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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online December 28, 2001 as doi:10.1096/fj.01-0368fje. |
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* Division of Cardiology, Department of Medicine, and
Department of Pathology, College of Physicians and Surgeons, Columbia University, New York, New York 10032, USA;
Laboratory of Ocular Therapeutics, National Eye Institute, National Institutes of Health, Bethesda, Maryland, USA; and
Pfizer Global Research and Development, Department of Cardiovascular and Metabolic Diseases, Groton, Connecticut 06340, USA
2Correspondence: Division of Cardiology, PH 10–403, College of P & S Columbia University, 630 W. 168th St., New York, NY 10032, USA. E-mail: rr260{at}Columbia.edu
SPECIFIC AIMS
Aldose reductase is monomeric, NADPH-dependent enzyme that is a member of the aldo-keto reductase family. This enzyme catalyzes the reduction of aldo sugars and other saturated and unsaturated aldehydes, and constitutes the first step of the polyol pathway. In this study, we have addressed the influence of ischemia on cardiac aldose reductase activity, as well as the effect of flux via aldose reductase on myocardial fatty acid and glucose metabolism.
PRINCIPAL FINDINGS
Ischemia increases myocardial aldose reductase activity
Aldose reductase activity increased during low-flow ischemia (Fig. 1
). Changes in NADPH absorption were monitored spectrophotometrically in the presence of DL-glyceraldehyde as substrate. To confirm that the reduction of glyceraldehyde was due to the presence of aldose reductase, the assay was performed with D-glucuronic acid or xylose as substrate. The lack of aldose reductase activity when glucuronic acid was used as a substrate clearly indicated the activity of aldose reductase in hearts (and not aldehyde reductase).
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Properties of aldose reductase from ischemic hearts
Since the activity of aldose reductase was higher in ischemic hearts, we performed kinetic studies of aldose reductase isolated and (200-fold) purified from control and ischemic rat heart extracts to examine for any potential differences in catalytic activity or turnover rate. The total amount of aldose reductase using the ELISA method was not significantly different in control and ischemic hearts. The Kcat and Vmax values were significantly higher in aldose reductase from ischemic hearts whereas Km values were not different. Although turnover rate (Kcat/Km) tends to be higher for aldose reductase from ischemic hearts, there was no statistically significant difference between the enzyme isolated from control and ischemic hearts. Control and ischemic hearts both clearly displayed immunostaining that corresponded exactly to that of purified aldose reductase. However, no obvious difference in the intensity of staining between the control and ischemic rats was observed. Similarly, data from Northern blot experiments demonstrated that aldose reductase mRNA expression was unchanged in control and ischemic hearts. Immunohistologic analysis of aldose reductase demonstrates the presence of aldose reductase antigen in myocytes with similar image intensity in control and ischemic hearts. These data suggest that increased aldose reductase activity in ischemic heart is not due to increased expression but to activation of the enzyme by endogenous factors.
Nitric oxide (NO) produced during ischemia influences myocardial aldose reductase activity
Since ischemia increases myocardial NO levels, it was examined whether this increase in NO might be responsible for activating aldose reductase. Figure 2
shows that inhibition of NO by L-NAME, an inhibitor of NO synthase, lowered aldose reductase activity. Incubation of homogenates from control hearts with an NO donor (SNAP) increased aldose reductase activity. These data suggest that NO produced during ischemia is one mechanism by which aldose reductase activation occurs.
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Aldose reductase inhibition improves myocardial glucose metabolism
Rates of glycolysis and glucose oxidation were significantly higher in zopolrestat-perfused hearts whereas rates of palmitate oxidation remained unaffected. Under ischemic conditions, rates of glycolysis remained significantly higher in aldose reductase-inhibited hearts (3696±351 in ARI vs. 2166±196 in control hearts, P=0.03). Consistent with increased glycolysis, ATP was significantly higher in aldose reductase-inhibited hearts during ischemia.
Lactate/pyruvate ratio, a measure of cytosolic redox state NADH/NAD+), was increased during ischemia, consistent with our earlier findings. Inhibition of aldose reductase with zopolrestat attenuated the increases in lactate/pyruvate ratio, suggesting that increases in cytosolic redox state are attenuated as well. One mechanism by which cytosolic redox state is likely to be attenuated by aldose reductase inhibition is by attenuating the flux of substrate through sorbitol dehydrogenase (which uses NAD+). Increases in flux via sorbitol dehydrogenase may decrease flux via glyceraldehyde-3-phosphate dehydrogenase by competing for NAD+. Lactate/pyruvate ratio data suggest that aldose reductase inhibition likely conserves NAD+ and aids glycolysis by reducing NAD+ use by sorbitol dehydrogenase.
Aldose reductase inhibition protects ischemic myocardium
The ability of aldose reductase inhibitor zopolrestat to reduce ischemic injury in the presence of fatty acids and insulin was determined in hearts perfused with physiological concentrations of palmitate and insulin. Creatine kinase release, a marker of ischemic injury, was significantly lower in aldose reductase-inhibited hearts, the magnitude of reduction in ischemic injury similar to those obtained earlier. Cardiac function was also improved in aldose reductase-inhibited subjected to ischemia-reperfusion. LVDP and EDP were similar in all groups under baseline conditions. During ischemia, the rise in EDP was greater in the control than in the aldose reductase-inhibited hearts. Upon reperfusion, the aldose reductase-inhibited hearts exhibited greater LVDP recovery than the untreated hearts.
CONCLUSIONS
Aldose reductase is a member of the aldo-keto reductase family with remarkably broad substrate specificity. Earlier studies from our laboratory demonstrated that pharmacological inhibition of aldose reductase protects perfused rat hearts from ischemic injury. Since aldose reductase inhibitors may inhibit aldehyde reductase (EC 1.1.1.2), another member of the aldo-keto reductase family, in addition to aldose reductase (EC 1.1.1.21), we investigated whether myocardial levels of aldose reductase changed under normal and ischemic conditions. We isolated and characterized myocardial aldose reductase and determined its influence on substrate metabolism and ischemic injury. To eliminate the NADPH reducing activity arising from aldehyde reductase, the activity of aldose reductase was assayed with an aldose D-xylose as substrate. Aldose reductase activity was higher in ischemic rat hearts than in control hearts. Aldose reductase was clearly detected on Western blots, but with no appreciable differences in the intensity of the protein from normal and ischemic hearts. In Northern blots, the signal for aldose reductase was much stronger than that for aldehyde reductase, again with no differences between the two normal and ischemic groups. The absence of any differences in Western and Northern blots of aldose reductase from normal and ischemic hearts suggests that the increased activity observed during ischemia is not due increased transcription or translation. However, these data clearly demonstrate the presence of aldose reductase in myocardial tissue under normal and ischemic conditions. The data also indicate that aldose reductase is the dominant aldo-keto reductase in the rat heart.
Aldose reductase has been known to exist in an activated form (oxidized form), with conversion to the activated form after purification as demonstrated in bovine aldose reductase and human placental aldose reductase. It is plausible that that similar conformational changes of aldose reductase may occur in rat hearts during ischemia. Our data were obtained on enzymes purified with the use of affinity chromatography and Orange A column. Since the activated (oxidized) enzyme does not bind to an Orange A column, the kinetic data presented here may have underestimated the effects of enzyme conversion to the oxidized form in ischemic hearts.
Reactive oxygen species have been shown to influence aldose reductase activity in cell culture. A recent study demonstrated that NO donors increase aldose reductase activity in cell culture. It is well known that ischemia increases myocardial NO levels. Hence, we performed ischemia experiments in the presence of NO synthase inhibitor to determine whether increases in aldose reductase activity is associated with increased NO production during ischemia. The data showed that inhibition of NO synthase attenuated the increases in aldose reductase activity during ischemia. Furthermore, we showed that incubation of cardiac homogenates with a NO donor increases aldose reductase activity. These data provide evidence that NO mediates activation of aldose reductase during ischemia.
The data demonstrating that ischemia increases myocardial aldose reductase activity and that pharmacological inhibition of aldose reductase is cardioprotective suggest that aldose reductase is a component of myocardial ischemic injury. Because of the complexities of cellular metabolic pathways, it is necessary to demonstrate that aldose reductase could affect substrate metabolism in response to ischemia. Pharmacological inhibition of aldose reductase increased myocardial glycolysis and glucose oxidation, as well as conserved ATP during ischemia. We also observed that aldose reductase inhibition in the presence of albumin-bound palmitate and insulin reduced ischemic injury, consistent with recent in vivo evidence. The cascade of events that result in reducing ischemic injury as a consequence of aldose reductase inhibition include attenuation of increases in cytosolic redox state, improved glycolytic metabolism and energy homeostasis, and attenuation of the rise in intracellular sodium and calcium during ischemia. The current studies indicate that aldose reductase is a key component of ischemic injury and provide a foundation for evaluating aldose reductase inhibitors as potential therapeutic adjuncts in treating patients with myocardial infarction.
In conclusion, aldose reductase was isolated from normal and ischemic myocardial tissue and its kinetic properties characterized. The enzyme activity was increased in ischemic hearts. Treatment with inhibitor of NO synthase attenuated ischemia induced increases in aldose reductase activity. Myocardial glycolysis and glucose oxidation were increased by inhibition of aldose reductase. In the presence of physiological concentrations of albumin-bound palmitate and insulin, inhibition of aldose reductase reduced ischemic injury and improved energy homeostasis in ischemic hearts. These data indicate that aldose reductase is a component of the myocardial response to ischemia and provide a foundation for evaluating aldose reductase inhibitors as potential therapeutic adjuncts in treating patients with myocardial infarction.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0368fje; to cite this article, use FASEB J. (December 28, 2001) 10.1096/fj.01-0368fje 
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