Aldose reductase modulates cardiac glycogen synthase kinase-3 ...

Am J Physiol Heart Circ Physiol 303: H297–H308, 2012. First published June 1, 2012; doi:10.1152/ajpheart.00999.2011.

Aldose reductase modulates cardiac glycogen synthase kinase-3␤ phosphorylation during ischemia-reperfusion Mariane Abdillahi,1,2 Radha Ananthakrishnan,1 Srinivasan Vedantham,1 Linshan Shang,2 Zhengbin Zhu,2 Rosa Rosario,1 Hylde Zirpoli,2 Kurt M. Bohren,3 Kenneth H. Gabbay,3 and Ravichandran Ramasamy1 1

Diabetes Research Program, Department of Medicine, New York University Langone Medical Center, New York, New York; Columbia University Medical Center, New York, New York; and 3Children’s Nutrition Research Center, Baylor College of Medicine, Houston, Texas

2

Submitted 19 October 2011; accepted in final form 25 May 2012

Abdillahi M, Ananthakrishnan R, Vedantham S, Shang L, Zhu Z, Rosario R, Zirpoli H, Bohren KM, Gabbay KH, Ramasamy R. Aldose reductase modulates cardiac glycogen synthase kinase-3␤ phosphorylation during ischemia-reperfusion. Am J Physiol Heart Circ Physiol 303: H297–H308, 2012. First published June 1, 2012; doi:10.1152/ajpheart.00999.2011.—Earlier studies have demonstrated that aldose reductase (AR) plays a key role in mediating ischemiareperfusion (I/R) injury. Our objective was to investigate if AR mediates I/R injury by influencing phosphorylation of glycogen synthase kinase-3␤ (p-GSK3␤). To investigate this issue, we used three separate models to study the effects of stress injury on the heart. Hearts isolated from wild-type (WT), human expressing AR transgenic (ARTg), and AR knockout (ARKO) mice were perfused with/ without GSK3␤ inhibitors (SB-216763 and LiCl) and subjected to I/R. Ad-human AR (Ad-hAR)-expressing HL-1 cardiac cells were exposed to hypoxia (0.5% O2) and reoxygenation (20.9% O2) conditions. I/R in a murine model of transient occlusion and reperfusion of the left anterior descending coronary artery (LAD) was used to study if p-GSK3␤ was affected through increased AR flux. Lactate dehydrogenase (LDH) release and left ventricular developed pressure (LVDP) were measured. LVDP was decreased in hearts from ARTg mice compared with WT and ARKO after I/R, whereas LDH release and apoptotic markers were increased (P ⬍ 0.05). p-GSK3␤ was decreased in ARTg hearts compared with WT and ARKO (P ⬍ 0.05). In ARKO, p-GSK3␤ and apoptotic markers were decreased compared with WT (P ⬍ 0.05). WT and ARTg hearts perfused with GSK3␤ inhibitors improved p-GSK3␤ expression and LVDP and exhibited decreased LDH release, apoptosis, and mitochondrial pore opening (P ⬍ 0.05). Ad-hAR-expressing HL-1 cardiac cells, exposed to hypoxia (0.5% O2) and reoxygenation (20.9% O2), had greater LDH release compared with control HL-1 cells (P ⬍ 0.05). p-GSK3␤ was decreased and correlated with increased apoptotic markers in Ad-hAR HL-1 cells (P ⬍ 0.05). Treatment with phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) inhibitor increased injury demonstrated by increased LDH release in ARTg, WT, and ARKO hearts and in Ad-hAR-expressing HL-1 cells. Cells treated with protein kinase C (PKC) ␣/␤ inhibitor displayed significant increases in p-Akt and p-GSK3␤ expression, and resulted in decreased LDH release. In summary, AR mediates changes in p-GSK3␤, in part, via PKC␣/␤ and Akt during I/R. aldose reductase; ischemia reperfusion; glycogen synthase kinase 3␤; cardioprotection

using thrombolytic or surgical approaches have helped salvage ischemic myocardium, damage observed during the reperfusion phase attenuates the ben-

WHILE RECANALIZATION THERAPY

Address for reprint requests and other correspondence: R. Ramasamy, Diabetes Research Program, Smilow Research Bldg. 901, 550 First Ave., New York, NY 10016 (e-mail: [email protected]). http://www.ajpheart.org

efits of these interventions (2, 45). Therapeutic strategies that target both the ischemic and reperfusion components of myocardial injury are likely to afford protection against acute myocardial ischemia-reperfusion (I/R). In the quest for novel therapeutic strategies for acute I/R injury, we have focused on interventions that protect against myocardial I/R injury by modulating substrate metabolism. In this context, we and others have demonstrated that the aldose reductase (AR) pathway contributes to myocardial I/R injury and that the inhibition of AR protects hearts from I/R damage (1–2, 16 –17, 19, 35–36, 40, 45). AR, a member of the aldo-keto reductase family, is a monomeric NADPH-dependent enzyme and the first, ratelimiting step in the polyol pathway (11, 17, 21, 34). Glucose substrate flux via AR increases under ischemic conditions, (17, 41) even in the absence of diabetes, and negatively impacts the myocardium by increasing oxidative stress, impairing ATP production as a result of altered glucose metabolism, and impairing calcium homeostasis, conditions that favor the opening of the mitochondrial permeability transition pore (mPTP) (1, 6, 13–14, 20). Preserving mitochondrial function is essential for normal recovery after I/R (1, 4, 14, 24). Opening of the mPTP has been implicated in upregulation of apoptotic and necrotic cell death mechanisms due to loss of membrane potential and ATP depletion (1, 6, 13). Studies have shown that use of cardioprotective agents increased the phosphorylation and inhibition of glycogen synthase kinase-3␤ (GSK3␤), resulting in a delayed opening of the mPTP. GSK3␤, a serine/threonine kinase, has been identified by several studies as a key signaling protein that mediates cardioprotection and reduces cell death (28 –29). GSK3␤ phosphorylates numerous substrates, including transcription factors and metabolic proteins, and is involved in several cellular processes such as gene transcription, apoptosis, and cell division (22–23). A wide spectrum of signaling pathways have been associated with the phosphorylation and inhibition of GSK3␤ (5, 32). We have previously demonstrated that increased metabolic flux through AR mediates I/R injury, in part, via changes in protein kinase C (PKC) ␣/␤-dependent signaling (18) and mPTP opening (1). Because phosphorylation of GSK3␤ is a key determinant of mPTP opening, we investigated if AR mediates I/R injury, in part, by adversely influencing GSK3␤ phosphorylation in murine hearts. We used three separate models to study whether AR mediates I/R injury by influencing phosphorylation of GSK3␤ (p-GSK3␤): HL-1 cardiomyocytes an in vitro I/R injury model, an ex vivo intact heart preparation subjected to I/R, and a transient occlusion and reperfusion of the left

0363-6135/12 Copyright © 2012 the American Physiological Society

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anterior descending coronary artery (LAD) in vivo model of I/R. Because mice AR activity is severalfold lower than that in rats and humans (16, 34), we employed transgenic mice overexpressing human aldose reductase (ARTg) as well as an aldose reductase knockout mouse model (ARKO) to determine whether altered flux via AR influenced GSK3␤ phosphorylation. MATERIALS AND METHODS

Animals. All animal experiments were approved by the Institutional Animal Care and Use Committees of Columbia University, New York University School of Medicine, and Baylor College of Medicine and conformed to the guidelines outlined in the National Institutes of Health Guide for Care and Use of Laboratory Animals (NIH Pub. No. 85–23, 1996). Male C57BL/6J mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and were used as control wild-type (WT) mice. Male mice weighing 25–30 g at age 12–14 wk were used in all experiments and maintained in a temperature-controlled room with alternating 12:12-h light-dark cycles. ARTg mice were obtained from Dr. Mitsuo Itakura (University of Tokushima), and a colony was established at our facility. Briefly, these transgenic mice were developed by injecting full-length human AR (hAR) cDNA with a mouse major histocompatibility antigen class I promoter (43). ARKO mice were generated as described recently (8). The hAR transgenic and ARKO mice have been backcrossed ⬎10 generations to develop a C57BL/6J background. Reagents. The primary antibodies used were anti-phospho-GSK3␤/ total GSK3␤, anti-phospho-protein kinase B (Akt; Thr308 and Ser473)/ total Akt IgG (Cell Signaling), anti-cytochrome c IgG (BD Pharmingen), and anti-␤-actin IgG (BD Biosciences Pharmingen). The secondary antibodies used were goat-anti-rabbit IgG-peroxidase antibody and rabbit-anti-mouse IgG-peroxidase antibody (Sigma). All primary antibodies were diluted 1:1,000 before use in Western blot studies. GSK3 inhibitors LiCl and SB-216763 and PKC␣/␤ inhibitor Gö-6976 were purchased from Sigma. Phosphatidylinositol 3-kinase (PI3K)/ Akt inhibitor LY-294002 was purchased from Calbiochem. Isolated perfused heart preparation. Experiments were performed using an isovolumic isolated heart preparation as published and modified for the use in mice hearts (1, 16). Mice were anesthetized using a mixture of ketamine (80 mg/kg) and xylazine (10 mg/kg). After deep anesthesia was achieved, hearts were rapidly excised, placed in iced saline, and retrogradely perfused at 37°C in a nonrecirculating mode through the aorta at a rate of 2.5 ml/min. Hearts were perfused with modified Krebs-Henseleit buffer containing (in mM) 118 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgCl2, 25 NaHCO3, 5 glucose, 0.4 palmitate, 0.4 BSA, and 70 mU/l insulin. The perfusate was equilibrated with a mixture of 95% O2-5% CO2, which maintained perfusate PO2 ⬎600 mmHg. Left ventricular developed pressure (LVDP) was measured using a latex balloon in the left ventricle. LVDP and coronary perfusion pressure were monitored continuously on a fourchannel Gould recorder. Surgical procedures. Surgical procedures relating to coronary artery ligation were carried out as previously described (16). Briefly, the LAD was ligated including the cannula for 30 min. After 30 min of ischemia, the LAD blood flow was restored. Heart samples from the affected ischemic area were further evaluated at 48 h of reperfusion. Appropriate sham-treated animals subjected to anesthesia and surgical procedure without occluding LAD were included. I/R protocol. Hearts from WT, ARTg, and ARKO hearts were perfused with Krebs-Henseleit buffer throughout the I/R protocol. After an equilibration period of 30 min, global ischemia was performed for 30 min followed by 60 min of reperfusion. Perfusate temperature was maintained at 37°C at all times during the protocol (i.e., during baseline, ischemia, and reperfusion).

To determine the link between AR and GSK3␤ after I/R injury, experiments were performed in the presence of the following GSK3␤ inhibitors: SB-216763 (3 ␮M) and LiCl (3 mM) as well as PI3K/Akt inhibitor (LY-294002, 10 ␮M). Hearts from WT and ARTg mice were perfused with modified Krebs-Henseleit buffer containing 3 ␮M SB-216763 or 3 mM LiCl. The doses of the inhibitors used in this study were based on publications in the literature (15, 31, 37). Inhibitors were present at the start of the equilibration period and continued throughout ischemia and reperfusion. AR activity. AR activity in the hearts of WT, hAR (ARTg), and AR null (ARKO) mice was evaluated by measuring NADPH consumption using D-glyceraldehyde as a substrate (17). One unit of AR activity is defined as nanomoles of NADPH consumed per milligram of heart tissue protein per minute. Mitochondrial swelling as a measure of mPTP opening. We employed the mitochondrial swelling as a measure of mPTP opening. For measurement of mPTP opening, mitochondria were suspended in freshly prepared swelling buffer (0.2 M sucrose, 10 mM Tris-MOPS, pH 7.4, 5 mM succinate, 1 mM phosphate, 2 ␮M rotenone, and 1.0 ␮M EGTA-Tris, pH 7.4) at 0.5 mg/ml, and swelling of mitochondria was monitored by decrease in absorbance at 540 nm in the presence of CaCl2 (5–100 ␮M). Extent of pore opening was expressed in terms of changes in absorbance at 540 nm/min in the presence and absence of Ca2⫹. Culture and transfection of HL-1 cardiomyocytes. Immortalized HL-1 cardiomyocytes were a gift from Dr. William Claycomb (Louisiana State University, New Orleans, LA) (42). HL-1 cell line was chosen because they were derived from mice cardiac muscle cells (42). Cells were plated on gelatin/fibronectin-coated six-well plates at a density of 4 ⫻ 105 cells/well. HAR was overexpressed in HL-1 cardiomyocytes through viral-mediated transfection. Appropriate empty vector was transfected as a control. For small-interfering RNA (siRNA) transfection, HL-1 cells were cultured in six-well plates and transfected with control or 40 nM GSK3␤ siRNA using Lipofectamine RNAiMax reagent (Invitrogen) according to the manufacturer’s instructions. HL-1 cardiomyocytes were subjected to hypoxic stress for 30 min (0.5% O2) using an In Vivo 400 hypoxic workstation maintained at 37°C, followed by 60 min of reoxygenation (20.9% O2). In specific experiments, cardiomyocytes were treated with either GSK3␤ siRNA and/or PI3K/Akt (10 ␮M) and PKC␣/␤ inhibitors (1 ␮M). Western blot analysis. The tissue and cell protein concentration was determined using a DC Protein Assay kit (Bio-Rad). Equal amounts of protein were separated by SDS-PAGE (4 –12% gradient gels), and proteins were transferred to a nitrocellulose membrane (Invitrogen). After blocking in 5% dry milk in TBST (20 mM Tris·HCl, pH 7.5, 250 mM NaCl, and 0.1% Tween 20), membranes were incubated overnight with target primary antibodies (1:1,000 dilution) according to the manufacturer’s instructions. Membranes were incubated sequentially with secondary antibody for 1 h. Blots were visualized with an ECL Horseradish Peroxidase Western Blot Detection System (Cell Signaling), and quantitative analysis was performed using Image Quant TL software (Amersham). LDH measurements. Cardiac injury due to I/R stress was assessed by measuring LDH release in the perfusates that were collected during 60 min of reperfusion. In HL-1 cardiomyocytes, injury due to hypoxia/reoxygenation (H/R) stress was measured in supernatants that were collected after H/R. Lactate dehydrogenase (LDH) was measured using the commercially available kits (Pointe Scientific) as published earlier (1, 37). Statistical analysis. All data are presented as means ⫾ SE. Statistical significance of differences between various groups of heart was determined by ANOVA. Post hoc comparisons were performed with Tukey or Dunnett procedures using GraphPad software as indicated. Statistical significance was ascribed to the data when P ⬍ 0.05.

AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00999.2011 • www.ajpheart.org


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Fig. 1. Glycogen synthase kinase-3␤ (GSK3␤) phosphorylation expression in wild-type (WT), aldose reductase transgenic (ARTg), and aldose reductase knockout (ARKO) mice. Western blot analysis for phospho (p)-GSK3␤-Ser9 [isolated heart (A) and LAD ligation (C)], p-GSK3␤-Tyr216 [isolated heart (B) and LAD ligation (D)], cytochrome c (E), and aldose reductase (AR) expression (F) in untreated WT, ARTg, and ARKO mice hearts subjected to ischemiareperfusion (I/R). ARTg hearts showed decreased p-GSK3␤ inhibition compared with WT hearts. ARKO hearts had significant increases in p-GSK3␤ (Ser9) (P ⬍ 0.05). ARTg hearts displayed a more than threefold increase in apoptosis compared with WT and ARKO hearts as was measured by cytochrome c expression (P ⬍ 0.05). ARKO hearts displayed significant decreases in apoptotic levels compared with WT (P ⬍ 0.05). ARTg hearts displayed a twofold increase in AR expression compared with WT (n ⫽ 4 –16 mice/group). AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00999.2011 • www.ajpheart.org

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Fig. 2. Pharmacological inhibition of GSK3␤ protects mice hearts upon I/R. Determination of myocardial ischemic injury and function with and without GSK3␤ inhibitor treatment as shown by left ventricular developed pressure (LVDP) recovery (A) and lactate dehydrogenase (LDH) release (B). Western blot analysis for p-GSK3␤-Ser9 (C), p-GSK3␤-Tyr216 (D), and cytochrome c (E) in WT and ARTg hearts perfused with and without GSK3␤ inhibitors SB-216763 and LiCl. Treatment with SB-216763 and LiCl protected WT and ARTg mice hearts, significantly increased p-GSK3␤ inhibition (P ⬍ 0.05), and decreased apoptosis (n ⫽ 4 –16 mice/group).


ALDOSE REDUCTASE MODULATES GLYCOGEN SYNTHASE KINASE-3␤ RESULTS

GSK3␤ phosphorylation expression in WT, ARTg, and ARKO mice. We used the isolated Langendorff perfusion system and intact transient occlusion and reperfusion of the LAD to establish the influence, if any, of AR-mediated ischemic injury on the phosphorylation and inhibition of GSK3␤. Previous studies have shown that the phosphorylation and inhibition of GSK3␤ is cardioprotective (28 –29). We first examined GSK3␤ phosphorylation at both the Ser9 and Tyr216 sites on GSK-3␤ after a 30-min baseline period only. Results show that there are no differences in GSK3␤ phosphorylation in WT, ARTg, and ARKO hearts after baseline (data not shown). We then determined whether GSK3␤ phosphorylation was influenced after I/R. Induction of I/R resulted in decreased inhibition of GSK3␤ demonstrated by decreased levels of p-GSK3␤ phosphorylated at Ser9 and increased levels of p-GSK3␤ phosphorylated at Tyr216 in ARTg mice hearts compared with WT mice (P ⬍ 0.05, Fig. 1, A and B). Phosphorylation of GSK3␤ on the Ser9 site was also significantly increased in ARKO hearts compared with WT (Fig. 1A). Similarly, 30 min of LAD occlusion followed by 48 h of reperfusion resulted in a decreased p-GSK3␤ expression on Ser9 and increased p-GSK3␤ on Tyr216 (Fig. 1, C and D). Furthermore, ARTg hearts displayed a more than threefold increase in apoptosis compared with WT and ARKO hearts as measured by cytochrome c expression in total lysates (P ⬍ 0.05, Fig. 1E). ARTg mice displayed a more than twofold increase in AR expression compared with WT mice, whereas ARKO hearts had negligent AR expression (Fig. 1F). Additionally, AR activity assay revealed that AR activity in ARTg mice hearts was greater, 16 nmol of NADPH consumed·min⫺1·mg of protein⫺1, than when compared with WT hearts, 1.63 nmol of NADPH consumed·min⫺1·mg of protein⫺1. ARKO hearts had no detectable units of AR activity. Pharmacological inhibition of GSK3␤ protects mice hearts upon I/R. Because ARTg hearts displayed decreased inhibition of GSK3␤, we next sought to further test the role of GSK3␤ in mediating I/R injury. WT and ARTg hearts were treated with GSK3␤ inhibitors SB-216763 and LiCl and were perfused under normoxic conditions for 30 min, followed by 30 min of ischemic conditions and 60 min of reperfusion conditions. LiCl targets the inhibitory phosphorylation site of GSK3␤-Ser9 and

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works to increase phosphorylation, whereas SB-216763 targets the stimulatory phosphorylation site GSK3␤-Tyr216 and works to decrease phosphorylation. Treatment with SB-216763 and LiCl protected WT and ARTg mice hearts significantly as shown in Fig. 2, A and B. Improvement of LVDP (P ⬍ 0.05, Fig. 2A) was observed in WT and ARTg hearts treated with both SB-216763 and LiCl. Similarly, Fig. 2B shows decreased release of LDH (P ⬍ 0.05, Fig. 2B) in WT and ARTg hearts upon treatment with both inhibitors. Treatment with LiCl and SB-216763 significantly inhibited GSK3␤ in both WT and ARTg hearts (P ⬍ 0.05, Fig. 2, C and D). WT and ARTg hearts perfused with both GSK3␤ inhibitors displayed markedly reduced apoptotic levels as was demonstrated by reduced cytochrome c (P ⬍ 0.05, Fig. 2E) expression. Pharmacological inhibition of GSK3␤ abolished mPTP pore opening in mitochondria from WT and ARTg hearts, as shown by the mitochondrial swelling changes in response to increasing amounts of added calcium (Fig. 3). PI3K/Akt is a well-established upstream kinase that has been shown to phosphorylate and inhibit GSK3␤ at the serine residue. We investigated Akt phosphorylation expression patterns in WT, ARTg, and ARKO hearts after I/R. Transient LAD occlusion for 30 min followed by 48 h of reperfusion resulted in significant decreases in p-Akt expression in ARTg hearts when compared with WT hearts (Fig. 4A). p-Akt expression in ARKO hearts was significantly lower compared with WT hearts (Fig. 4A). To determine whether Akt is a key player by which AR mediates I/R injury, we used the PI3K/Akt inhibitor LY-294002 during I/R. Functional recovery was impaired in WT, ARTg, and ARKO hearts in the presence of LY-294002 as was demonstrated by significant decreases in LVDP (P ⬍ 0.05, Fig. 4B). As shown in Fig. 4C, LDH release was increased significantly in WT, ARTg, and ARKO hearts subjected to I/R stress in the presence of LY-294002. Marked reductions in p-Akt levels were observed in WT, ARTg, and ARKO hearts treated with LY-294002 (P ⬍ 0.05, Fig. 4D). Consequentially, p-GSK3␤ levels were decreased in all three groups after I/R when in the presence of the PI3K/Akt inhibitor (P ⬍ 0.05, Fig. 4E). Inhibition of the PI3K/Akt pathway resulted in significant increases in apoptotic levels as was measured by cytochrome c expression (P ⬍ 0.05, Fig. 4F).

Fig. 3. Mitochondrial permeability in WT and ARTg hearts treated with and without GSK3␤ inhibitors SB-216763 and LiCl and subjected to I/R. Mitochondrial permeability transition pore opening (mPTP) was determined by monitoring swelling of mitochondria by measuring light scattering at 540 nm in the presence and absence of 50 ␮M (A) and 100 ␮M (B) Ca2⫹. Pore opening was reduced in WT and ARTg hearts treated with SB-216763 and LiCl in response to increasing calcium concentrations (P ⬍ 0.05) (n ⫽ 4 –16/group).


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Fig. 4. p-Protein kinase B (Akt). Western blot analysis for p-Akt in LAD ligation of WT, ARTg, and ARKO hearts (A), determination of myocardial ischemic injury and function, as shown by LVDP (B) and LDH release (C) in WT, ARTg, and ARKO perfused with and without phosphatidylinositol 3-kinase (PI3K)/AKT inhibitor LY-294002. Western blot analysis for p-Akt (D), p-GSK3␤-Ser9 (E), and cytochrome c (F) in WT, ARTg, and ARKO hearts perfused with and without PI3K/Akt inhibitor LY-294002. Treatment with LY-294002 decreased p-Akt and p-GSK3␤ levels in all 3 groups (P ⬍ 0.05). Cytochrome c levels were increased in WT and ARKO hearts (P ⬍ 0.05), but not ARTg upon perfusion with LY-294002 (n ⫽ 4 –16 mice/group).



These data indicate that reduction in GSK3␤ phosphorylation is in part due to reduced PI3K/Akt phosphorylation. Akt and GSK3␤ phosphorylation expression in hAR-overexpressing HL-1 cells. We employed the murine cardiomyocyte HL-1 cell line to identify where the signaling mechanisms are occurring in the myocardium. We opted to use the HL-1 cardiomyocyte cell line due to the problems we encountered in

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our previous attempts to isolate adult cardiomyocytes, including cell death within a very short period of time, which made transfection studies extremely difficult, and failed attachment of the cells to culture plates. We used siRNA to inhibit GSK3␤ and to determine whether GSK3␤ knock down would decrease cellular injury and apoptosis. Different concentrations of GSK3␤ siRNA were used to determine the least amount

Fig. 5. GSK3␤ small-interfering RNA (siRNA) knock down in HL-1 cells. Western blot analysis for total GSK3␤ dose-dependent siRNA knockdown (A) and AR expression (B). Determination of cardiac injury as determined by LDH release in control and human AR (hAR) siRNA knock down of GSK3␤ (C). D: Western blot analysis of cytochrome c expression in control and hAR-overexpressing cells transfected with GSK3␤ siRNA. No change in LDH release was observed in control and hAR-overexpressing cells exposed to 30 min of normoxia alone. Thirty minutes of hypoxia followed by 60 min of reoxygenation increased LDH release (P ⬍ 0.05), whereas inhibiting GSK3␤ with siRNA significantly reduced LDH release and injury (P ⬍ 0.05). hAR-transfected cells, as expected, had greater AR expression than control cells. There was no difference in AR expression between HL-1 control cells subjected to normoxic and H/R conditions. GSK3␤ siRNA knock down significantly reduced GSK3␤ expression in both control and hAR-overexpressing cells. Inhibition of GSK3␤ significantly reduced apoptotic levels in both control and hAR-overexpressing cells. NS, not significant. Error bars not visible. Control normoxia: SE ⫾ 0.003; control hAR: SE ⫾ 0.006; control GSK3␤ siRNA: ⫾0.005. AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00999.2011 • www.ajpheart.org

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necessary for efficient GSK3␤ knock down. Approximately 60% GSK3␤ knock down was achieved in hAR-overexpressing HL-1 cardiomyocytes with a 40 nM siRNA concentration (Fig. 5A). Cells were transiently transfected with hAR virus (Fig. 5B). LDH measured in the supernatants after GSK3␤ siRNA transfection and H/R stress was markedly decreased in both control and hAR-expressing cells (Fig. 5C). As was expected, GSK3␤ siRNA knock down in control and hARexpressing cells significantly reduced cytochrome c levels (P ⬍ 0.05, Fig. 5D). HL-1 cells expressing hAR and empty control vectors were exposed to H/R stress to determine p-Akt and p-GSK3␤ patterns. Similar to what was found in ARTg hearts during I/R, the expression of p-Akt was reduced signif-

icantly in Ad-hAR-expressing cells during H/R stress (P ⬍ 0.05, Fig. 6A). Decreased expression of p-Akt corresponded with decreased phosphorylation and inhibition of GSK3␤. We analyzed the expression of p-GSK3␤ at both the Ser9 and Tyr216 residues in hAR-overexpressing cells and found that phosphorylation of GSK3␤ was decreased significantly on the Ser9 site and increased on the Tyr216 site (P ⬍ 0.05, Fig. 6, B and C) after H/R. As was observed in ex vivo heart I/R injury, decreased phosphorylation and inhibition of GSK3␤ correlated with increased cell death after H/R as was measured by the increased expression of cytochrome c in hAR-overexpressing cells (Fig. 6D). Additionally, cardiac injury as measured by LDH release in supernatants from hAR-overexpressing cells

Fig. 6. HL-1 cardiomyocyte studies. Western blot analysis for p-Akt (A), p-GSK3␤-Ser9 (B), p-GSK3␤-Tyr216 (C), and cytochrome c (D) in control and hAR-overexpressing HL-1 cells subjected to H/R alone. E: determination of cardiomyocyte H/R injury as shown by LDH release in control and hARoverexpressing HL-1 cell supernatants. F: LDH release in control and hAR-overexpressing HL-1 cells treated with and without PI3K/Akt inhibitor LY-294002. Western blot analysis for p-GSK3␤-Ser9 (G) and cytochrome c (H) in control and hAR-overexpressing HL-1 cells treated with and without PI3K/Akt inhibitor LY-294002. Cells were incubated with LY-294002 (10 ␮M) or its vehicle control dimethyl sulfoxide (DMSO) for 1 h, followed by 30 min of hypoxia and 1 h reoxygenation (H/R). Treatment with LY-294002 decreased p-Akt levels, decreased inhibition of GSK3␤, and increased apoptosis in both control and hAR-overexpressing cells. LDH release in supernatants in both control and hAR-overexpressing cells was increased with PI3K/Akt inhibitor LY-294002 treatment. Error bars not visible. Control: SE ⫾ 0.006; and hAR: SE ⫾ 0.011. AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00999.2011 • www.ajpheart.org


after H/R was increased significantly (Fig. 6E). HL-1 cells overexpressing hAR and control cells were treated with the PI3K/Akt signaling inhibitor LY-294002 during H/R to establish if Akt phosphorylation is a key event that mediates injury. As shown in Fig. 6F, both control and hAR-overexpressing cells treated with the Akt signaling inhibitor displayed marked increases in LDH release after H/R, which was parallel to experiments in the ex vivo I/R heart preparations. In addition to the increased LDH release, LY-294002 treatment significantly reduced p-GSK3␤ expression (P ⬍ 0.05, Fig. 6G) and resulted in increased apoptotic levels as shown by increased cytochrome c levels (P ⬍ 0.05, Fig. 6H). To further elaborate and clarify the role of PKC in ARmediated I/R injury, we have previously demonstrated that increased metabolic flux through AR mediates I/R injury, in part, via changes in PKC␣/␤-dependent signaling. Flux via the polyol pathway increases the cytosolic ratio of NADH/NAD⫹, which results in increased levels of diacylglycerol and, subsequently, increased PKC activation (18). We have also shown earlier that flux via AR leads to activation of PKC␣/␤ and that inhibition of PKC␣/␤ with Gö-6976 attenuates I/R injury (18). In addition, the PI3K/Akt pathway has been shown to be a downstream target of PKC␣/␤ (9). In the current study, to

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investigate if AR-driven changes in PKC␣/␤ influence GSK3␤ phosphorylation, we treated HL-1 cells with PKC␣/␤ inhibitor Gö-6976 and subjected them to H/R stress. PKC inhibition resulted in increased p-Akt (P ⬍ 0.05, Fig. 7A) and p-GSK3␤ (P ⬍ 0.05, Fig. 7B) in Ad-hAR-expressing HL-1 cells. Furthermore, treatment of HL-1 cells with Gö-6976 resulted in a marked reduction of injury as shown by decreased LDH release (P ⬍ 0.05, Fig. 7C). These data establish that AR modulates p-GSK3␤, in part, via PKC␣/␤ in cardiac cells. DISCUSSION

Previous studies demonstrated the important role of AR in mediating myocardial I/R injury (1, 16 –19, 34 –36, 40). Our goal in this study was to investigate if AR-mediated I/R injury is linked to changes in phosphorylation of GSK3␤ and whether these changes would correlate with the impaired ability of the myocardium to recover after an ischemic insult. Consistent with earlier results, we show that transgenic mice expressing hAR have increased I/R injury (1, 18). Hearts from ARKO mice demonstrated a significant increase in LVDP recovery compared with WT mice. Our earlier studies showed that inhibition of AR in WT mice reduced injury and also improved

Fig. 7. Protein kinase C (PKC) ␣/␤ inhibition. Western blot analysis for p-Akt (A) and p-GSK3␤ (B). C: determination of cardiomyocyte injury as shown by LDH release. Treatment with Gö-6976 resulted in decreased cellular injury and an increase in p-GSK3␤. Cells were incubated with Gö-6976 (1 ␮M) or its vehicle control DMSO for 1 h, followed by 30 min of hypoxia and 1 h reoxygenation (H/R). AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00999.2011 • www.ajpheart.org

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function. We have previously shown that, after treatment with Zopolrestat, an aldose reductase inhibitor, WT mice had significant decreases in the opening of the mPTP. This can be explained in part because flux via AR increases after ischemia (34). Here, we show that inhibition of AR (ARKO) significantly protects the myocardium as a result of increased phosphorylation of GSK3␤ and a reduction in apoptosis. We demonstrate that AR mediates I/R injury, in part, via decreased phosphorylation of GSK3␤ (Ser9) thereby impairing mitochondrial function, as well as functional recovery of the heart. In these studies, impaired mitochondrial function in ARTg hearts after I/R resulted in both necrotic and apoptotic cell death mechanisms as demonstrated by increased mPTP opening and increases in cytochrome c expression. Phosphorylation of GSK3␤ was decreased significantly in ARTg mouse hearts as well as in hAR-overexpressing HL-1 cells. Further experiments indicated that, upon pharmacological inhibition of GSK3␤, significantly higher levels of p-GSK3␤ were observed in ARTg hearts subjected to I/R and hAR-overexpressing cells subjected to H/R. The increases in levels of p-GSK3␤ correlated with decreased apoptosis in GSK3␤-inhibited ARTg hearts. Our efforts to further elucidate the signaling mechanisms that contribute to the decreased phosphorylation of GSK3␤ highlight signaling via Akt, which functions upstream of GSK3␤ as a key player in mediating I/R injury. Inhibition of the PI3K/Akt pathway resulted in significant cardiac injury and impairment of cardiac recovery function. Treatment with PI3K/Akt inhibitor decreased p-Akt and p-GSK3␤ levels. Two mammalian isoforms of GSK3 have been identified [GSK3␣ and GSK3␤ (29)]. The GSK3␤ isoform regulates numerous cell processes, including apoptosis and proliferation, and has been shown to enhance cardioprotection in several studies (3, 10, 12). The activity of GSK3␤ is regulated by phosphorylation (22–23, 29). The phosphorylation and inhibition of GSK3␤ is a major component of its cardioprotective role. Several studies have shown that the inhibition of GSK3␤ reduces apoptosis and confers cardioprotection depending on the degree and duration of inhibition (29, 32). The use of cardioprotective agents in a cardiomyocyte model has been shown to phosphorylate and inhibit GSK3␤ and further protects by inhibiting mPTP opening (23). Cardiomyocytes from mice with a constitutively active GSK3␤ were not protected upon treatment with cardioprotective agents. Other studies have shown a reduction in infarct size and an improvement in postischemic cardiac function with the use of GSK3␤ inhibitors (23, 27, 29, 39). Consistent with these data of cardioprotection achieved by inhibiting the activity of GSK3␤, our data show significantly enhanced phosphorylated GSK3␤ upon treatment with GSK3␤ inhibitors after I/R injury, which correlated with enhanced cardioprotection and a reduction in apoptosis in ARTg mice hearts. Analogous to heart ex vivo I/R experiments, siRNA knock down of GSK3␤ in hAR-expressing cells was protected against H/R stress. Several kinases upstream converge and inhibit GSK3␤. GSK3␤ has emerged as the integration point of many of these pathways and plays a central role in transferring protective signals downstream to target(s) (23, 26). One of these upstream regulators includes Akt/PKB, a Ser/Thr kinase that has been shown to phosphorylate and inhibit GSK3␤ on the serine residue (5). Fujio et al. (7) have shown that Akt functions to promote cardiomyocyte survival and protects against I/R in the

mouse hearts. Our results show that the metabolic flux through AR mediates I/R injury, in part, via decreased Akt activation. Furthermore, in hearts from WT and ARKO mice perfused with PI3K/Akt signaling inhibitors, we observed marked increases in LDH and impaired functional recovery during reperfusion. The PI3K/Akt inhibitor resulted in marked decrease in the levels of phosphorylated GSK3␤ that consequentially led to increased cell death and apoptosis. These data lead us to believe that Akt and GSK3␤ are key kinases that participate in the signaling mechanisms that mediate functional and cardiac recovery after I/R injury as a result of increased flux via AR. Decreased phosphorylation of these kinases resulted in the impairment of cardiac recovery that contributed to the vulnerability of the myocardium to recover after an ischemic insult. Studies by us (18) and others (33, 38) have demonstrated that increased flux via AR leads to activation of PKC (␣/␤) in euglycemic and hyperglycemic conditions. Furthermore, previous reports in the literature have alluded to the selective inhibition of PI3K/Akt as a result of PKC activation in endothelial cells (30). The mechanism of PI3K/Akt inhibition has been attributed to activation of the PKC␤ isoform in particular, due to hyperglycemia. Hence, we investigated the role of PKC activation and GSK3␤ phosphorylation. Naruse et al. (30) have demonstrated that inhibition of PKC␤ resulted in improved glomerular endothelial cell function and improved insulin action. Furthermore, translocation of PKC␤II from the cytosol to membrane has been shown to contribute to I/R injury, and either genetic or pharmacological inhibition of PKC␤II protects ischemic hearts (25). Additionally, activation of PKC␣ has been shown to contribute to cardiac hypertrophy, and inhibition of this PKC isoform led to attenuation of inflammation, cardiomyocyte growth, and cardiac hypertrophy (44). Here, we demonstrate reductions in LDH release upon treatment with PKC␣/␤ inhibitor Gö-6976. Furthermore, we demonstrate that Akt and GSK3␤ phophorylation was increased in both control and Ad-hAR HL-1 cells treated with Gö-6976. Taken together, our data demonstrate that increases in AR flux

Fig. 8. Schematic diagram. Increased flux via AR after I/R resulted in generation of diacylglycerol (DAG) and activation of PKC␣ and -␤ isoforms, leading to decreased phosphorylation of Akt and GSK3␤ kinases. Decreased GSK3␤ ultimately decreased cardioprotection via increased apoptotic mechanisms and mitochondrial permeability opening.



lead to PKC␣/␤ activation followed by decreases in Akt and GSK3␤ phosphorylation and is linked to increases in I/R injury (Fig. 8). In summary, our data demonstrate decreased levels of p-GSK3␤ in ARTg mice hearts compared with WT hearts. Inhibition of GSK3␤ increased levels of p-GSK3␤ and was associated with decreased injury, improved functional recovery, and decreased apoptosis after I/R in ARTg hearts. ARKO mice had increased levels of p-GSK3␤, improved functional recovery, and decreased apoptosis after I/R compared with WT and ARTg hearts. Furthermore, we show that AR modulates changes in levels of p-GSK3␤ via the Akt pathway. Taken together, our data demonstrate that AR mediates I/R injury, in part, via modulation of GSK3␤ phosphorylation.

12. 13. 14. 15. 16.

17. ACKNOWLEDGMENTS We thank Latoya Woods for assistance in manuscript preparation.

18.

GRANTS This work was supported by National Institutes of Health Grants AG026467, HL-61783, HL-60901, and HL-102022, the Harry B. & Aileen B. Gordon Foundation (K. H. Gabbay), and the Jacob & Louise Gabbay Foundation (K. H. Gabbay).

19. 20.

DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the authors. AUTHOR CONTRIBUTIONS

21. 22.

M.A., R.A., L.S., Z.Z., R. Rosario, and H.Z. performed experiments; M.A., R.A., L.S., Z.Z., and H.Z. analyzed data; M.A., S.V., H.Z., and R. Ramasamy interpreted results of experiments; M.A. prepared figures; S.V., K.M.B., K.H.G., and R. Ramasamy edited and revised manuscript; R. Ramasamy conception and design of research; R. Ramasamy drafted manuscript; R. Ramasamy approved final version of manuscript.

23. 24.

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