Exploring the interactions between cardiomyocytes and endothelial cells to induce cardiac revascularization and regeneration

None. The adult heart is unable to regenerate after ischemic injury, primarily due to the incapacity of cardiomyocyte (CM) to re-enter in the cell cycle and endothelial cells (EC) to form new vessels. Interestingly, our recent data have unveiled that the decline of CM proliferation after birth is paralleled by a reduction in angiogenic potential. Nevertheless, the precise mechanisms governing angiogenesis inhibition in the adult heart remain elusive. We hypothesize that the terminal differentiation of CMs after birth leads to the paracrine suppression of EC proliferation. This study aims to identify membrane or secreted factors produced by adult CMs that inhibit EC proliferation and vessel formation in order to create new therapeutic strategies to induce cardiac revascularization and regeneration. To validate our hypothesis, ECs were co-cultured with CMs at distinct differentiation stages. Leveraging existing RNA sequencing datasets, we generated an interactome between ligands produced by CMs and receptors expressed by cardiac ECs. Through gain and loss of function studies, we identified pivotal anti-angiogenic factors and subsequently knocked-out the corresponding genes in Cas9 mice, with the aim of rescue the angiogenic potential of the adult heart. Concurrently, we assessed the ability of miR199a, a pro-proliferative miRNA known for inducing CM de-differentiation, to elicit EC proliferation and angiogenesis when delivered to the adult heart together with VEGF-A, utilizing adeno-associated viral vectors. Our findings revealed that adult CMs impaired EC proliferation. Comprehensive analysis identified 20 membrane and secreted factors, establishing 118 ligand-receptor interactions between CMs and ECs. Notably, KCNJ11 and CD74 emerged as key factors, as validated through gain and loss of function experiments. In vivo knockout of these genes in CMs led to significant increase in EC proliferation and the formation of neo-vessels. Furthermore, CM de-differentiation induced by miR199a demonstrated the capacity to promote EC proliferation and neo-vessel formation upon VEGF-A stimulation. Collectively, this work demonstrated that fully mature CMs have a role in limiting angiogenic potential of the adult heart, likely explaining the failure of therapeutic angiogenesis approaches attempted so far. Our results suggest that innovative strategies targeting CM-EC cross-talk or inducing partial CM de-differentiation may open new therapeutic avenues in the field of cardiac regeneration.Adult CMs inhibit EC proliferationCMdedifferentiation rescues angiogenesis