S-nitrosoglutathione reductase (GSNOR) deficiency accelerates cardiomyocyte differentiation of induced pluripotent stem cells

Abstract

Introduction: Induced pluripotent stem cells (iPSCs) provide a model of cardiomyocyte (CM) maturation. Nitric oxide signaling promotes CM differentiation and maturation, although the mechanisms remain controversial.

Aim: The study tested the hypothesis that in the absence of S-nitrosoglutathione reductase (GSNOR), a denitrosylase regulating protein S-nitrosylation, the resultant increased S-nitrosylation accelerates the differentiation and maturation of iPSC-derived cardiomyocytes (CMs).

Methods and Results: iPSCs derived from mice lacking GSNOR (iPSC^GSNOR-/-) matured faster than wildtype iPSCs (iPSC^WT) and demonstrated transient increases in expression of murine Snail Family Transcriptional Repressor 1 gene (Snail), murine Snail Family Transcriptional Repressor 2 gene (Slug) and murine Twist Family BHLH Transcription Factor 1 gene (Twist), transcription factors that promote epithelial-to-mesenchymal transition (EMT) and that are regulated by Glycogen Synthase Kinase 3 Beta (GSK3β). Murine Glycogen Synthase Kinase 3 Beta (Gsk3β) gene exhibited much greater S-nitrosylation, but lower expression in iPSC^GSNOR-/-. S-nitrosoglutathione (GSNO)-treated iPSC^WT and human (h)iPSCs also demonstrated reduced expression of GSK3β. Nkx2.5 expression, a CM marker, was increased in iPSC^GSNOR-/- upon directed differentiation toward CMs on Day 4, whereas murine Brachyury (t), Isl1, and GATA Binding Protein (Gata4) mRNA were decreased, compared to iPSC^WT, suggesting that GSNOR deficiency promotes CM differentiation beginning immediately following cell adherence to the culture dish-transitioning from mesoderm to cardiac progenitor.

Conclusion: Together these findings suggest that increased S-nitrosylation of Gsk3β promotes CM differentiation and maturation from iPSCs. Manipulating the post-translational modification of GSK3β may provide an important translational target and offers new insight into understanding of CM differentiation from pluripotent stem cells.

One sentence summary: Deficiency of GSNOR or addition of GSNO accelerates early differentiation and maturation of iPSC-cardiomyocytes.

Keywords

GSNOR / GSK3β / differentiation / EMT / cardiomyocytes / iPSCs

References

[1] Hodge AJ,Lipke EA.Enhanced stem cell-derived cardiomyocyte differentiation in suspension culture by delivery of nitric oxide using S-nitrosocysteine.Biotechnol Bioeng2016;113:882-94

[2] Wu B,Wang Y.Peroxiredoxin-2 nitrosylation facilitates cardiomyogenesis of mouse embryonic stem cells via XBP-1s/PI3K pathway.Free Radic Biol Med2016;97:179-91

[3] Dulce RA,Rangel EB,Hare JM.Interaction between neuronal nitric oxide synthase signaling and temperature influences sarcoplasmic reticulum calcium leak: role of nitroso-redox balance.Circ Res2015;116:46-55 PMCID:PMC4282621

[4] Haldar SM.S-nitrosylation: integrator of cardiovascular performance and oxygen delivery.J Clin Invest2013;123:101-10 PMCID:PMC3533273

[5] Cao Y,Rangel EB.S-nitrosoglutathione reductase-dependent PPARγ denitrosylation participates in MSC-derived adipogenesis and osteogenesis.J Clin Invest2015;125:1679-91 PMCID:PMC4396480

[6] Beigi F,Minhas KM.Dynamic denitrosylation via S-nitrosoglutathione reductase regulates cardiovascular function.Proc Natl Acad Sci U S A2012;109:4314-9 PMCID:PMC3306718

[7] Chung HS,Venkatraman V.Dual labeling biotin switch assay to reduce bias derived from different cysteine subpopulations: a method to maximize S-nitrosylation detection.Circ Res2015;117:846-57 PMCID:PMC4979977

[8] Liu L,Zeng M.Essential roles of S-nitrosothiols in vascular homeostasis and endotoxic shock.Cell2004;116:617-28

[9] Hatzistergos KE,Dulce RA.S-nitrosoglutathione reductase deficiency enhances the proliferative expansion of adult heart progenitors and myocytes post myocardial infarction.J Am Heart Assoc2015;4:e001974 PMCID:PMC4608081

[10] Ohta S,Tachibana K,Yamada G.Cessation of gastrulation is mediated by suppression of epithelial-mesenchymal transition at the ventral ectodermal ridge.Development2007;134:4315-24

[11] Thiery JP.Complex networks orchestrate epithelial-mesenchymal transitions.Nat Rev Mol Cell Biol2006;7:131-42

[12] Zhou BP,Xia W.Dual regulation of snail by GSK-3beta-mediated phosphorylation in control of epithelial-mesenchymal transition.Nat Cell Biol2004;6:931-40

[13] Burridge PW,Shukla P.Chemically defined generation of human cardiomyocytes.Nat Methods2014;11:855-60 PMCID:PMC4169698

[14] Kattman SJ,Gagliardi M.Stage-specific optimization of activin/nodal and BMP signaling promotes cardiac differentiation of mouse and human pluripotent stem cell lines.Cell Stem Cell2011;8:228-40

[15] Wang SB,Crowgey EL.Protein S-nitrosylation controls glycogen synthase Kinase 3β function independent of its phosphorylation state.Circ Res2018;122:1517-31 PMCID:PMC6047766

[16] Uosaki H,Seo K.Identification of chemicals inducing cardiomyocyte proliferation in developmental stage-specific manner with pluripotent stem cells.Circ Cardiovasc Genet2013;6:624-33 PMCID:PMC3898889

[17] Guide for the care and use of laboratory animals. 8th ed, National Academies Press, 2010.

[18] Lian X,Wilson G.Robust cardiomyocyte differentiation from human pluripotent stem cells via temporal modulation of canonical Wnt signaling.Proc Natl Acad Sci U S A2012;109:E1848-57 PMCID:PMC3390875

[19] Forrester MT,Foster MW,Moseley MA.Proteomic analysis of S-nitrosylation and denitrosylation by resin-assisted capture.Nat Biotechnol2009;27:557-9 PMCID:PMC2891235

[20] Bloch W,Lorke DE.Nitric oxide synthase expression and role during cardiomyogenesis.Cardiovasc Res1999;3:675-84

[21] Takahashi K,Ohnuki M.Induction of pluripotent stem cells from adult human fibroblasts by defined factors.Cell2007;131:861-72

[22] Li HX,Bernier M.Krüppel-like factor 4 promotes differentiation by transforming growth factor-beta receptor-mediated Smad and p38 MAPK signaling in vascular smooth muscle cells.J Biol Chem2010;285:17846-56 PMCID:PMC2878548

[23] Tejedo JR,Mora-Castilla S.Low concentrations of nitric oxide delay the differentiation of embryonic stem cells and promote their survival.Cell Death Dis2010;1:e80 PMCID:PMC3035898

[24] Saraiva RM,Raju SV.Deficiency of neuronal nitric oxide synthase increases mortality and cardiac remodeling after myocardial infarction: role of nitroso-redox equilibrium.Circulation2005;112:3415-22

[25] Fu K,Soufi A.Comparison of reprogramming factor targets reveals both species-specific and conserved mechanisms in early iPSC reprogramming.BMC Genomics2018;19:956 PMCID:PMC6303873

[26] Bachelder RE,Franci C,Mercurio AM.Glycogen synthase kinase-3 is an endogenous inhibitor of Snail transcription: implications for the epithelial-mesenchymal transition.J Cell Biol2005;168:29-33 PMCID:PMC2171685

[27] Wang Y,Ying X,Zhou BP.Twist-mediated epithelial-mesenchymal transition promotes breast tumor cell invasion via inhibition of hippo pathway.Sci Rep2016;6:24606 PMCID:PMC4837350

[28] Otani R,Shin M.Brachyury gene copy number gain and activation of the PI3K/Akt pathway: association with upregulation of oncogenic Brachyury expression in skull base chordoma.J Neurosurg2018;128:1428-37

[29] Yan X,Li H.Inhibition of glycogen synthase kinase 3 beta suppresses the growth and survival of skull base chordoma cells by downregulating brachyury expression.Onco Targets Ther2019;12:9783-91 PMCID:PMC6874116

[30] Zhu J,Mackem S.Putative oncogene brachyury (T) is essential to specify cell fate but dispensable for notochord progenitor proliferation and EMT.Proc Natl Acad Sci U S A2016;113:3820-5 PMCID:PMC4833266

[31] Bedada FB,Metzger SK.Acquisition of a quantitative, stoichiometrically conserved ratiometric marker of maturation status in stem cell-derived cardiac myocytes.Stem Cell Reports2014;3:594-605 PMCID:PMC4223713

[32] Weber N,Greten S.Stiff matrix induces switch to pure β-cardiac myosin heavy chain expression in human ESC-derived cardiomyocytes.Basic Res Cardiol2016;111:68

[33] Mesaeli N,Zvaritch E.Calreticulin is essential for cardiac development.J Cell Biol1999;144:857-68 PMCID:PMC2148186

[34] Li J,Perez-Terzic C.Calreticulin reveals a critical Ca(2+) checkpoint in cardiac myofibrillogenesis.J Cell Biol2002;158:103-13 PMCID:PMC2173019

[35] Mora-Castilla S,Hmadcha A.Nitric oxide repression of Nanog promotes mouse embryonic stem cell differentiation.Cell Death Differ2010;17:1025-33

[36] Benhar M,Stamler JS.Protein denitrosylation: enzymatic mechanisms and cellular functions.Nat Rev Mol Cell Biol2009;10:721-32

[37] Kohr MJ,Sun J.Characterization of potential S-nitrosylation sites in the myocardium.Am J Physiol Heart Circ Physiol2011;300:H1327-35 PMCID:PMC3075037

[38] Lickert H,Kanzler B,Taketo MM.Formation of multiple hearts in mice following deletion of β-catenin in the embryonic endoderm.Dev Cell2002;3:171-81

[39] Sato N,Skaltsounis L,Brivanlou AH.Maintenance of pluripotency in human and mouse embryonic stem cells through activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor.Nat Med2004;10:55-63

[40] McCubrey JA,Bertrand FE.GSK-3 as potential target for therapeutic intervention in cancer.Oncotarget2014;5:2881-911 PMCID:PMC4102778

[41] Nakamura M,Husain S.Glycogen Synthase kinase-3α promotes fatty acid uptake and lipotoxic cardiomyopathy.Cell Metab2019;29:1119-1134.e12 PMCID:PMC6677269

[42] Hödar C,Colombres M.Genome-wide identification of new Wnt/beta-catenin target genes in the human genome using CART method.BMC Genomics2010;11:348 PMCID:PMC2996972

[43] Huang TS,Moalim-Nour L.A regulatory network involving β-catenin, e-cadherin, PI3k/Akt, and slug balances self-renewal and differentiation of human pluripotent stem cells in response to wnt signaling.Stem Cells2015;33:1419-33 PMCID:PMC5297972

[44] Baritaki S,Sahakyan A.Mechanisms of nitric oxide-mediated inhibition of EMT in cancer: inhibition of the metastasis-inducer Snail and induction of the metastasis-suppressor RKIP.Cell Cycle2010;9:4931-40 PMCID:PMC3233484

[45] Mirzoev TM,Shenkman BS.The role of GSK-3β in the regulation of protein turnover, myosin phenotype, and oxidative capacity in skeletal muscle under disuse conditions.Int J Mol Sci2021;22:5081 PMCID:PMC8151958

[46] Woulfe KC,Lal H.Glycogen synthase kinase-3beta regulates post-myocardial infarction remodeling and stress-induced cardiomyocyte proliferation in vivo.Circ Res2010;106:1635-45 PMCID:PMC2895414

[47] Matsuda T,Maejima Y.Distinct roles of GSK-3alpha and GSK-3beta phosphorylation in the heart under pressure overload.Proc Natl Acad Sci U S A2008;105:20900-5 PMCID:PMC2634936

[48] Singh AP,Guo Y,Gupte M.Inhibition of GSK-3 to induce cardiomyocyte proliferation: a recipe for in situ cardiac regeneration.Cardiovasc Res2019;115:20-30 PMCID:PMC6555088

[49] Jho EH,Domon C,Freund JN.Wnt/beta-catenin/Tcf signaling induces the transcription of Axin2, a negative regulator of the signaling pathway.Mol Cell Biol2002;22:1172-83 PMCID:PMC134648

[50] Kang HE,Yun JS.Metformin and niclosamide synergistically suppress Wnt and YAP in APC-mutated colorectal cancer.Cancers (Basel)2021;13:3437 PMCID:PMC8308039

[51] Ahn SY,Lee K.Niclosamide is a potential therapeutic for familial adenomatosis polyposis by disrupting Axin-GSK3 interaction.Oncotarget2017;8:31842-55 PMCID:PMC5458252

[52] Ding VW,McCormick F.Differential regulation of glycogen synthase kinase 3beta by insulin and Wnt signaling.J Biol Chem2000;275:32475-81

[53] Lui TT,Ahmed SM.The ubiquitin-specific protease USP34 regulates axin stability and Wnt/β-catenin signaling.Mol Cell Biol2011;31:2053-65 PMCID:PMC3133363

[54] Kehat I,Snir M.Human embryonic stem cells can differentiate into myocytes with structural and functional properties of cardiomyocytes.J Clin Invest2001;108:407-14 PMCID:PMC209357

[55] Weinberger F,Friedrich FW.Localization of Islet-1-positive cells in the healthy and infarcted adult murine heart.Circ Res2012;110:1303-10 PMCID:PMC5559221

[56] Kunieda T,Miura K.Reduced nitric oxide causes age-associated impairment of circadian rhythmicity.Circ Res2008;102:607-14

[57] Murray CI,Uhrigshardt H,Van Eyk JE.Site-mapping of in vitro S-nitrosation in cardiac mitochondria: implications for cardioprotection.Mol Cell Proteomics2011;10:M110.004721 PMCID:PMC3047159

[58] Gonzalez DR,Sun QA,Hare JM.S-nitrosylation of cardiac ion channels.J Cardiovasc Pharmacol2009;54:188-95 PMCID:PMC3390783

[59] Petroff MG,Pepe S.Endogenous nitric oxide mechanisms mediate the stretch dependence of Ca²⁺ release in cardiomyocytes.Nat Cell Biol2001;3:867-73

[60] Xu L,Meissner G.Activation of the cardiac calcium release channel (ryanodine receptor) by poly-S-nitrosylation.Science1998;279:234-7

[61] Gonzalez DR,Treuer AV.Deficient ryanodine receptor S-nitrosylation increases sarcoplasmic reticulum calcium leak and arrhythmogenesis in cardiomyocytes.Proc Natl Acad Sci U S A2007;104:20612-7 PMCID:PMC2154479

[62] Ahmed RE,Chanthra N.A brief review of current maturation methods for human induced pluripotent stem cells-derived cardiomyocytes.Front Cell Dev Biol2020;8:178 PMCID:PMC7096382

[63] Acun A,Zorlutuna P.In vitro aged, hiPSC-origin engineered heart tissue models with age-dependent functional deterioration to study myocardial infarction.Acta Biomater2019;94:372-91 PMCID:PMC6779061

[64] Rizza S,Montagna C.S-nitrosylation drives cell senescence and aging in mammals by controlling mitochondrial dynamics and mitophagy.Proc Natl Acad Sci U S A2018;115:E3388-97 PMCID:PMC5899480