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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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Ogt is at least partially permissive for proliferation and migration.A: Representative image (magnification 10x) showing cell staining for a transwell migration assay performed after enhancing O-GlcNAc levels, and B: Quantification of the migrated cells demonstrates that increasing protein O-GlcNAcylation does not alter cell motility relative to untreated cells. Negative controls are also shown. C: Cell staining is shown (magnification 10x) for cells carrying floxed Ogt and the effect of gene deletion in a migration assay, quantified in D: demonstrating that loss-of-function for Ogt significantly reduces the number of cells that can migrate. This effect is also borne out in negative controls. Individual data points are plotted and the horizontal line represents the mean, n = 3/group, *p < 0.05 vs Veh/OGT FL.
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pone.0142939.g009: Ogt is at least partially permissive for proliferation and migration.A: Representative image (magnification 10x) showing cell staining for a transwell migration assay performed after enhancing O-GlcNAc levels, and B: Quantification of the migrated cells demonstrates that increasing protein O-GlcNAcylation does not alter cell motility relative to untreated cells. Negative controls are also shown. C: Cell staining is shown (magnification 10x) for cells carrying floxed Ogt and the effect of gene deletion in a migration assay, quantified in D: demonstrating that loss-of-function for Ogt significantly reduces the number of cells that can migrate. This effect is also borne out in negative controls. Individual data points are plotted and the horizontal line represents the mean, n = 3/group, *p < 0.05 vs Veh/OGT FL.

Mentions: Cellular migration/chemotaxis is another important index of cell competence. We observed no significant migratory difference in cells with high O-GlcNAc levels (due to TMG) compared with Veh treated cells (Fig 9A and 9B). Conversely, half as many Ogt deficient cells migrated to the stimulus (Fig 9C and 9D). Unlike the modest differences observed with Ogt deletion in the proliferation experiments, Ogt apparently plays a more significant role in the ability of the cells to migrate to chemotactic stimuli.


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)

Ogt is at least partially permissive for proliferation and migration.A: Representative image (magnification 10x) showing cell staining for a transwell migration assay performed after enhancing O-GlcNAc levels, and B: Quantification of the migrated cells demonstrates that increasing protein O-GlcNAcylation does not alter cell motility relative to untreated cells. Negative controls are also shown. C: Cell staining is shown (magnification 10x) for cells carrying floxed Ogt and the effect of gene deletion in a migration assay, quantified in D: demonstrating that loss-of-function for Ogt significantly reduces the number of cells that can migrate. This effect is also borne out in negative controls. Individual data points are plotted and the horizontal line represents the mean, n = 3/group, *p < 0.05 vs Veh/OGT FL.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0142939.g009: Ogt is at least partially permissive for proliferation and migration.A: Representative image (magnification 10x) showing cell staining for a transwell migration assay performed after enhancing O-GlcNAc levels, and B: Quantification of the migrated cells demonstrates that increasing protein O-GlcNAcylation does not alter cell motility relative to untreated cells. Negative controls are also shown. C: Cell staining is shown (magnification 10x) for cells carrying floxed Ogt and the effect of gene deletion in a migration assay, quantified in D: demonstrating that loss-of-function for Ogt significantly reduces the number of cells that can migrate. This effect is also borne out in negative controls. Individual data points are plotted and the horizontal line represents the mean, n = 3/group, *p < 0.05 vs Veh/OGT FL.
Mentions: Cellular migration/chemotaxis is another important index of cell competence. We observed no significant migratory difference in cells with high O-GlcNAc levels (due to TMG) compared with Veh treated cells (Fig 9A and 9B). Conversely, half as many Ogt deficient cells migrated to the stimulus (Fig 9C and 9D). Unlike the modest differences observed with Ogt deletion in the proliferation experiments, Ogt apparently plays a more significant role in the ability of the cells to migrate to chemotactic stimuli.

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