Abstract 285: High Throughput Profiling of Gsk3α Regulated Fibroblast Kinome Reveals Raf as a Mediator of Fibrosis in Failing Heart

Abstract

Background: Heart failure is the leading cause of mortality, morbidity, and healthcare expenditures worldwide. Numerous studies have implicated Glycogen Synthase Kinase-3 (GSK-3) as a promising therapeutic target for cardiovascular diseases. GSK-3 isoforms appear to play overlapping, unique, and even opposing functions in the heart. Recently our group has identified cardiac fibroblast (CF) GSK3β as a negative regulator of fibrotic remodeling in the ischemic heart. However, the role of CF-GSK3α in cardiac pathophysiology is unknown. Methods and Results: GSK3α was deleted specifically from cardiac fibroblasts or myofibroblasts with tamoxifen-inducible TCF21- or periostin- promoter-driven Cre recombinase. At 2 months of age, WT and KO mice were subjected to cardiac injury, and heart functions were monitored by serial echocardiography. Histological analysis and morphometric studies were performed at 8 weeks post-injury. In both settings, GSK3α deletion restricted fibrotic remodeling and improved cardiac function. To investigate underlying mechanisms, we examined the effect of GSK3α deletion on myofibroblast transformation and pro-fibrotic TGFβ1-SMAD3 signaling in vitro . WT and KO mouse embryonic fibroblasts (MEFs) were treated with TGFβ1. Indeed, a significant reduction in cell migration, collagen gel contraction, and α-SMA expression in TGFβ1 treated KO MEFs confirmed that GSK3α is required for myofibroblast transformation. Surprisingly, GSK3α deletion had no effect on SMAD3 activation, indicating the pro-fibrotic role of GSK3α is SMAD3 independent. At 4 weeks post-injury, total proteins were isolated from CFs of WT and KO animals, and kinome profiling was performed by utilizing PamStation®12 high throughput microarray platform. The upstream kinase analysis identified the downregulation of RAF family kinase activity in GSK3α-KO-CFs. Moreover, mapping of significantly altered kinases against literature annotated interactions generated ERK-centric networks. These findings are consistent with previous studies that implicated ERK in fibrotic diseases across multiple organs. Conclusion: CF-GSK3α plays a causal role in the cardiac pathophysiology that could be therapeutically targeted for future clinical applications.