Discovering small molecules as Wnt inhibitors that promote heart regeneration and injury repair

There are intense interests in discovering pro regenerative medicine leads that can promote cardiac differentiation and regeneration,as well as repair damaged heart tissues.We have combined zebrafish embryo-based screens with cardiomyogenesis assays to discover selective small molecules that modulate heart development and regeneration with minimal adverse effects.Two related compounds with novel structures,named as Cardiomogen 1 and 2(CDMG1 and CDMG2),were identified for their capacity to promote myocardial hyperplasia through expansion of the cardiac progenitor cell population.We find that Cardiomogen acts as a Wnt inhibitor by targeting p-catenin and reducing Tcf/Lef-mediated transcription in cultured cells.CDMG treatment of amputated zebrafish hearts reduces nuclear p-catenin in injured heart tissue,increases cardiomyocyte(CM)proliferation,and expedites wound healing,thus accelerating cardiac muscle regeneration.Importantly,Cardiomogen can alleviate the functional deterioration of mammalian hearts after myocardial infarction.Injured hearts exposed to CDMG1 display increased newly formed CMs and reduced fibrotic scar tissue,which are in part attributable to the^-catenin reduction.Our findings indicate Cardiomogen as a Wnt inhibitor in enhancing injury-induced CM proliferation and heart regeneration,highlighting the values of embryo-based small molecule screens in discovery of effective and safe medicine leads.

Induction of Wnt signaling antagonists and p21-activated kinase enhances cardiomyocyte proliferation during zebrafish heart regeneration

Heart regeneration occurs by dedifferentiation and proliferation of pre-existing cardiomyocytes(CMs).However,the signaling mechanisms by which injury induces CM renewal remain incompletely understood.Here,we find that cardiac injury in zebrafish induces expression of the secreted Wnt inhibitors,including Dickkopf 1(Dkkl),Dkk3,secreted Frizzled-related protein 1(sFrpl),and sFrp2,in cardiac tissue adjacent to injury sites.Experimental blocking of Wnt activity via Dkkl overexpression enhances CM proliferation and heart regeneration,whereas ectopic activation of Wnt8 signaling blunts injury-induced CM dedifferentiation and proliferation.Although Wnt signaling is dampened upon injury,the cytoplasmic β-catenin is unexpectedly increased at disarrayed CM sarcomeres in myocardial wound edges.Our analyses indicated that p21-activated kinase 2(Pak2)is induced at regenerating CMs,where it phosphorylates cytoplasmic β-catenin at Ser 675 and increases its stability at disassembled sarcomeres.Myocardial-specific induction of the phospho-mimeticβ-catenin(S675E)enhances CM dedifferentiation and sarcomere disassembly in response to injury.Conversely,inactivation of Pak2 kinase activity reduces the Ser 675-phosphorylatedβ-catenin(pS675-β-catenin)and attenuates CM sarcomere disorganization and dedifferentiation・Taken together,these findings demonstrate that coordination of Wnt signaling inhibition and Pak2/pS675-βYatenin signaling enhances zebrafish heart regeneration by supporting CM dedifferentiation and proliferation.