Abstract 18934: Inhibition of Wnt6 by Sfrp2 Regulates CPC Differentiation by Differential Modulation of Canonical and Non Canonical Wnt Pathways

Abstract

Introduction: We have previously shown that in vivo administration of Sfrp2, an inhibitor of canonical Wnt signaling, into injured myocardium enhanced cardiac regeneration. In vitro, Sfrp2 selectively bound to Wnt6 expressed in cardiac progenitor cells (CPCs) and induced cell cycle arrest and cardiac differentiation. Here, we explore the role of Wnt6 inhibition by Sfrp2 in modulating Wnt pathways and regulation of CPC proliferation and differentiation. We also employ lineage tracing to investigate the contribution of CPCs to the in vivo effects of Sfrp2. Methods: Sca1+ CPCs were established and treated with recombinant Sfrp2 or Wnt6. Wnt pathway activation was measured by Western blotting. Proliferation was assessed by BrdU. Differentiation was documented by Nkx2.5 and Gata4 staining. Recombinant Sfrp2 protein was injected in Sca1-eGFP mice after ischemia/reperfusion injury. Co-staining of GFP with cardiac markers was assessed by confocal microscopy. Cardiac function was evaluated by echocardiography. Results: Treatment with Wnt6 increased canonical Wnt signaling and induced a 3.9 ± 0.9 fold increase in CPC proliferation. Simultaneous addition of Sfrp2 abolished the effects of Wnt6. Sfrp2 treatment alone inhibited canonical and activated non canonical Wnt signaling through β-catenin and JNK, respectively. JNK inhibitor SP600125 repressed Sfrp2 induced differentiation. To further explore the dual role of Sfrp2 in regulating Wnt signaling, CPCs modified to express constitutive active β-catenin (CA-CPCs) were treated with Sfrp2 and found to be resistant to both cell cycle arrest and differentiation. Interestingly, Sfrp2 treatment of CA-CPCs did not result in JNK activation suggesting Sfrp2 regulates JNK indirectly. Preliminary in vivo data indicate that exogenous Sfrp2 increased CPC differentiation and restored fractional shortening and contractility 2 months post infarction. Conclusion: Inhibition of Wnt6 by Sfrp2 differentially regulates canonical and non canonical Wnt pathways in CPCs, permitting further lineage commitment towards cardiomyocytes. Along with its cytoprotective and antifibrotic effects, Sfrp2 enhances cardiac regeneration in vivo thus highlighting its value as a potential therapeutic agent for cardiac disease.