Abstract Thu029: Neutrophils Promote Cardiomyocyte Proliferation and Heart Regeneration by Regulating Macrophage Polarization and Angiogenesis

Background: Irreversible cardiomyocyte loss is a key driver of heart failure. Enhancing cardiomyocyte proliferation and cardiac regeneration is a promising therapeutic strategy. While inflammation is well-recognized in cardiac regeneration, particularly via macrophages and regulatory T (Treg) cells, the role of neutrophils remains unclear. Methods: Neonatal mice were treated with anti-Ly6G antibodies for neutrophil depletion, while controls received isotype antibodies. A myocardial infarction (MI) model was established in 2-day-old mice. Cardiac regeneration and fibrosis were assessed via Masson’s staining at 28 days post-MI, and cardiac function was evaluated by echocardiography. Flow cytometry at 6 hours, 1 day, and 3 days post-MI analyzed immune cell populations. RT-qPCR assessed M1/M2 macrophage markers and cytokines. Cardiomyocyte proliferation was evaluated via immunofluorescence at days 4 and 7, and RT-qPCR quantified cell cycle genes and angiogenesis-related genes. Results: Neutrophil depletion impaired cardiac regeneration, increased fibrosis, and reduced contractile function. Total macrophage numbers remained unchanged; however, Ly6C high macrophages decreased while Ly6C low macrophages increased early post-MI. RT-qPCR revealed a downregulation of M1 macrophage markers and pro-inflammatory cytokines, accompanied by an upregulation of M2 markers and anti-inflammatory cytokines, indicating that neutrophils orchestrate early macrophage polarization. Neutrophil depletion also reduced cardiomyocyte proliferation, cell cycle gene expression, and angiogenesis-related gene expression. Conclusions: Neutrophils are critical for cardiomyocyte proliferation and cardiac regeneration, primarily by shaping early macrophage polarization. They promote M1 macrophages and cytokine secretion (IL-6, OSM) to enhance cardiomyocyte proliferation. Additionally, neutrophils facilitate cardiac repair by promoting angiogenesis. These findings establish neutrophils as key regulators of neonatal heart regeneration.