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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online March 3, 2005 as doi:10.1096/fj.04-2780fje. |
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* Division of Surgical Science, Department of Surgery, College of Physicians and Surgeons, Columbia University, New York, New York, USA; and
Vascular Biology Center, Medical College of Georgia, Augusta, Georgia, USA
1 Correspondence: Division of Surgical Science, P& S 17-401 Columbia University, 630 West 168th St., New York, NY 10032, USA. E-mail; rr260{at}columbia.edu
PRINCIPAL AIMS
A number of mechanisms with diverse etiologies have been postulated to contribute to myocardial damage due to acute ischemia. In the ongoing efforts to reduce cell injury and death during an acute episode of ischemia and reperfusion, we and others have identified myocardial aldose reductase pathway as an important target for adjunctive therapeutic intervention. In this pathway aldose reductase (AR) catalyzes conversion of glucose to sorbitol in the presence of NADPH whereas sorbitol dehydrogenase (SDH) converts sorbitol to fructose in the presence of NAD+. Blockade of AR pathway at either AR or SDH protected ischemic myocardium and was associated with improved energy metabolism. A common feature of blocking AR or SDH was the attenuation of lactate/pyruvate (L/P) ratio in ischemic hearts, a measure of cytosolic NADH/NAD+ ratio. Numerous signaling molecules have been postulated to be affected by changes in NADH/NAD+ ratio, including protein kinase C (PKC). The aim of these studies was to investigate whether AR pathway influences JAK-STAT signaling by influencing NADH/NAD+ ratio and PKC activity. Using novel human AR transgenic mice and blockers of AR and SDH, we demonstrate for the first time that AR pathway induces JAK-STAT signaling in ischemic hearts and that these changes are due in part to increased NADH/NAD+ ratio and PKC activity. These data reveal a novel axis of signaling in ischemic myocardium originating from flux via AR pathway.
PRINCIPAL FINDINGS
1. Aldose reductase pathway influences JAK-STAT signaling
We used the isolated perfused rat heart protocol to determine the potential effect of AR pathway blockade on JAK-STAT signaling in ischemic hearts. Previous studies have demonstrated JAK-STAT signaling in ischemic hearts. We first explored the expression of JAK and its phosphorylated forms in normoxic and ischemic hearts (Fig. 1
A, B). Induction of global ischemia resulted in phosphorylation of JAK-2 as published earlier. Compared with untreated rat hearts, AR inhibition using zopolrestat blocked phosphorylation of JAK-2 in ischemic hearts. Phosphorylation of JAK-2 has been shown to activate a number of STAT proteins. Phosphorylation of STAT-5 was observed in ischemic hearts (Fig. 1A, C
) whereas STAT-1 and -3 were not phosphorylated. Blockade of AR resulted in attenuation of STAT-5 phosphorylation (Fig. 1A, C
).
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2. JAK-STAT activation dependent on changes in NADH/NAD+ and protein kinase C
The continuation of flux via SDH requires NAD+; hence substrate reduction by sorbitol dehydrogenase results in increased NADH, thus affecting the cytosolic NADH/NAD+ ratio. Lactate/pyruvate ratio, a measure of cytosolic NADH/NAD+, was reduced by inhibiting aldose reductase or sorbitol dehydrogenase (30–40% reduction in cytosolic redox ratio). To determine whether the above AR-associated changes in JAK-STAT phosphorylation requires flux via SDH, hearts were perfused with sorbitol dehydrogenase inhibitor CP-470,711. Western blots shown in Fig. 1A-C
demonstrate that SDH inhibition reduces phosphorylation of JAK-2 and STAT-5.
Niacin, a precursor of NAD+, was used to alter the cytosolic NADH/NAD+ ratio independent of the aldose reductase pathway. Western blot studies demonstrated that niacin blocks the phosphorylation of JAK-2 and STAT-5 (data not shown). Data from hearts perfused with SDH inhibitor or niacin indicate that the JAK-STAT pathway is affected by changes in cytosolic NADH/NAD+.
Changes in NADH/NAD+ ratio due to aldose reductase pathway flux can influence diacylglycerol levels, thus affecting PKC activity. Diacylglycerol levels (expressed as pmol/mg protein) were lower in ARI (49±8) and SDHI (56±12) -treated hearts compared with untreated hearts (104±19, P<0.05). Membrane-bound myocardial PKC activity was reduced by 55% in ARI- and by 62% in SDHI-treated ischemic hearts. These data indicate that flux via aldose reductase leads to increases in PKC activity via changes in DAG. Perfusion of hearts with Gö 6976, an inhibitor of PKC-
and -ß, or chelerythrine (inhibits most isoforms) blocked JAK-2 and STAT-5 phosphorylation in ischemic rat hearts. These data indicate that aldose reductase-mediated JAK-STAT signaling involves PKC- and -ß.
3. Pharmacological inhibition of JAK-2 protects ischemic rat hearts
The above studies demonstrate that activation of JAK-2 in ischemic hearts. Since this pathway is an important signal in events leading to cell death, we determined whether inhibition of JAK-2 would protect ischemic myocardium. Hearts perfused with JAK-2 inhibitor AG-490 displayed reduced ischemic injury (creatine kinase release, IU/g dry wt, during reperfusion was 1189±278 in untreated vs. 683Å125 in AG-490-treated, P<0.05) and improved left ventricular functional recovery upon reperfusion (38±15 in untreated vs. 83±10 in AG490-treated hearts, P<0.05), indicating that JAK-STAT pathway is an important component of ischemia-reperfusion injury.
4. JAK-STAT activation in aldose reductase transgenic mice
We used mice overexpressing human aldose reductase (ARTg) to further dissect the role of AR in JAK-STAT signaling. These ARTg mice showed levels of AR similar to those in humans. JAK-2 and STAT-5 phopshorylation was observed in ARTg mouse hearts under normoxic conditions (Fig. 2
A), which was further enhanced upon induction of global ischemia. JAK-2 and STAT-5 phosphorylation was significantly higher in ARTg mouse hearts than nontransgenic littermates under all perfusion conditions. Perfusion with inhibitors of AR (zopolrestat) or PKC (Gö 6976, an inhibitor of PKC- and -ß) reduced phosphorylation of JAK-2 and STAT-5 under baseline and ischemic conditions (Fig. 2B
). Membrane-bound PKC activity was greater in ARTg hearts (12.6±2.2 pmol of Pi transferred/mg protein/min) than in littermate (6.9±1.3 pmol of Pi transferred/mg protein/min) hearts (P<0.05) under baseline conditions; under ischemic conditions, the differences were 2.4-fold greater in ARTg vs. littermates. Baseline PKC activities were reduced in ARTg mouse hearts treated with ARI (5.6±2.8 pmol of Pi transferred/mg protein/min) and SDHI (4.8±3.1 pmol of Pi transferred/mg protein/min). Diacylglycerol levels were 2.1- and 2.6-fold higher in ARTg hearts than in littermates under baseline and ischemia, respectively (P<0.05). Inhibition of AR or SDH attenuated these diacylglycerol changes in ARTg mouse hearts. The changes in diacylglycerol and PKC activity were associated with increases in lactate/pyuvate ratio in ARTg hearts. These data indicate that increases in JAK2 and STAT5 phosphorylation in ARTg hearts are due in part to increases in PKC activity.
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To determine whether the JAK-STAT pathway is associated with increased ischemic injury in ARTg mice, JAK-2 inhibitor AG-490 was perfused in ARTg mouse hearts subjected to I/R. JAK-2 inhibition using AG490 reduced ischemic injury and improved functional recovery in ARTg mouse hearts (Fig. 2C
). Inhibition of PKC-ß with Gö 6976 protected hearts from ischemic injury in ARTg mice (Fig. 2C
). These data provide further evidence that aldose reductase-mediated JAK-STAT signaling involves PKC- and -ß.
CONCLUSIONS AND SIGNIFICANCE
The presence and importance of aldose reductase pathway in mediating ischemic injury have been demonstrated in recent studies. It was shown that flux via aldose reductase pathway in ischemic hearts increases cytosolic NADH/NAD+ ratio, impairs energy metabolism, reduces Na+,K+-ATPase activity, and impairs intracellular sodium and calcium homeostasis. Interventions that inhibit aldose reductase and or sorbitol dehydrogenase activity have been shown to reduce ischemic injury and improve energy homeostasis. The data presented in this study indicate that inhibition of aldose reductase or sorbitol dehydrogenase affords protection of ischemic myocardium by influencing cytosolic redox, PKC, and JAK-STAT signaling.
In this study, we demonstrate that ischemia in rat and mouse hearts induce JAK-2 and STAT-5 phosphorylation and that this activation is blocked by inhibiting aldose reductase or sorbitol dehydrogenase. Data indicate that lowering of cytosolic NADH/NAD+ is an important step whereby inhibition of aldose reductase or sorbitol dehydrogenase blocks JAK-STAT phosphorylation. Niacin, known to lower cytosolic NADH/NAD+ independent of the aldose reductase pathway, blocked JAK-STAT activation. These data indicate that changes in cytosolic NADH/NAD+ constitute an important event by which ischemia influences JAK-STAT signaling.
Several studies have shown that ischemia increases membrane PKC activity and expression. Inhibition of certain PKC isoforms has been shown to reduce myocardial injury due to ischemia-reperfusion. Here we show that inhibitors of aldose reductase or sorbitol dehydrogenase reduce membrane-bound PKC activity during ischemia and is associated with changes in diacylglycerol and cytosolic NADH/NAD+. It was demonstrated that inhibitors of PKC (/ß) block JAK-STAT activation in ischemia hearts. These results indicate that involvement of PKC-(/ß) in the aldose reductase pathway mediated JAK-STAT signaling. The beneficial effects of blocking JAK-2 or PKC-(/ß) or aldose reductase or sorbitol dehydrogenase clearly demonstrated signaling events that underlie ischemic injury. The data presented here show for the first time that flux via aldose reductase pathway mediates JAK-STAT signaling in ischemic hearts.
This paper demonstrates for the first time that the aldose reductase pathway influences JAK-2 and STAT-5 activation in ischemic hearts and that these changes are associated with increases in cytosolic NADH/NAD+ ratio, and PKC activity (Fig. 3
).
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2780fje;
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