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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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5-Azacytidine-induced differentiation.A: Densitometry showing significant reduction in O-GlcNAcylation at day 7 upon initiating differentiation stimuli in CSCs. B: Conversely, an enhanced O-GlcNAc status influences commitment to a cardiogenic lineage in terms of significantly reduced mRNA expression of an early cardiac transcription factor Nkx2.5. C: During this time, O-GlcNAc levels were maintained at an elevated degree as depicted in the immunoblot. D: Densitometric quantification showing significantly elevated O-GlcNAcylation due to TMG. Individual data points are plotted and the horizontal line represents the mean, n>/ = 3/group, *p < 0.05 vs day 0/-TMG.
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pone.0142939.g003: 5-Azacytidine-induced differentiation.A: Densitometry showing significant reduction in O-GlcNAcylation at day 7 upon initiating differentiation stimuli in CSCs. B: Conversely, an enhanced O-GlcNAc status influences commitment to a cardiogenic lineage in terms of significantly reduced mRNA expression of an early cardiac transcription factor Nkx2.5. C: During this time, O-GlcNAc levels were maintained at an elevated degree as depicted in the immunoblot. D: Densitometric quantification showing significantly elevated O-GlcNAcylation due to TMG. Individual data points are plotted and the horizontal line represents the mean, n>/ = 3/group, *p < 0.05 vs day 0/-TMG.

Mentions: Protein O-GlcNAc levels diminished early (7 d; Fig 3A) and remained low over the period of the differentiation assay (21 d). A decline in Nkx2.5 manifested early (5 d; Fig 3B) coincident with augmented O-GlcNAc levels (TMG; Fig 3C and 3D). Significant induction of Nkx2.5 was evidenced upon exposure to 5-Azacytidine (21 d; Fig 4A); however, enhancing protein O-GlcNAcylation (TMG) suppressed this effect (21 d; Fig 4A). Moreover, the differentiation-induced expression of connexin 40 (Cx40), which is regulated by Nkx2.5, displayed a parallel reduction in response to elevated O-GlcNAc levels (Fig 4B).


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)

5-Azacytidine-induced differentiation.A: Densitometry showing significant reduction in O-GlcNAcylation at day 7 upon initiating differentiation stimuli in CSCs. B: Conversely, an enhanced O-GlcNAc status influences commitment to a cardiogenic lineage in terms of significantly reduced mRNA expression of an early cardiac transcription factor Nkx2.5. C: During this time, O-GlcNAc levels were maintained at an elevated degree as depicted in the immunoblot. D: Densitometric quantification showing significantly elevated O-GlcNAcylation due to TMG. Individual data points are plotted and the horizontal line represents the mean, n>/ = 3/group, *p < 0.05 vs day 0/-TMG.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0142939.g003: 5-Azacytidine-induced differentiation.A: Densitometry showing significant reduction in O-GlcNAcylation at day 7 upon initiating differentiation stimuli in CSCs. B: Conversely, an enhanced O-GlcNAc status influences commitment to a cardiogenic lineage in terms of significantly reduced mRNA expression of an early cardiac transcription factor Nkx2.5. C: During this time, O-GlcNAc levels were maintained at an elevated degree as depicted in the immunoblot. D: Densitometric quantification showing significantly elevated O-GlcNAcylation due to TMG. Individual data points are plotted and the horizontal line represents the mean, n>/ = 3/group, *p < 0.05 vs day 0/-TMG.
Mentions: Protein O-GlcNAc levels diminished early (7 d; Fig 3A) and remained low over the period of the differentiation assay (21 d). A decline in Nkx2.5 manifested early (5 d; Fig 3B) coincident with augmented O-GlcNAc levels (TMG; Fig 3C and 3D). Significant induction of Nkx2.5 was evidenced upon exposure to 5-Azacytidine (21 d; Fig 4A); however, enhancing protein O-GlcNAcylation (TMG) suppressed this effect (21 d; Fig 4A). Moreover, the differentiation-induced expression of connexin 40 (Cx40), which is regulated by Nkx2.5, displayed a parallel reduction in response to elevated O-GlcNAc levels (Fig 4B).

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