Abstract 12033: The IFNb-Gbp7 Pathway Plays an Important Role in Activating Cardiomyocyte Genes

Abstract

We discovered that reprogramming scar fibroblasts into cardiomyocytes requires activation of innate immunity. Recently, we found that innate immunity exerts its influence on fibroblast to cardiomyocyte reprogramming via the RNA-sensing receptor Rig1. However, the mechanism was not identified. Rig1 activation, whether by classical agonists or via rationally designed RNA molecules, enhanced the efficacy of cardiac reprogramming with cardiomyocyte-specific gene expression increasing by 10 to 50 fold (Actn2, Myh6, Tnni3; N=6; P<0.01) and cardiomyocyte generation increasing 5 fold (N=3, P<0.01). RNA-seq indicated that Rig1 activation in fibroblasts induced IFNβ signaling pathways. To verify a role for IFNβ, knockdown studies were performed. Transfection of fibroblasts with an IFNβ targeting siRNA completely ablated the effects of Rig1 agonists with respect to cardiac reprogramming (N=5, P<0.01). Further studies with specific blocking antibodies and media replacement showed that IFNβ was acting in a paracrine manner. To determine the mechanism whereby IFNβ regulated cardiomyocyte gene expression, we screened a number of candidates identified in the RNA-seq: Ccl5, Ccl10, Gbp5, and Gbp7. Knockdown of Ccl5, Ccl10, or Gbp5 had no effect on the ability of IFNβ to enhance cardiac reprogramming efficacy. In contrast, the ability of IFNβ to enhance cardiac reprogramming was lost in fibroblasts lacking Gbp7 (N=5, P<0.01). To conclude, the IFNβ-Gbp7 pathway plays a critical role in cardiac reprogramming. Manipulation of the IFNβ-Gbp7 pathway may enhance the effectiveness of cardiac reprogramming in vivo.