Increased O-GlcNAc levels during reperfusion lead to improved functional recovery and reduced calpain proteolysis

Abstract

We have previously shown that preischemic treatment with glucosamine improved cardiac functional recovery following ischemia-reperfusion, and this was mediated, at least in part, via enhanced flux through the hexosamine biosynthesis pathway and subsequently elevated O-linked N-acetylglucosamine (O-GlcNAc) protein levels. However, preischemic treatment is typically impractical in a clinical setting; therefore, the goal of this study was to investigate whether increasing protein O-GlcNAc levels only during reperfusion also improved recovery. Isolated perfused rat hearts were subjected to 20 min of global, no-flow ischemia followed by 60 min of reperfusion. Administration of glucosamine (10 mM) or an inhibitor of O-GlcNAcase, O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc; 200 microM), during the first 20 min of reperfusion significantly improved cardiac functional recovery and reduced troponin release during reperfusion compared with untreated control. Both interventions also significantly increased the levels of protein O-GlcNAc and ATP levels. We also found that both glucosamine and PUGNAc attenuated calpain-mediated proteolysis of alpha-fodrin as well as Ca(2+)/calmodulin-dependent protein kinase II during reperfusion. Thus two independent strategies for increasing protein O-GlcNAc levels in the heart during reperfusion significantly improved recovery, and this was correlated with attenuation of calcium-mediated proteolysis. These data provide further support for the concept that increasing cardiac O-GlcNAc levels may be a clinically relevant cardioprotective strategy and suggest that this protection could be due, at least in part, to inhibition of calcium-mediated stress responses.

Figure 1

Typical left ventricular pressure (LVP) tracing in hearts before and after 20 min global no flow ischemia in A) control B) glucosamine (10 mM) for first 20 min of reperfusion; C) PUGNAc (200 μM) for 20 min of reperfusion; D) 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; E) End diastolic pressure (EDP) at the end of reperfusion; F) Total cardiac troponin I (cTnI) release during reperfusion in control (n=6), glucosamine (n=6) and PUGNAc (n=4) groups; *= p<0.05 compared to control group; one-way ANOVA with Bonfferoni’s Comparison Test.

Figure 2

A) ATP and B) UDP-GlcNAc levels at the end of reperfusion in control (n=6), glucosamine (n=6) and PUGNAc (n=4) groups; *= p<0.05 compared to I/R CTL group; one-way ANOVA with Bonfferoni’s Multiple Comparison Test.

Figure 3

Comparison of cardiac protein O-GlcNAc levels at the end of reperfusion in A) control (n=6) and glucosamine (n=6) treated groups and B) control (n=6) and PUGNAc (n=4) treated groups. In the upper panels are CTD110 immunoblots of solubilized proteins (left) and the mean intensities quantified by densitometric analysis of the immunoblots normalized to the mean intensities of control group (right). In the lower panels are the coomassie blue staining showing uniform protein loading of samples between groups. *= p<0.05 compared with control group; unpaired Students T-Test.

Figure 4

Western blot analysis of total and phosphorylated CaMKII and α-tubulin as a protein loading control in A) normoxic perfused hearts (n=4); untreated control (CTL) (n=5) and glucosamine (GlcN) (n=6) treated groups after ischemia/reperfusion and B) normoxic perfused hearts (n=4); untreated control (CTL) and (n=6) and PUGNAc (n=4) treated groups after ischemia/reperfusion; C) and D) are the mean intensities of total CaMKII; E) and F) are the mean intensities of phosphorylated CaMKII quantified by densitometric analysis of the immunoblots normalized to α-tubulin and presented relative to normoxic control; G) and H) are the ratios of phosphorylated to total CaMKII. *= p<0.05 compared with I/R control group; one-way ANOVA with Bonfferoni’s Multiple Comparison Test.

Figure 5

Western blot analysis of α-fodrin with α-tubulin as a protein loading control in A) normoxic perfused hearts (n=4); untreated control (CTL) (n=5) and glucosamine (GlcN) (n=6) treated groups after ischemia/reperfusion and B) untreated control (CTL) and (n=6) and PUGNAc (n=4) treated groups after ischemia/reperfusion. On the left are α-fodrin immunoblots and on the right are the mean intensities quantified by densitometric analysis of the 145/150 kD fragment; data are normalized to the mean intensities of normoxic control group. *= p<0.05 compared with I/R control group; one-way ANOVA with Bonfferoni’s Multiple Comparison Test.

Figure 6

Correlations between A) RPP and B) EDP and the 145/150 kDa α-fodrin fragment and between C) cTnI release and O-GlcNAc levels at the end of reperfusion. Data are presented for all control, glucosamine and PUGNAc groups.