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

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.

Myocardial Reparative Properties of Cardiac Mesenchymal Cells Isolated on the Basis of Adherence

“…Genetic fate-mapping studies showed that endogenous cardiac c-kit POS cells contributed minimally to myocytes; instead, they differentiated into endothelium or mesenchyma (48). Moreover, c-kit–sorted cardiac cells also express mesenchymal markers, and some studies showed that these cells have properties similar to bone marrow mesenchymal cells, with ability to differentiate into bone, cartilage, and adipose cells (10,13,18,19,21,22). Thus, considerable evidence suggests that c-kit POS cardiac cells might be a population of CMCs (3).…”

“…As is the case for virtually all other cell types, the vast majority of transplanted c-kit POS CPCs disappeared soon after transplantation (9–12,14,17). Interestingly, several studies suggested that c-kit POS CPCs possess mesenchymal stromal characteristics and express classic mesenchymal stromal markers, including CD90, CD105, CD29, and CD73 (13,18–22), intimating that c-kit POS CPCs might, in fact, be a subset of mesenchymal stromal cells. If this is the case, their reparative actions after MI might relate to the general properties of mesenchymal cells rather than to the expression of c-kit per se.…”

A New Method to Stabilize C-Kit Expression in Reparative Cardiac Mesenchymal Cells

“…Whether maintenance of c-kit expression is required for the beneficial effects of cell therapy with c-kit sorted cells remains unanswered. In fact, few studies involving c-kit sorted cells clearly report the maintenance of c-kit expression after passage; our experience has been that expression is lost (Zafir et al, 2013 , 2015 ; Salabei et al, 2015 ). If c-kit expression is necessary for the effectiveness of c-kit cells, we should be intentional about identifying a reproducible approach to stabilize c-kit expression during passage—this was a primary goal of the present study.…”

The dual role of the hexosamine biosynthetic pathway in cardiac physiology and pathophysiology

“…Created with BioRender.com. adult and embryonic cardiac stem cells (169)(170)(171). Evidence supports the shift away from oxidative metabolism and towards biosynthetic pathways to provide substrates for anabolic processes (170).…”

“…The impact of the HBP on in vitro cell differentiation is a lesser investigated area of research. The HBP is integral in mesenchymal stromal cell differentiation which extends to adult and embryonic cardiac stem cells ( 169 – 171 ). Evidence supports the shift away from oxidative metabolism and towards biosynthetic pathways to provide substrates for anabolic processes ( 170 ).…”