Protein O-GlcNAcylation Is a Novel Cytoprotective Signal in Cardiac Stem Cells

Abstract

Clinical trials demonstrate the regenerative potential of cardiac stem cell (CSC) therapy in the post-infarcted heart. Despite these encouraging preliminary clinical findings, the basic biology of these cells remains largely unexplored. The principal requirement for cell transplantation is to effectively prime them for survival within the unfavorable environment of the infarcted myocardium. In the adult mammalian heart, the β-O-linkage of N-acetylglucosamine (i.e., O-GlcNAc) to proteins is a unique post-translational modification that confers cardioprotection from various otherwise lethal stressors. It is not known whether this signaling system exists in cardiac stem cells. In the present study, we demonstrate that protein O-GlcNAcylation is an inducible stress response in adult murine Sca-1+/lin− CSCs and exerts an essential pro-survival role. Post-hypoxic CSCs responded by time-dependently increasing protein O-GlcNAcylation upon reoxygenation. We utilized pharmacological interventions for loss- and gain-of-function, i.e., enzymatic inhibition of OGT (adds the O-GlcNAc modification to proteins) by TT04, or inhibition of OGA (removes O-GlcNAc) by thiamet-G (ThG). Reduction in the O-GlcNAc signal (via TT04, or OGT gene deletion using Cre-mediated recombination) significantly sensitized CSCs to post-hypoxic injury, whereas augmenting O-GlcNAc levels (via ThG) enhanced cell survival. Diminished O-GlcNAc levels render CSCs more susceptible to the onset of post-hypoxic apoptotic processes via elevated PARP cleavage due to enhanced caspase-3/7 activation, whereas promoting O-GlcNAcylation can serve as a pre-emptive anti-apoptotic signal regulating the survival of CSCs. Thus, we report the primary demonstration of protein O-GlcNAcylation as an important pro-survival signal in CSCs, which could enhance CSC survival prior to in vivo autologous transfer.

Type 2 Diabetes Dysregulates Glucose Metabolism in Cardiac Progenitor Cells

“…1A). As reported previously, the cells retained only ϳ5-10% positivity for c-kit after sorting and culture (30,31), and both nondiabetic and diabetic cells were Ͼ90% positive for the mesenchymal cell marker CD29 and Sca1 and Ͻ5% positive for CD45 (Fig. 1, B and D).…”

“…Murine CPCs were isolated from 2.5-3-month-old male B6.BKS(D)-Lepr db /J (db/db) mice and C57BL/6J (WT) (The Jackson Laboratory, Bar Harbor, ME). After outgrowth, the CPCs were subjected to sequential sorting for c-kit ϩ /lin Ϫ markers using magnetic immunobeads (29,30). As described previously (30,31), the CPCs were cultured in growth medium.…”

“…After outgrowth, the CPCs were subjected to sequential sorting for c-kit ϩ /lin Ϫ markers using magnetic immunobeads (29,30). As described previously (30,31), the CPCs were cultured in growth medium. Passages 3-9 were used for these studies.…”

O-GlcNAcase deficiency suppresses skeletal myogenesis and insulin sensitivity in mice through the modulation of mitochondrial homeostasis

“…Enhanced global O-GlcNAcylation has been found in the liver tissue of db/db mice and HFD-induced diabetic mouse models [33], and in the cardiac muscle and coronary endothelial cells of STZinduced diabetic mouse models [13,34], which is consistent with our observations in skeletal muscle. Meanwhile, the cytoprotective potential of increased O-GlcNAc signalling in the cardiovascular system has also been proposed in several studies [35,36], suggesting that O-GlcNAc signalling has multiple roles in regulating physiological activities. More efforts are required to elucidate the details of the involvement of O-GlcNAc signalling in diabetes progression.…”

MicroRNA-539 Is Up-regulated in Failing Heart, and Suppresses O-GlcNAcase Expression

“…In Vitro Hypoxia-reoxygenation Injury-To determine whether additional insults, such as hypoxia-reoxygenation alter miR-539 levels, NRCMs were subjected to hypoxia-reoxygenation as described (9). In brief, cells were subjected to 3 h hypoxia in Esumi lethal ischemia medium for glucose and nutrient deprivation (containing 117 mmol/liter of NaCl, 12 mmol/liter of KCl, 0.9 mmol/liter of CaCl 2 , 0.49 mmol/liter of MgCl 2 , 4 mmol/liter of HEPES, 20 mmol/liter of sodium lactate, and 5.6 mmol/liter of L-glucose; pH 6.2) in a sealed humidified hypoxic chamber (Billups-Rothenberg, Inc.) flushed with 5% CO 2 and 95% N 2 , and maintained at 37°C.…”