Glutamine-induced protection of isolated rat heart from ischemia/reperfusion injury is mediated via the hexosamine biosynthesis pathway and increased protein O-GlcNAc levels - PubMed (original) (raw)

Glutamine-induced protection of isolated rat heart from ischemia/reperfusion injury is mediated via the hexosamine biosynthesis pathway and increased protein O-GlcNAc levels

Jia Liu et al. J Mol Cell Cardiol. 2007 Jan.

Abstract

It has been shown that glutamine protects the heart from ischemia/reperfusion (I/R) injury; however, the mechanisms underlying this protection have not been identified. Glutamine:fructose-6-phosphate amidotransferase (GFAT) regulates the entry of glucose into the hexosamine biosynthesis pathway (HBP), and activation of this pathway has been shown to be cardioprotective. Glutamine is required for metabolism of glucose via GFAT; therefore, the goal of this study was to determine whether glutamine cardioprotection could be attributed to increased flux through the HBP and elevated levels of O-linked N-acetylglucosamine (O-GlcNAc) on proteins. Hearts from male rats were isolated and perfused with Krebs-Henseliet buffer containing 5 mM glucose, and global, no-flow ischemia was induced for 20 min followed by 60 min of reperfusion. Thirty-minute pre-treatment with 2.5 mM glutamine significantly improved functional recovery (RPP: 15.6+/-5.7% vs. 59.4+/-6.1%; p<0.05) and decreased cardiac troponin I release (25.4+/-3.0 vs. 4.7+/-1.9 ng/ml; p<0.05) during reperfusion. This protection was associated with a significant increase in the levels of protein O-GlcNAc and ATP. Pre-treatment with 80 muM azaserine, an inhibitor of GFAT, completely reversed the protection seen with glutamine and prevented the increase in protein O-GlcNAc. O-GlcNAc transferase (OGT) catalyzes the formation of O-GlcNAc, and inhibition of OGT with 5 mM alloxan also reversed the protection associated with glutamine. These data support the hypothesis that in the ex vivo perfused heart glutamine cardioprotection is due, at least in part, to enhanced flux through the HBP and increased protein O-GlcNAc levels.

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Figures

Figure 1

Figure 1

A) Typical left ventricular pressure (LVP) in hearts before and after 20 min global zero flow ischemia in control, untreated heart; heart treated with 2.5 mM glutamine 30 min prior to ischemia; heart treated with 80 μM azaserine 15 minutes before glutamine treatment; and heart treated with 5 mM alloxan 15 minutes before glutamine treatment; B) Functional recovery of heart rate (HR), left ventricular developed pressure (LVDP), rate-pressure product (RPP) and positive and negative rates of pressure change (± dP/dt) following 20 minutes ischemia and 60 minutes reperfusion as a % of pre-ischemic values; C) End diastolic pressure (EDP) at the end of ischemia; D) EDP at the end of reperfusion; E)Total cardiac troponin I (cTnI) release during reperfusion in control hearts (n=7), hearts perfused with 2.5 mM glutamine (n=7), 2.5 mM glutamine + 80 μM azaserine (n=7), and 2.5 mM glutamine + 5 mM alloxan (n=4). *= p<0.05 compared to glutamine group; ANOVA with Dunnett’s Multiple Comparison Test.

Figure 2

Figure 2

A) UDP-GlcNAc levels and B) ATP levels at the end of reperfusion in control hearts (n=7), hearts perfused with 2.5 mM glutamine (n=7), 2.5 mM glutamine + 80 μM azaserine (n=7), and 2.5 mM glutamine + 5 mM alloxan (n=4). *= p<0.05 compared to glutamine group; ANOVA with Dunnett’s Multiple Comparison Test.

Figure 3

Figure 3

Comparison of protein O-GlcNAc levels at the end of reperfusion in A) control hearts (n=7) and hearts perfused with 2.5 mM glutamine (n=7); B) hearts perfused with 2.5 mM glutamine and 2.5 mM glutamine + 80 μM azaserine (n=7), C) hearts perfused with 2.5 mM glutamine and 2.5 mM glutamine + 5 mM alloxan (n=4). On the left are CTD110 immunoblots of solubilized proteins from left ventricles of hearts and on the right are the mean intensities quantified by densitometric analysis of the immunoblots; in A) data are normalized to the mean intensities of control group in C) and D) data are normalized to the mean intensities of glutamine group; *= p<0.05, (unpaired Students T-Test).

Figure 4

Figure 4

A) Functional recovery of heart rate (HR), left ventricular developed pressure (LVDP), rate-pressure product (RPP) and positive and negative rates of pressure change (± dP/dt) following 20 minutes ischemia and 60 minutes reperfusion as a % of pre-ischemic values in untreated control hearts; hearts treated with 80μM azaserine (n= 4) or 5mM alloxan (n=4). Note that the control data are the same as that shown in Fig 1 and are shown here for comparison purposes only; B) Comparison of protein O-GlcNAc levels at the end of reperfusion in control hearts (n=7) and hearts treated with 80μM azaserine (n= 4) or 5mM alloxan (n=4); C) Comparison of protein O-GlcNAc levels following time-control normoxic perfusions in untreated control hearts (n=5) and hearts treated with 80μM azaserine (n= 4) or 5mM alloxan (n=4).

Figure 5

Figure 5

A) Functional recovery of heart rate (HR), left ventricular developed pressure (LVDP), rate-pressure product (RPP) and positive and negative rates of pressure change (±dP/dt) following 20 minutes ischemia and 60 minutes reperfusion as a % of pre-ischemic values in control hearts (CTL) (n=4), hearts perfused with 10 mM glucosamine (n=5), and hearts perfused with 10 mM glucosamine + 80 μM azaserine (n=4). B) End diastolic pressure (EDP) at the end of reperfusion. #= p<0.05 compared to control; ANOVA with Dunnett’s Multiple Comparison Test.

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