Inhibition of O-GlcNAcase in perfused rat hearts by NAG-thiazolines at the time of reperfusion is cardioprotective in an O-GlcNAc-dependent manner

Abstract

Acute increases in O-linked β-N-acetylglucosamine (O-GlcNAc) levels of cardiac proteins exert protective effects against ischemia-reperfusion (I/R) injury. One strategy to rapidly increase cellular O-GlcNAc levels is inhibition of O-GlcNAcase (OGA), which catalyzes O-GlcNAc removal. Here we tested the cardioprotective efficacy of two novel and highly selective OGA inhibitors, the NAG-thiazoline derivatives NAG-Bt and NAG-Ae. Isolated perfused rat hearts were subjected to 20 min global ischemia followed by 60 min reperfusion. At the time of reperfusion, hearts were assigned to the following four groups: 1) untreated control; 2) 50 μM NAG-Bt; 3) 100 μM NAG-Bt; or 4) 50 μM NAG-Ae. All treatment groups significantly increased total O-GlcNAc levels (P < 0.05 vs. control), and this was significantly correlated with improved contractile function and reduced cardiac troponin I release (P < 0.05). Immunohistochemistry of normoxic hearts showed intense nuclear O-GlcNAc staining and higher intensity at Z-lines with colocalization of O-GlcNAc and the Z-line proteins desmin and vinculin. After I/R, there was a marked loss of both cytosolic and nuclear O-GlcNAcylation and disruption of normal striated Z-line structures. OGA inhibition largely preserved structural integrity and attenuated the loss of O-GlcNAcylation; however, nuclear O-GlcNAc levels remained low. Immunoblot analysis confirmed ∼50% loss in both nuclear and cytosolic O-GlcNAcylation following I/R, which was significantly attenuated by OGA inhibition (P < 0.05). These data provide further support for the notion that increasing cardiac O-GlcNAc levels by inhibiting OGA may be a clinically relevant approach for ischemic cardioprotection, in part, by preserving the integrity of O-GlcNAc-associated Z-line protein structures.

Fig. 1.

Effect of 1,2 dideoxy-2′-methyl-α-d-glycopyranoso-[2,1-d]-Δ2′-thiazoline (NAG-thiazoline) treatment during reperfusion on functional recovery after 20 min zero-flow ischemia. A: time course of changes in rate-pressure product (RPP) during 60 min reperfusion. B: functional recoveries of coronary flow, heart rate (HR), left ventricular developed pressure (LVDP), positive and negative rates of left ventricular pressure change (±dP/dt), and RPP following 60 min reperfusion as a percent of preischemic values in untreated ischemia-reperfusion hearts (control; n = 7) and hearts treated with 50 μM 1,2 dideoxy-2′-propyl-α-d-glycopyranoso-[2,1-d]-Δ2′-thiazoline [NAG-Bt (NBt50); n = 7], 100 μM NAG-Bt (NBt100; n = 5), and 50 μM 1,2 dideoxy-2′-ethylamino-α-d-glucopyranoso-[2,1-d]-Δ2′-thiazoline [NAG-Ae (NAe); n = 3]. P < 0.05 vs. control (*), vs. NBt50 (†), and vs. NBt100 (‡).

Fig. 2.

Effect of NAG-thiazoline treatment on cardiac troponin I (cTnI) release (A), ATP levels (B), and UDP-N-acetylhexosamine (HexNAc) concentrations (C) in untreated, time-control, normoxic hearts (Norm, n = 5) at the end of 110 min normoxic perfusion and at the end of 60 min reperfusion after 20 min zero-flow ischemia in untreated ischemia-reperfusion hearts (control, n = 7) and NBt50 (n = 7), NBt100 (n = 5), and NAe (n = 3) hearts. P < 0.05 vs. control (*), vs. NBt50 (†), vs. NBt100 (‡), and vs. Norm (§).

Fig. 3.

Effect of NAG-thiazoline treatment on O-linked β-N-acetylglucosamine (O-GlcNAc) levels of the perfused rat heart. Representative anti-O-GlcNAc (CTD110.6) immunoblot (A) and densitometric results of protein-associated cardiac O-GlcNAc levels (B) after time-control, normoxic perfusions (Norm); and after 20 min zero-flow ischemia and 60 min reperfusion (I/R) in the absence (control) and in the presence of NBt50, NBt100, and NAe treatments. Densitometric analyses were performed using original anti-O-GlcNAc immunoblots to compare all experimental groups (n = 3–5 hearts/group). The entire lane mean intensities are normalized to calsequestrin levels shown as protein loading control, and are relative to the control group. P < 0.001 vs. control (*), vs. NBt50 (†), vs. NBt100 (‡), and vs. Norm (§).

Fig. 4.

Correlations of cardiac O-GlcNAc levels and RPP (A), maximum rate of left ventricular pressure changes (max dP/dt; B), and cTnI release (C) at the end of reperfusion. D: correlation of cTnI levels and RPP after reperfusion. Data of cardiac functions are expressed as %preischemic values and are presented for all ischemia-reperfusion hearts in the untreated group (control; n = 7) and following treatments in the NBt50 (n = 7), NBt100 (n = 5), and NAe (n = 3) groups.

Fig. 5.

Immunohistochemistry of rat myocardium after normoxic perfusions (Normoxia); after 20 min zero-flow ischemia (Ischemia); at the end of 60 min reperfusion without treatment (I/R); and after reperfusion following treatment with 50 μM NAG-Bt (I/R + NBt). A: O-GlcNAc immunohistochemistry (green) and merged images with nuclei staining (blue; 4′,6-diamidino-2-phenylindole, DAPI); note that ischemia and I/R result in alterations in nuclear O-GlcNAc distribution with appearance of punctate staining (arrowheads) and perinuclear staining of otherwise O-GlcNAc-negative nuclei (arrows). B: desmin immunohistochemistry (red) and its cross-striated colocalization with O-GlcNAc (green) at Z-line. C: vinculin immunohistochemistry (red) and colocalization of O-GlcNAc (green) with vinculin at Z-line, but not intercalated disc (*) (×40 magnification).

Fig. 6.

Immunohistochemistry of nuclear O-GlcNAcylation in response to ischemia, I/R, I/R + NBt. In normoxic hearts (Normoxia), intensity of O-GlcNAc (green) staining is predominantly concentrated in the nuclei of cardiomyocytes. Ischemia leads to prominent changes in nuclear O-GlcNAc distribution with increased punctate staining (arrowheads) and perinuclear staining of otherwise O-GlcNAc-negative nuclei (arrows).

Fig. 7.

Effect of I/R and NAG-thiazoline treatment on O-GlcNAc levels and O-GlcNAc transferase (OGT) protein content in nuclear and cytosolic compartments of the perfused rat heart. A: representative anti-O-GlcNAc (CTD110.6) immunoblots, and densitometric analyses, demonstrate the changes in nuclear and cytosolic levels of O-GlcNAc. Note that, given the marked differences in overall O-GlcNAc levels between nuclear and cytosolic fractions, different exposure times as indicated were used for the gel images. Consequently, the mean data are shown normalized to the intensity of the untreated group [control (Ctr)] for each fraction; thus, comparison of O-GlcNAc levels between the two fractions cannot be made. B and C: OGT (B) and high-molecular-weight OGT (C) immunoreactive bands in the cytosolic fraction in hearts after time-control, normoxic perfusions (Norm) and after I/R in the untreated group (control) and the treated NBt50, NBt100, and NAe groups (n = 3–4 hearts/group). TATA-binding protein and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are shown as purity controls. P < 0.05, nuclear vs. cytosolic (*), vs. cytosolic control (†), vs. nuclear control (‡), vs. cytosolic Norm (§), and vs. nuclear Norm (#).

Fig. 8.

Immunoblot analyses of cardiac O-GlcNAc and OGT (A), phospho-Tyr⁸²² vinculin and total vinculin (B), and desmin (C) levels in the crude membrane fraction (n = 3 hearts/group) after time-control, normoxic perfusions (Norm) and after I/R in untreated hearts (control) and hearts from NBt50, NBt100, and NAe groups. Data are expressed as %control group. P < 0.05 vs. control (*), vs. NBt50 (†), and vs. Norm (§).