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O-GlcNAcylation Negatively Regulates Cardiomyogenic Fate in Adult Mouse Cardiac Mesenchymal Stromal Cells.

Zafir A, Bradley JA, Long BW, Muthusamy S, Li Q, Hill BG, Wysoczynski M, Prabhu SD, Bhatnagar A, Bolli R, Jones SP - PLoS ONE (2015)

Bottom Line: Differentiation significantly decreased the abundance of O-GlcNAcylated proteins.Although elevated O-GlcNAc levels did not significantly affect fibroblast and endothelial marker expression, acquisition of cardiomyocyte markers was limited.We conclude that O-GlcNAcylation contributes significantly to cardiac mesenchymal stromal cell lineage and function.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Cardiology; Diabetes and Obesity Center, Department of Medicine, Division of Cardiovascular Medicine, University of Louisville, Louisville, Kentucky, United States of America.

ABSTRACT
In both preclinical and clinical studies, cell transplantation of several cell types is used to promote repair of damaged organs and tissues. Nevertheless, despite the widespread use of such strategies, there remains little understanding of how the efficacy of cell therapy is regulated. We showed previously that augmentation of a unique, metabolically derived stress signal (i.e., O-GlcNAc) improves survival of cardiac mesenchymal stromal cells; however, it is not known whether enhancing O-GlcNAcylation affects lineage commitment or other aspects of cell competency. In this study, we assessed the role of O-GlcNAc in differentiation of cardiac mesenchymal stromal cells. Exposure of these cells to routine differentiation protocols in culture increased markers of the cardiomyogenic lineage such as Nkx2.5 and connexin 40, and augmented the abundance of transcripts associated with endothelial and fibroblast cell fates. Differentiation significantly decreased the abundance of O-GlcNAcylated proteins. To determine if O-GlcNAc is involved in stromal cell differentiation, O-GlcNAcylation was increased pharmacologically during the differentiation protocol. Although elevated O-GlcNAc levels did not significantly affect fibroblast and endothelial marker expression, acquisition of cardiomyocyte markers was limited. In addition, increasing O-GlcNAcylation further elevated smooth muscle actin expression. In addition to lineage commitment, we also evaluated proliferation and migration, and found that increasing O-GlcNAcylation did not significantly affect either; however, we found that O-GlcNAc transferase--the protein responsible for adding O-GlcNAc to proteins--is at least partially required for maintaining cellular proliferative and migratory capacities. We conclude that O-GlcNAcylation contributes significantly to cardiac mesenchymal stromal cell lineage and function. O-GlcNAcylation and pathological conditions that may affect O-GlcNAc levels (such as diabetes) should be considered carefully in the context of cardiac cell therapy.

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Related in: MedlinePlus

O-GlcNAc-primed CSCs maintain a normal growth profile, but loss of Ogt suppresses proliferation.A: Growth curves of CSCs maintained in culture with TMG demonstrate that augmented O-GlcNAc levels do not affect the characteristics of normal population growth. B:Ogt deletion results in significantly reduced cell proliferation. Inset indicates loss of OGT protein and diminished O-GlcNAc levels in the OGT KO cells. n = 3/group, *p < 0.05 vs OGT FL.
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pone.0142939.g008: O-GlcNAc-primed CSCs maintain a normal growth profile, but loss of Ogt suppresses proliferation.A: Growth curves of CSCs maintained in culture with TMG demonstrate that augmented O-GlcNAc levels do not affect the characteristics of normal population growth. B:Ogt deletion results in significantly reduced cell proliferation. Inset indicates loss of OGT protein and diminished O-GlcNAc levels in the OGT KO cells. n = 3/group, *p < 0.05 vs OGT FL.

Mentions: Given our previous publication showing that enhanced O-GlcNAcylation promotes cell survival, and our present results indicating that O-GlcNAcylation antagonizes commitment to a cardiomyogenic fate, it was important to determine whether other aspects of cell competence might also be affected. To this end, we treated cells with TMG or Veh and monitored cell proliferation (Fig 8A), and found no significant differences between the two groups as depicted by comparable cell numbers in the lag and exponential phases, with slight variability in the stationary phase. Next, we generated Ogt deficient cells to determine whether Ogt was required for cell proliferation, and found a mild, but statistically significant, impairment in proliferation (Fig 8B). Thus, Ogt performs a minimally permissive role in cell proliferation; however, increasing O-GlcNAcylation does not affect cell proliferation.


O-GlcNAcylation Negatively Regulates Cardiomyogenic Fate in Adult Mouse Cardiac Mesenchymal Stromal Cells.

Zafir A, Bradley JA, Long BW, Muthusamy S, Li Q, Hill BG, Wysoczynski M, Prabhu SD, Bhatnagar A, Bolli R, Jones SP - PLoS ONE (2015)

O-GlcNAc-primed CSCs maintain a normal growth profile, but loss of Ogt suppresses proliferation.A: Growth curves of CSCs maintained in culture with TMG demonstrate that augmented O-GlcNAc levels do not affect the characteristics of normal population growth. B:Ogt deletion results in significantly reduced cell proliferation. Inset indicates loss of OGT protein and diminished O-GlcNAc levels in the OGT KO cells. n = 3/group, *p < 0.05 vs OGT FL.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4643874&req=5

pone.0142939.g008: O-GlcNAc-primed CSCs maintain a normal growth profile, but loss of Ogt suppresses proliferation.A: Growth curves of CSCs maintained in culture with TMG demonstrate that augmented O-GlcNAc levels do not affect the characteristics of normal population growth. B:Ogt deletion results in significantly reduced cell proliferation. Inset indicates loss of OGT protein and diminished O-GlcNAc levels in the OGT KO cells. n = 3/group, *p < 0.05 vs OGT FL.
Mentions: Given our previous publication showing that enhanced O-GlcNAcylation promotes cell survival, and our present results indicating that O-GlcNAcylation antagonizes commitment to a cardiomyogenic fate, it was important to determine whether other aspects of cell competence might also be affected. To this end, we treated cells with TMG or Veh and monitored cell proliferation (Fig 8A), and found no significant differences between the two groups as depicted by comparable cell numbers in the lag and exponential phases, with slight variability in the stationary phase. Next, we generated Ogt deficient cells to determine whether Ogt was required for cell proliferation, and found a mild, but statistically significant, impairment in proliferation (Fig 8B). Thus, Ogt performs a minimally permissive role in cell proliferation; however, increasing O-GlcNAcylation does not affect cell proliferation.

Bottom Line: Differentiation significantly decreased the abundance of O-GlcNAcylated proteins.Although elevated O-GlcNAc levels did not significantly affect fibroblast and endothelial marker expression, acquisition of cardiomyocyte markers was limited.We conclude that O-GlcNAcylation contributes significantly to cardiac mesenchymal stromal cell lineage and function.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Cardiology; Diabetes and Obesity Center, Department of Medicine, Division of Cardiovascular Medicine, University of Louisville, Louisville, Kentucky, United States of America.

ABSTRACT
In both preclinical and clinical studies, cell transplantation of several cell types is used to promote repair of damaged organs and tissues. Nevertheless, despite the widespread use of such strategies, there remains little understanding of how the efficacy of cell therapy is regulated. We showed previously that augmentation of a unique, metabolically derived stress signal (i.e., O-GlcNAc) improves survival of cardiac mesenchymal stromal cells; however, it is not known whether enhancing O-GlcNAcylation affects lineage commitment or other aspects of cell competency. In this study, we assessed the role of O-GlcNAc in differentiation of cardiac mesenchymal stromal cells. Exposure of these cells to routine differentiation protocols in culture increased markers of the cardiomyogenic lineage such as Nkx2.5 and connexin 40, and augmented the abundance of transcripts associated with endothelial and fibroblast cell fates. Differentiation significantly decreased the abundance of O-GlcNAcylated proteins. To determine if O-GlcNAc is involved in stromal cell differentiation, O-GlcNAcylation was increased pharmacologically during the differentiation protocol. Although elevated O-GlcNAc levels did not significantly affect fibroblast and endothelial marker expression, acquisition of cardiomyocyte markers was limited. In addition, increasing O-GlcNAcylation further elevated smooth muscle actin expression. In addition to lineage commitment, we also evaluated proliferation and migration, and found that increasing O-GlcNAcylation did not significantly affect either; however, we found that O-GlcNAc transferase--the protein responsible for adding O-GlcNAc to proteins--is at least partially required for maintaining cellular proliferative and migratory capacities. We conclude that O-GlcNAcylation contributes significantly to cardiac mesenchymal stromal cell lineage and function. O-GlcNAcylation and pathological conditions that may affect O-GlcNAc levels (such as diabetes) should be considered carefully in the context of cardiac cell therapy.

Show MeSH
Related in: MedlinePlus