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

Heart failure, aprogressive clinical syndrome and major global health challenge, often developsfrom pre-existing ischaemic heart disease (IHD), where coronary arterynarrowing leads to cardiomyocyte (CM) loss and myocardial damage. Currenttherapies cannot prevent CM death or promote cardiac regeneration, highlightingthe need for pro-regenerative strategies. Recent studies indicate that adultCMs can dedifferentiate toward a foetal-like state, reactivating the heart’sintrinsic regenerative potential. The transcription factor ZEB2 is a keyregulator 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 translationalpotential, we tested AAV-ZEB2 in living myocardial slices (LMS) from porcineand human hearts, observing enhanced angiogenesis and activation ofdedifferentiation programs, consistent with murine findings. We also investigatedZeb2 opposite strand (Zeb2os), a natural antisense transcript involvedin the regulation of Zeb2 expression. Zeb2os ishypoxia-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 selectivelyenhances ZEB2 under hypoxic conditions. In vitro, Zeb2ossilencing robustly increases ZEB2, suggesting a complementary therapeuticapproach to AAV delivery. In parallel, we developed an improved single-nucleusRNA sequencing (snRNA-seq) method to dissect disease-related changes andevaluate cell-specific therapeutic effects. Altogether, the data presented inthis thesis establish ZEB2 as a promising target for cardiac regeneration andprovide tools to advance preclinical research toward clinical application.