O-GlcNAc signaling attenuates ER stress-induced cardiomyocyte death

Abstract

We previously demonstrated that the O-linked beta-N-acetylglucosamine (O-GlcNAc) posttranslational modification confers cardioprotection at least partially through mitochondrial-dependent mechanisms, but it remained unclear if O-GlcNAc signaling interfered with other mechanisms of cell death. Because ischemia/hypoxia causes endoplasmic reticulum (ER) stress, we ascertained whether O-GlcNAc signaling could attenuate ER stress-induced cell death per se. Before induction of ER stress (with tunicamycin or brefeldin A), we adenovirally overexpressed O-GlcNAc transferase (AdOGT) or pharmacologically inhibited O-GlcNAcase [via O-(2-acetamido-2-deoxy-d-glucopyranosylidene) amino-N-phenylcarbamate] to augment O-GlcNAc levels or adenovirally overexpressed O-GlcNAcase to reduce O-GlcNAc levels. AdOGT significantly (P < 0.05) attenuated the activation of the maladaptive arm of the unfolded protein response [according to C/EBP homologous protein (CHOP) activation] and cardiomyocyte death (reflected by percent propidium iodide positivity). Moreover, pharmacological inhibition of O-GlcNAcase significantly (P < 0.05) mitigated ER stress-induced CHOP activation and cardiac myocyte death. Interestingly, overexpression of GCA did not alter ER stress markers but exacerbated brefeldin A-induced cardiomyocyte death. We conclude that enhanced O-GlcNAc signaling represents a partially proadaptive response to reduce ER stress-induced cell death. These results provide new insights into a possible interaction between O-GlcNAc signaling and ER stress and may partially explain a mechanism of O-GlcNAc-mediated cardioprotection.

Fig. 1.

Cardiac myocytes were subjected to hypoxia-reoxygenation, and activation of ER stress was evaluated. A: hypoxia-activated endoplasmic reticulum (ER) stress as reflected by immunoblots showing augmented Grp94, Grp78, and calreticulin levels. B: densitometric quantification of immunoblots showed significant posthypoxic upregulation of Grp94, Grp78, calreticulin (Cal), and PARP cleavage [expressed as cleaved/uncleaved poly(ADP-ribose) polymerase-1 (PARP) in bar graph]. C: hypoxia-induced cardiac myocyte death [according to lactate dehydrogenase (LDH) release]. *P < 0.05 vs. normoxia; n = 6/group.

Fig. 2.

Normoxic cardiac myocytes were infected with adenovirus carrying the green fluorescent protein (AdGFP), rat OGT (AdOGT), or rat O-GlcNAcase (AdGCA) gene and subjected to ER stress [with brefeldin A (BfA) or tunicamycin (TM)]. Whole cell lysates were immunoblotted for O-GlcNAc. A: OGT overexpression significantly augmented O-GlcNAc levels over those induced by BfA as shown in immunoblot and densitometric quantification of immunoblots. The BfA-induced increase in O-GlcNAc levels in GCA-overexpressed cells was less than baseline O-GlcNAc levels. B: OGT overexpression significantly augmented O-GlcNAc levels over those induced by TM. The TM-induced increase in O-GlcNAc levels in GCA-overexpressed cells did not reach baseline O-GlcNAc levels. *P < 0.05 vs. AdGFP; **P < 0.05 vs. AdGFP + BfA or AdGFP + TM; n ≥ 5/group.

Fig. 3.

Normoxic cardiac myocytes were treated with either AdGFP, AdOGT, or AdGCA and subjected to ER stress (with BfA or TM), and whole cell lysates were immunoblotted for Grp94, Grp78, calreticulin, PARP, and CHOP. A: total protein was isolated from selected cultures and immunoblotted for ER stress indicators. OGT overexpression significantly attenuated, while GCA overexpression did not change, BfA-induced ER stress according to immunoblotting. B: densitometric quantification of immunoblots showed significant reduction in BfA-induction of Grp94, Grp78, and CHOP with OGT overexpression despite no change with GCA overexpression. OGT overexpression did not affect calreticulin expression or PARP cleavage (expressed as cleaved PARP/uncleaved PARP). C: OGT overexpression significantly mitigated, while GCA overexpression did not change, TM-induced ER stress according to immunoblotting. D: densitometric quantification of immunoblots showed significant reduction in TM induction of Grp94, Grp78, and CHOP with OGT overexpression despite no change with GCA overexpression. E: to evaluate cell death, similarly treated cultures were stained with Hoechst and propidium iodide (PI). OGT overexpression significantly attenuated, while GCA overexpression exacerbated ER stress-induced cardiac myocyte death according to PI positivity. F: OGT or GCA overexpression did not change ER stress-induced cardiomyocyte apoptosis. G: OGT overexpression also significantly reduced ER stress-induced cardiac myocyte death despite no change with GCA overexpression according to PI positivity. H: neither OGT nor GCA overexpression affected TM-induced cardiomyocyte apoptosis. **P < 0.05 vs. AdGFP + BfA or TM; n ≥ 5/group.

Fig. 4.

Normoxic cardiac myocytes were treated with either vehicle or O-GlcNAcase inhibitor [O-(2-acetamido-2-deoxy-d-glucopyranosylidene) amino-N-phenylcarbamate (PUGNAc)] and subjected to ER stress (with BfA or TM). A: Western blots of whole cell lysates showed augmented O-GlcNAc levels with O-GlcNAcase inhibition compared with vehicle. O-GlcNAcase inhibition augmented O-GlcNAc levels above those induced by BfA. B: O-GlcNAcase inhibition significantly increased O-GlcNAc levels above those induced by TM. *P < 0.05 vs. vehicle, ** P < 0.05 vs. BfA or TM; n = 4/group.

Fig. 5.

Normoxic cardiac myocytes were pretreated with either vehicle or O-GlcNAcase inhibitor (PUGNAc) and subjected to ER stress with BfA or TM. A: Western blots of whole cell lysates showed significant reduction in BfA-mediated activation of Grp94, Grp78, calreticulin, and CHOP protein levels with O-GlcNAcase inhibition. B: densitometric quantification of immunoblots showed significantly reduced Grp94, Grp78, calreticulin, and CHOP protein levels with O-GlcNAcase inhibition. PUGNAc treatment did not affect the BfA-mediated increase in calreticulin expression or PARP cleavage (expressed as cleaved PARP/uncleaved PARP). O-GlcNAcase inhibition also significantly reduced the TM-mediated increase in Grp78, Grp94, and CHOP protein levels compared with BfA alone according to representative immunoblots (C) and densitometric quantification (D). E: similarly treated cultures were stained with Hoechst and PI for cell death. O-GlcNAcase inhibition attenuated BfA-induced cardiomyocyte death. F: O-GlcNAcase inhibition did not affect BfA-induced cardiomyocyte apoptosis. G: O-GlcNAcase inhibition attenuated TM-induced cardiomyocyte death. H: O-GlcNAcase inhibition did not affect TM-induced cardiomyocyte apoptosis. *P < 0.05 vs. control; **P < 0.05 vs. BfA or TM; n ≥ 5/group.