Small-molecule cocktail 5SM induces heart regeneration by upregulating TGFβ/BMP signaling

Zebrafish and neonatal mammals possess a remarkable capacity for cardiac regeneration following injury, a property that is largely absent in adult mammals. We have recently identified a cocktail of five small molecules (5SM) that promote adult cardiomyocyte (CM) proliferation and heart regeneration. However, the underlying mechanisms through which 5SM induces CM proliferation remain incompletely understood. In this study, we demonstrated an essential role for TGFβ/BMP signaling in mediating 5SM-induced heart regeneration. Harmine, one component of 5SM, plays a dominant role in upregulating TGFβ/BMP signaling by inhibiting DYRK1B. Inhibition of DYRK1B releases PRKACA, which then activates CREB phosphorylation, subsequently upregulating the expression of Tgfβ2, Tgfβ3, Bmp2, and Bmp7 in CMs. Treatment with exogenous TGFβ2, TGFβ3, BMP2, or BMP7 enabled cultured CMs to re-enter the cell cycle, while pharmacological inhibition of either the TGFβ or BMP pathways markedly diminished the effect of 5SM on CM proliferation in vitro and in vivo. CM-specific knockout of Smad4 impaired the regenerative effects of 5SM following myocardial infarction (MI) in adult mice. Based on the autocrine loop and pro-proliferative effects of TGFβ/BMP signaling, the ligands induced by 5SM ultimately bind to their receptors in CMs, leading to CM proliferation. In summary, our work establishes that TGFβ/BMP signaling mediates the effect of Harmine in promoting CM proliferation and gains novel insights into the DYRK1B-PKA-CREB axis in heart regeneration.