S-nitrosylation Promotes Cell Cycle, Cell Viability and Proliferation by Activating the Snail/Slug Pathway in miPSC-Derived CM

Introduction: Epithelial-to-mesenchymal transition (EMT), is a crucial step in cardiac differentiation. Snail and Slug are increased and E-cadherin is decreased during EMT. Expression of Snail and Slug is reduced by GSK3β, a key regulator of the Wnt/β-catenin signaling pathway. S-nitrosylation (S-NO) of GSK3β inhibits its activity. Objective: We hypothesized that in the absence of S-nitrosoglutathione reductase (GSNOR), the Snail/Slug pathway is activated in miPSC-derived cardiomyocytes. Methods: We compared the initial growth and differentiation of iPSCs derived from GSNOR –/– (iPSC GSNOR-/- ) and wild type (WT [control], iPSC WT ) mice on days (D) 0-6 in cell culture. Cell morphology was assessed and MTT, BrdU and transwell invasion assays performed to determine cell proliferation, invasion and migration. EMT-related transcription factors and members of the GSK3β pathway were measured at D0-D6. Results: During early differentiation, iPSC GSNOR-/- cells exhibited accelerated loss of pluripotency markers and, from D4, greater proliferation/differentiation, and apparent EMT compared to iPSC WT . MTT, BrdU and migration assays demonstrated that loss of GSNOR stimulated cell proliferation and migration. Slug and Snail were upregulated and E-Cadherin was downregulated in iPSC GSNOR-/- suggesting that increased NO levels reduced GSK3β activity in iPSC GSNOR-/- . Conclusions: Our results suggest that the deletion of GSNOR affects early CM differentiation and promote EMT. These findings have important implications for regenerative medicine and provide new targets for iPSC-based therapy.