Glucosamine protects neonatal cardiomyocytes from ischemia-reperfusion injury via increased protein O-GlcNAc and increased mitochondrial Bcl-2

Abstract

We have previously reported that glucosamine protected neonatal rat ventricular myocytes against ischemia-reperfusion (I/R) injury, and this was associated with an increase in protein O-linked-N-acetylglucosamine (O-GlcNAc) levels. However, the protective effect of glucosamine could be mediated via pathways other that O-GlcNAc formation; thus the initial goal of the present study was to determine whether increasing O-GlcNAc transferase (OGT) expression, which catalyzes the formation of O-GlcNAc, had a protective effect similar to that of glucosamine. To better understand the potential mechanism underlying O-GlcNAc-mediated cytoprotection, we examined whether increased O-GlcNAc levels altered the expression and translocation of members of the Bcl-2 protein family. Both glucosamine (5 mM) and OGT overexpression increased basal and I/R-induced O-GlcNAc levels, significantly decreased cellular injury, and attenuated loss of cytochrome c. Both interventions also attenuated the loss of mitochondrial membrane potential induced by H2O2 and were also associated with an increase in mitochondrial Bcl-2 levels but had no effect on Bad or Bax levels. Compared with glucosamine and OGT overexpression, NButGT (100 microM), an inhibitor of O-GlcNAcase, was less protective against I/R and H2O2 and did not affect Bcl-2 expression, despite a 5- to 10-fold greater increase in overall O-GlcNAc levels. Decreased OGT expression resulted in lower basal O-GlcNAc levels, prevented the I/R-induced increase in O-GlcNAc and mitochondrial Bcl-2, and increased cellular injury. These results demonstrate that the protective effects of glucosamine are mediated via increased formation of O-GlcNAc and suggest that this is due, in part, to enhanced mitochondrial Bcl-2 translocation.

Fig. 1

Increasing global O-GlcNAc protein levels by glucosamine (GlcN) reduced cellular injury during ischemia and reperfusion injury. A) Cell injury assessed by determining lactate dehydrogenase (LDH) release as a percentage of total LDH; B) Representative CTD110.6 and RL2 immunoblot of O-linked N-acetylglucosamine (O-GlcNAc) proteins and mean intensity of all O-GlcNAc proteins determined by densitometric analysis. Equal protein loading was normalized by densitometric analysis of β-actin levels. O-GlcNAc levels are normalized to normoxic control untreated cells. Control untreated cells and glucosamine (5 mM)-treated cells were incubated under normoxic condition for 6 h or subjected to 4 h of ischemia followed by 2 h of reperfusion (I₄/R₂). Data are presented as means±SEM of more than 3 experiments. # = P< 0.05 Vs untreated, † P< 0.05 Vs normoxia.

Fig. 2

Glucosamine had no effect on Bcl-2 family proteins, Bcl-2, Bax, and Bad, but enhanced mitochondrial Bcl-2 translocation. A) Representative Bcl-2, Bax, and Bad immunoblots of the whole cell lysate of NRVMs. Equal protein loading was confirmed by β-actin levels; B) Representative Bcl-2, Bax, and Bad of the mitochondrial and post-mitochondrial (cytosolic/microsomal) fraction of NRVMs. Purity of each fraction was assessed by GAPDH and COX4, respectively; C) Mean intensity of mitochondrial Bcl-2 determined by densitometric analysis. Levels are normalized to normoxic control untreated cells. Control untreated cells and glucosamine (5 mM)-treated cells were incubated under normoxic condition for 6 h or subjected to 4 h of ischemia followed by 2 h of reperfusion (I₄/R₂). Data are presented as means±SEM of more than 3 experiments. # P< 0.05 Vs untreated, † P< 0.05 Vs normoxia.

Fig. 3

Increasing O-GlcNAc transferase (OGT) and blocking O-GlcNAcase by NButGT had different effects on O-GlcNAc protein modification, cellular survival and Bcl-2 translocation. A) Representative immunoblotting CTD110.6 and OGT and mean intensity of all O-GlcNAc proteins and OGT of the total whole cell lysate determined by densitometric analysis. Equal protein loading was normalized by densitometric analysis of β-actin levels. Proteins are normalized to normoxic control untreated cells; B) Cell injury assessed by determining lactate dehydrogenase (LDH) release as a percentage of total LDH; C) Representative immunoblotting of Bcl-2 expression levels in total cell lysate and mitochondrial fractions and mean densitometric analysis of mitochondrial Bcl-2. Purity of mitochondrial fraction was assessed by COX4 and GAPDH. Control untreated cells, O-GlcNAc transferase (OGT) adenovirus infected cells, NButGT (100 µM)-treated cells were incubated under normoxic condition for 6 h or subjected to 4 h of ischemia followed by 2 h of reperfusion (I₄/R₂). Data are presented as means±SEM of more than 3 experiments. # =P< 0.05 Vs untreated, † P< 0.05 Vs normoxia, * P< 0.05 Vs OGT overexpression.

Fig. 4

Increasing global O-GlcNAc proteins protected the loss of cytochrome C. A) Representative immunofluorescence of cytochrome C andO-GlcNAc proteins (CTD 110.6) under normoxia and following ischemia/reperfusion; B) Mean intensity of cytochrome C determined by IPLab analysis software was normalized with the control untreated cells. Data are presented as means±SEM of 150–200 cells. Control untreated cells, glucosamine (5 mM)-treated cells, O-GlcNAc transferase (OGT) adenovirus infected cells, NButGT (100 µM)-treated cells were incubated under normoxic condition for 6 h or subjected to 4 h of ischemia followed by 2 h of reperfusion (I₄/R₂). # = P< 0.05 Vs untreated, * P< 0.05 Vs NButGT.

Fig. 5

Increased O-GlcNAc levels attenuated the loss of mitochondrial membrane potential (MMP). A) Representative fluorescent merged images with red and green filters of JC-1 staining NRVMs treated with hydrogen peroxide at 1 mM from 0 to 20 mins; B) Mean intensity of mitochondrial JC-1 staining (Red filter) determined by IPLab analysis software and normalized with cells at starting time. Data are presented as means±SEM of 30 cells. Control untreated cells, glucosamine (5 mM)-1 hour pretreated cells, O-GlcNAc transferase (OGT) adenovirus infected cells, NButGT (100 µM)-1 hour pretreated cells were incorporated with JC-1 to monitor MMP. # = P< 0.05 Vs untreated, * P< 0.05 Vs NButGT.

Fig. 6

O-GlcNAc modification is essential for cellular survival and Bcl-2 translocation. A) Representative immunoblotting CTD110.6 and OGT and mean intensity of all O-GlcNAc proteins and OGT of the total whole cell lysate determined by densitometric analysis. Equal protein loading was normalized by densitometric analysis of β-actin levels. Proteins are normalized to normoxic control untreated cells; B) Cell injury assessed by determining lactate dehydrogenase (LDH) release as a percentage of total LDH; C) Representative immunoblotting of Bcl-2 expression levels in total cell lysate and mitochondrial fractions and mean densitometric analysis of mitochondrial Bcl-2. Purity of mitochondrial fraction was assessed by COX4 and GAPDH. Control, si-Negative oligonuleotides transfected cells (si-Neg) and si-OGT oligonucleotides transfected cells (si-OGT) cells were incubated under normoxic condition for 6 h or subjected to 4 h of ischemia followed by 2 h of reperfusion (I₄/R₂). Data are presented as means±SEM of more than 3 experiments. # =P< 0.05 Vs untreated, † P< 0.05 Vs normoxia.

Fig. 7

Decreasing global O-GlcNAc proteins exacerbated the loss of cytochrome C and apoptosis. A) Representative immunofluorescence of cytochrome C and O-GlcNAc proteins (CTD 110.6) under normoxia and following ischemia/reperfusion; B) Representative immunofluorescent of apoptosis (TUNEL), O-GlcNAc proteins (CTD 110.6), and nuclear staining (Hoechst). Control, si-Negative oligonuleotides transfected cells (si-Neg) and si-OGT oligonucleotides transfected cells (si-OGT) cells were incubated under normoxic condition for 6 h or subjected to 4 h of ischemia followed by 2 h or 16 h of reperfusion.