Zeb2os Hinders Cardiac Healing by Suppressing ZEB2 Reactivation and Cardiomyocyte Dedifferentiation

Background: Long noncoding RNAs (lncRNAs) have emerged as critical regulators in cardiovascular biology, influencing cardiac development, remodeling, and regeneration. Zeb2os, a natural antisense transcript (NATs) of the Zeb2 gene, has been linked to these processes in various organs. While ZEB2 promotes cardiac repair, the role of Zeb2os in these processes remains unclear. This study investigates the role of Zeb2os in modulating ZEB2 expression and cardiac remodelling after ischemic injury. Methods: We used adeno-associated virus (AAV9) vectors to overexpress Zeb2os in mouse models of cardiac IR injury. RNA sequencing, immunofluorescence, and high-resolution respirometry were employed to evaluate the effects of Zeb2os delivery on gene expression, ZEB2 reactivation, cardiomyocyte phenotype, scar composition and mitochondrial function. Experiments in cultured cardiomyocytes under hypoxia further explored the regulatory dynamics between Zeb2os and Zeb2. Results: We identified Zeb2os as a hypoxia-responsive lncRNA that displays an inverse and oscillatory expression pattern with Zeb2 in both in vitro and in vivo models of cardiac injury. Functional experiments revealed that Zeb2os negatively regulates ZEB2 expression, impairing the cardiomyocyte dedifferentiation and metabolic remodelling necessary for effective repair. AAV9-mediated delivery of Zeb2os resulted in preserved sarcomere structure, altered scar composition, reduced expression of regenerative genes, and diminished cardiac function following injury. In contrast, silencing of Zeb2os increased ZEB2 protein expression, suggesting a potential therapeutic strategy to enhance repair. Mechanistically, modulation of Zeb2os levels inversely regulated ZEB2 protein expression, whereas ZEB2 modulation had no effect on Zeb2os levels, indicating a unidirectional regulatory axis between the two transcripts. Conclusions: Our findings identify Zeb2os as a stress-responsive inhibitor of ZEB2 reactivation that limits cardiomyocyte plasticity and hinders repair following ischemic injury. Given its specific activity under ischemic conditions, targeting os may represent a novel therapeutic strategy to enhance endogenous cardiac regeneration