Deciphering the signals driving cardiomyocyte proliferation: advancing genuine cardiac regeneration

Abstract Background Acute myocardial infarction (MI) remains one of the leading causes of morbidity, with over 10 million new cases annually in Europe. Despite advances in reperfusion therapies and secondary prevention, the fundamental problem remains unsolved: adult human cardiomyocytes (CMs) lack meaningful regenerative capacity. Current standard-of-care therapies can slow disease progression, but they cannot replace lost myocardium or restore contractile function. This raises an urgent need for transformative therapies that address the root cause of myocardial dysfunction - irreversible loss of CMs. Remarkably, CMs in the infarct border zone show do show signs of dedifferentiation, evidenced by sarcomere disassembly, re-expression of fetal genes, and a metabolic shift toward glucose utilization. Purpose We hypothesize that combinatorial modulation of the Wnt, Akt–FoxO, and N-Cadherin (cell-cell contact inhibition) pathways using multiple small molecules will synergistically enhance proliferation of human stem cell-derived CMs (hiPSC-CMs). Prior studies from our groups and others suggest that single-pathway interventions can partially stimulate cell-cycle re-entry, but robust and stable CM renewal requires the simultaneous activation of fetal growth signals and disruption of contact-mediated inhibition. Methods We used our 2-dimensional (2D) 96-well screening platform, provided with beating day 12 hiPSC-CMs and (semi-)automated readouts for Trop T intensity, Ki67+ cells, pHH3+ cells and total nuclei. After identification of 4 compounds, acting all through different mechanisms, capable of boosting CM proliferation (Laduviglusib (CHIR99021), Insulin, AS1842856 and Exherin (ADH-1)), we used the platform for combinatory screening of these compounds and to identify potential synergies. Next, we used the ideal cocktail of compounds to grow large scale 3-dimensional (3D) cardiac organoids. Downstream characterizations were performed to confirm the expansion of phenotypical hiPSC-CMs. Results Our results revealed three synergistic regenerative pathways: the Wnt signaling cascade, the Insulin–Akt–FoxO axis, and N-cadherin–mediated cell–cell adhesion in promoting CM proliferation. Each of these pathways plays a distinct and complementary role in cardiac repair: Wnt modulation can reactivate developmental programs and stimulate CM proliferation; Akt–FoxO signaling can enhance cell survival, metabolic adaptation, and stress resistance; and N-cadherin engagement can restore structural coupling between cells, ensuring integration of newly formed CMs in 2D and 3D. Conclusion Prior studies from our groups and others suggest that single-pathway interventions can partially stimulate cell-cycle re-entry, but robust and stable CM renewal requires the simultaneous activation of fetal growth signals and disruption of contact-mediated inhibition, providing the basis for a therapy capable of driving functional heart regeneration.