Effect of Oxygen on Cardiac Differentiation in Mouse iPS Cells: Role of Hypoxia Inducible Factor-1 and Wnt/Beta-Catenin Signaling

Tanya L Medley,Milena Furtado,David M Kaye,Nicholas T Lam,Rejhan Idrizi,David Williams,Mauro Costa,Paul J Verma,Joseph Najbauer

doi:10.1371/journal.pone.0080280

Abstract

BackgroundDisturbances in oxygen levels have been found to impair cardiac organogenesis. It is known that stem cells and differentiating cells may respond variably to hypoxic conditions, whereby hypoxia may enhance stem cell pluripotency, while differentiation of multiple cell types can be restricted or enhanced under hypoxia. Here we examined whether HIF-1alpha modulated Wnt signaling affected differentiation of iPS cells into beating cardiomyocytes.ObjectiveWe investigated whether transient and sustained hypoxia affects differentiation of cardiomyocytes derived from murine induced pluripotent stem (iPS) cells, assessed the involvement of HIF-1alpha (hypoxia-inducible factor-1alpha) and the canonical Wnt pathway in this process.MethodsEmbryoid bodies (EBs) derived from iPS cells were differentiated into cardiomyocytes and were exposed either to 24 h normoxia or transient hypoxia followed by a further 13 days of normoxic culture.ResultsAt 14 days of differentiation, 59±2% of normoxic EBs were beating, whilst transient hypoxia abolished beating at 14 days and EBs appeared immature. Hypoxia induced a significant increase in Brachyury and islet-1 mRNA expression, together with reduced troponin C expression. Collectively, these data suggest that transient and sustained hypoxia inhibits maturation of differentiating cardiomyocytes. Compared to normoxia, hypoxia increased HIF-1alpha, Wnt target and ligand genes in EBs, as well as accumulation of HIF-1alpha and beta-catenin in nuclear protein extracts, suggesting involvement of the Wnt/beta-catenin pathway.ConclusionHypoxia impairs cardiomyocyte differentiation and activates Wnt signaling in undifferentiated iPS cells. Taken together the study suggests that oxygenation levels play a critical role in cardiomyocyte differentiation and suggest that hypoxia may play a role in early cardiogenesis.

Highlights

Cardiomyogenesis from specific progenitor cells is critically dependent on the presence of a tightly regulated biochemical and mechanical microenvironment
At 14 days of differentiation, 5962% of normoxic Embryoid bodies (EBs) were beating, whilst transient hypoxia abolished beating at 14 days and EBs appeared immature
Hypoxia impairs cardiomyocyte differentiation and activates Wnt signaling in undifferentiated induced pluripotent stem (iPS) cells

Summary

Introduction

Cardiomyogenesis from specific progenitor cells is critically dependent on the presence of a tightly regulated biochemical and mechanical microenvironment. An appropriate level of tissue oxygen tension is one of the most critical factors for the formation of the heart and its subsequent structural development In this context, hypoxia is recognized to cause a range of cardiac malformations, including myocardial and valvular hypoplasia [1,2,3]. HIFs are a family of transcription factors consisting of two subunits: the oxygen sensitive alpha subunit, and the constitutively expressed beta subunit, which binds to all HIF alpha combinations (HIF-1alpha, HIF-2alpha, HIF-3alpha, IPAS and NEPAS) Both HIF-1 and HIF-2 protein complexes are expressed in cardiac tissue [5], with a larger body of evidence demonstrating the role of HIF-1alpha expression in myocardial remodeling and coronary vessel formation. We examined whether HIF1alpha modulated Wnt signaling affected differentiation of iPS cells into beating cardiomyocytes

Methods

Results

Conclusion