Abstract Body: Background: Recent studies demonstrate that promoting cell cycle reentry in adult cardiomyocytes (CM) enhances cardiac rejuvenation by influencing paracrine signaling. We previously demonstrated that the inhibition of two cell cycle inhibitors, Retinoblastoma 1 (Rb1) and Meis homeobox 2 (Meis2), in the adult CM enhances angiogenesis and cardiac function following ischemic injury, but the underlying mechanisms have yet to be elucidated. The goal of this study is to determine the mechanisms by which inhibition of senescence pathways promotes cardiac rejuvenation and cardioprotection.
Methods: Myocardial infarction (MI) was induced in adult C57/BL6 mice via permanent left anterior descending (LAD) artery occlusion, followed by direct intramyocardial injection of a siCocktail (siRb1 + siMeis2) or control. Cardioprotection was assessed by evaluating heart function, infarct size, vascular density, and fibrosis. Further, RNA sequencing was performed on isolated adult CMs after Rb1 and Meis2 knockdown. MCM2 overexpression was achieved through intramyocardial injection of an MCM2-expressing plasmid, and cardioprotection was evaluated as described above. The mechanisms underlying MCM2-mediated cardioprotection were further explored by identifying MCM2-interacting proteins, as well as performing transcriptomic and proteomic analyses of MCM2-overexpressing CMs and their extracellular vesicles (EVs).
Results: We observed a significant reduction in infarct size and improved cardiac function following the knockdown of Rb1 and Meis2 in the adult heart after MI. Transcriptomics analysis identified MCM2 as a key mediator of the cardioprotective effects associated with Rb1 and Meis2 inhibition. Notably, re-expression of MCM2 in the adult heart post-MI reduced scar formation, attenuated fibrosis, and enhanced vascular density. Proteomic analysis of MCM2 interacting proteins revealed that MCM2 directly interacts with proteins with a known role in cardioprotection. Additionally, transcriptomic profiling showed that MCM2 overexpression modulated critical cardioprotective pathways, including angiogenesis, fibrosis, and CM survival. Proteomic analysis of EVs from MCM2-overexpressing CMs further emphasizes MCM2’s role in mediating pro-angiogenic, antifibrotic, and wound healing signaling.
Conclusion: Collectively, these findings suggest that re-expression of MCM2 promotes cardiac rejuvenation associated paracrine signaling, and cardioprotection after MI in the adult heart.