Exploring cardiomyocyte plasticity & regeneration A multi-species approach to cardiac repair

Heart failure, a progressive clinical syndrome and major global health challenge, often develops from pre-existing ischaemic heart disease (IHD), where coronary artery narrowing leads to cardiomyocyte (CM) loss and myocardial damage. Current therapies cannot prevent CM death or promote cardiac regeneration, highlighting the need for pro-regenerative strategies. Recent studies indicate that adult CMs can dedifferentiate toward a foetal-like state, reactivating the heart’s intrinsic regenerative potential. The transcription factor ZEB2 is a key regulator of CM dedifferentiation and repair, transiently reactivated (in mice) after ischaemic injury. Prolonged ZEB2 reactivation via adeno-associated virus (AAV)-mediated overexpression improves CM survival, stimulates angiogenesis, limits fibrosis, and enhances cardiac function. To assess translational potential, we tested AAV-ZEB2 in living myocardial slices (LMS) from porcine and human hearts, observing enhanced angiogenesis and activation of dedifferentiation programs, consistent with murine findings. We also investigated Zeb2 opposite strand (Zeb2os), a natural antisense transcript involved in the regulation of Zeb2 expression. Zeb2os is hypoxia-responsive and shows an inverse, oscillatory expression relative to Zeb2. Our results demonstrate that Zeb2os suppresses ZEB2 reactivation, limiting CM dedifferentiation and regeneration, and its inhibition selectively enhances ZEB2 under hypoxic conditions. In vitro, Zeb2os silencing robustly increases ZEB2, suggesting a complementary therapeutic approach to AAV delivery. In parallel, we developed an improved single-nucleus RNA sequencing (snRNA-seq) method to dissect disease-related changes and evaluate cell-specific therapeutic effects. Altogether, the data presented in this thesis establish ZEB2 as a promising target for cardiac regeneration and provide tools to advance preclinical research toward clinical application.