Limits...
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.

Show MeSH

Related in: MedlinePlus

O-GlcNAcylation favors induction of smooth muscle actin.A: Immunoblot (with mouse heart lysate as positive control), and B: densitometric analysis demonstrating a robust early (day 5) and significant increase in smooth muscle actin (SMA) expression during differentiation. SMA remained elevated for extended periods of differentiation (7 to 21 d). C: Immunoblot, and D: densitometry revealing that higher O-GlcNAc levels (maintained with TMG) induced a significantly higher level of SMA expression 5 days post-differentiation than Veh treated differentiating cells. Individual data points are plotted and the horizontal line represents the mean, n>/ = 3/group, *p < 0.05 vs day 0/Control; #: p < 0.05 vs Veh +5AZA.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4643874&req=5

pone.0142939.g006: O-GlcNAcylation favors induction of smooth muscle actin.A: Immunoblot (with mouse heart lysate as positive control), and B: densitometric analysis demonstrating a robust early (day 5) and significant increase in smooth muscle actin (SMA) expression during differentiation. SMA remained elevated for extended periods of differentiation (7 to 21 d). C: Immunoblot, and D: densitometry revealing that higher O-GlcNAc levels (maintained with TMG) induced a significantly higher level of SMA expression 5 days post-differentiation than Veh treated differentiating cells. Individual data points are plotted and the horizontal line represents the mean, n>/ = 3/group, *p < 0.05 vs day 0/Control; #: p < 0.05 vs Veh +5AZA.

Mentions: To further ascertain terminal differentiation, whole cell lysates were harvested at various time points for protein expression patterns. Neither VEGF2 nor cTnI were detected in differentiated or undifferentiated cells by immunoblotting, relative to whole mouse hearts (data not shown); however, smooth muscle actin (SMA) was found robustly upregulated by 5 days post-differentiation and persisted at later time points (7 d, 21 d; Fig 6A and 6B). Notably, enhancing protein O-GlcNAcylation (via TMG) resulted in a further upregulation of SMA relative to Veh treated differentiating cells (Fig 6C and 6D).


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-GlcNAcylation favors induction of smooth muscle actin.A: Immunoblot (with mouse heart lysate as positive control), and B: densitometric analysis demonstrating a robust early (day 5) and significant increase in smooth muscle actin (SMA) expression during differentiation. SMA remained elevated for extended periods of differentiation (7 to 21 d). C: Immunoblot, and D: densitometry revealing that higher O-GlcNAc levels (maintained with TMG) induced a significantly higher level of SMA expression 5 days post-differentiation than Veh treated differentiating cells. Individual data points are plotted and the horizontal line represents the mean, n>/ = 3/group, *p < 0.05 vs day 0/Control; #: p < 0.05 vs Veh +5AZA.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0142939.g006: O-GlcNAcylation favors induction of smooth muscle actin.A: Immunoblot (with mouse heart lysate as positive control), and B: densitometric analysis demonstrating a robust early (day 5) and significant increase in smooth muscle actin (SMA) expression during differentiation. SMA remained elevated for extended periods of differentiation (7 to 21 d). C: Immunoblot, and D: densitometry revealing that higher O-GlcNAc levels (maintained with TMG) induced a significantly higher level of SMA expression 5 days post-differentiation than Veh treated differentiating cells. Individual data points are plotted and the horizontal line represents the mean, n>/ = 3/group, *p < 0.05 vs day 0/Control; #: p < 0.05 vs Veh +5AZA.
Mentions: To further ascertain terminal differentiation, whole cell lysates were harvested at various time points for protein expression patterns. Neither VEGF2 nor cTnI were detected in differentiated or undifferentiated cells by immunoblotting, relative to whole mouse hearts (data not shown); however, smooth muscle actin (SMA) was found robustly upregulated by 5 days post-differentiation and persisted at later time points (7 d, 21 d; Fig 6A and 6B). Notably, enhancing protein O-GlcNAcylation (via TMG) resulted in a further upregulation of SMA relative to Veh treated differentiating cells (Fig 6C and 6D).

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