, harboring mutations in the A-band region of the protein, and one healthy donor. Biochemical characterization by western blot confirmed reduced titin protein levels, while gene expression analysis revealed reduced wild-type titin levels, with unchanged N-terminal transcripts and a significant decrease in the C-terminal region. TTNtv-hiPSC-CMs displayed sarcomeric disarray and impaired proliferative capacity. We then proceeed to set up Engineered Heart Tissues in vitro culture, in order to deeply characterize the cell lines in a tridimensional asset, better resembling the in vivo conditions. When assembled into three-dimensional engineered heart tissues (EHTs), TTNtv-derived CMs exhibited increased contractile force and beating frequency, alongside decreased proliferative capacity. To explore the translational potential of our model, we tested the efficacy of several compounds routinely used in DCM treatment, including next-generation myosin modulators (mavacamten, omecamtiv mecarbil, danicamtiv) and standard-of-care drugs (bisoprolol, ramipril), evaluating their effects on sarcomere organization, contractility, and diastolic function on TTNtv-derived EHTs. While omecamtiv mecarbil produced comparable effects in both TTNtv and control tissues, Mavacamten markedly restored the physiological contractility parameters in patient-derived cells, demonstrating that sarcomeric disruption selectively alters the sensitivity to myosin inhibition. These findings establish TTNtv-derived EHTs as a valuable platform for patient-specific drug testing in titin-related cardiomyopathy. TTNtv cardiomyocytes displayed markedly impaired proliferation, prompting us to investigate the underlying signaling defects. We employed AAV6-based fluorescent reporters to track the activity of Notch and Hippo/YAP pathways, both known regulators of cardiomyocyte proliferation. Control hiPSC-CMs showed robust activation of both pathways, whereas TTNtv cells exhibited substantially reduced signaling. Co-immunoprecipitation experiments from nuclear fractions revealed a physical interaction between Notch1 intracellular domain and YAP, with reciprocal binding of YAP to RBP-JK and Notch1-ICD to TEAD1. Chromatin immunoprecipitation confirmed that Notch1 was enriched on canonical YAP target promoters, and YAP on canonical Notch targets, while TEAD1 appeared ubiquitously present. These findings point to a direct crosstalk between the two pathways at the chromatin level. To test whether restoring this signaling axis could promote cardiomyocyte proliferation, we overexpressed both Notch1-ICD and constitutively active YAP in adult mouse hearts. Concomitant activation of the two pathways led to a marked increase in cardiomyocyte proliferation and a dramatic thickening of the ventricular wall, but also triggered pathological hypertrophy and proved lethal within two weeks. This work combines patient-derived iPSC models, three-dimensional engineered tissues, and pathway-specific reporters to dissect the molecular consequences of titin truncations. Our data demonstrate that TTNtv mutations impair both sarcomeric integrity and proliferative signaling, and identify a Notch-YAP axis as a key regulator of cardiomyocyte proliferation with potential implications for cardiac regeneration. The differential pharmacological response observed in patient tissues suggests that engineered heart models may serve