A

RKa

b

c

d

a

AA

KAIEUM

1

iaboaidbsl

Bf

0d

Chemico-Biological Interactions 178 (2009) 242–249

Contents lists available at ScienceDirect

Chemico-Biological Interactions

journa l homepage: www.e lsev ier .com/ locate /chembio int

ldose reductase decreases endoplasmic reticulum stress in ischemic hearts

achel J. Keitha,b, Petra Haberzettl a, Elena Vladykovskayaa, Bradford G. Hilld,arin Kaiserovaa, Sanjay Srivastavaa, Oleg Barskia, Aruni Bhatnagara,b,c,∗

Institute of Molecular Cardiology, University of Louisville, Louisville, KY, United StatesDepartment of Physiology and Biophysics, University of Louisville, Louisville, KY, United StatesDepartment of Biochemistry and Molecular Biology, University of Louisville, Louisville, KY, United StatesDepartment of Pathology, University of Alabama at Birmingham, Birmingham, AL, United States

r t i c l e i n f o

rticle history:vailable online 11 November 2008

eywords:ldose reductase

schemiaR stressnfolded protein responseouse

a b s t r a c t

Aldose reductase (AR) is a multi-functional AKR (AKR1B1) that catalyzes the reduction of a wide rangeof endogenous and xenobiotic aldehydes and their glutathione conjugates with high efficiency. Previ-ous studies from our laboratory show that AR protects against myocardial ischemia–reperfusion injury,however, the mechanisms by which it confers cardioprotection remain unknown. Because AR metab-olizes aldehydes generated from lipid peroxidation, we tested the hypothesis that it protects againstischemic injury by preventing ER stress induced by excessive accumulation of aldehyde-modified proteinsin the ischemic heart. In cell culture experiments, exposure to model lipid peroxidation aldehydes—4-hydroxy-trans-2-nonenal (HNE), 1-palmitoyl-2-oxovaleroyl phosphatidylcholine (POVPC) or acrolein ledto an increase in the phosphorylation of ER stress markers PERK and eIF2-� and an increase in ATF3. Thereduced metabolite of POVPC 1-palmitoyl-2-hydroxyvaleroyl phosphatidylcholine (PHVPC) was unableto stimulate JNK phosphorylation. No increase in phospho-eIF2-�, ATF3 or phospho-PERK was observed

in cells treated with the reduced HNE metabolite 1,4-dihydroxynonenol (DHN). Lysates prepared fromisolated perfused mouse hearts subjected to 15 min of global ischemia followed by 30 min of reperfusionex vivo showed greater phosphorylation of PERK and eIF2-� than hearts subjected to aerobic perfusionalone. Ischemia-induced increases in phospho-PERK and phospho-eIF2-� were diminished in the heartsof cardiomyocyte-specific transgenic mice overexpressing the AR transgene. These observations supportthe notion that by removing aldehydic products of lipid peroxidation, AR decreases ischemia–reperfusion

tress

sfsds

aR[

injury by diminishing ER s

. Introduction

Acute myocardial infarction (MI) is the leading cause of deathn the US and Europe [1,2]. Clinical symptoms of MI appear uponbrupt interruption of blood flow due to plaque rupture and throm-otic occlusion of major coronary arteries. Sudden interruptionf blood flow to myocardial tissue establishes an energy deficitnd prevents removal of metabolic waste. These changes resultn several pathophysiological alterations that increase [Ca2+]i and

ecrease intracellular pH. Ischemic injury is further exacerbatedy redox changes. Interruption of blood flow due to vessel occlu-ion decreases oxidative phosphorylation leading to mitochondrialeakage, which results in an increased production of reactive oxygen

∗ Corresponding author at: University of Louisville, 580 S. Preston St., Delia Baxteruilding, Rm 421, Louisville, KY 40202, United States. Tel.: +1 502 852 2477;

ax: +1 502 852 3663.E-mail address: aruni@louisville.edu (A. Bhatnagar).

purhtatm

m

009-2797/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.cbi.2008.10.055

.© 2008 Elsevier Ireland Ltd. All rights reserved.

pecies (ROS) [3]. If reperfusion occurs, ROS production is increasedurther, leading to extensive reperfusion injury. Several studies havehown that inhibition of ROS production or an increase in antioxi-ant defense decreases reperfusion injury and increases myocardialalvage after ischemia–reperfusion [3–5].

Although highly reactive and toxic, most ROS are short-livednd it has been suggested that part of the tissue injury induced byOS may be mediated by secondary products of lipid peroxidation6]. Oxidation of unsaturated lipids in lipoproteins and membranehospholipids generates a variety of toxic species, of which unsat-rated aldehydes such as 4-hydroxy-trans-2-nonenal (HNE) areeactive [6] and are generated in high abundance in the ischemiceart [7,8]. Additionally, in isolated myocytes HNE has been showno cause ATP depletion, changes in sodium and potassium current

nd to induce cell death [9]. Despite these findings, the contribu-ion of HNE and related products of lipid peroxidation products toyocardial ischemia–reperfusion injury are unknown.

To assess the contribution of lipid-derived aldehdyes toyocardial ischemia–reperfusion injury, we studied biochemical

ical In

ptt[SoAaaHoHis[aiar

rpscpnrgst

Eltpd(Uiaeetbssoia

2

2

dwfBJaCAPC

Sr

2

lmfciooa

2

SMcIcwWfbSc

owPat6H2(AwtAPa

2

dmCM2ewBpdown. Ca2+ was reintroduced in five steps. After 2 h incubation in

R.J. Keith et al. / Chemico-Biolog

athways that metabolize and detoxify unsaturated aldehydes inhe heart. These studies showed that HNE is metabolized viahree major pathways catalyzed by aldose reductase (AR; AKR1B)10,11], aldehyde dehydrogenase (ALDH) [12] and glutathione--transferases (GSTs) [13,14]. The GSTs catalyze the conjugationf unsaturated aldehydes with reduced glutathione, whereasLDH oxidizes the aldehyde using NAD+ as a cofactor. AR cat-lyzes the reduction of both the free aldehyde as well as theldehyde–glutathione conjugate. In the myocardial metabolism ofNE, AR appears to be a critical component because as shown byur previous work inhibition of AR increases the accumulation ofNE in the heart [15]. AR inhibition also abolishes the late phase of

schemic pre-conditioning in rabbit heart [15] and increases infarctize in rat hearts subjected to coronary ligation and reperfusion16]. Although these data are consistent with the view that AR playsn important, non-redundant role in myocardial protection againstschemia–reperfusion, specific mechanisms by which AR protectsgainst myocardial injury induced by HNE and related aldehydesemain unclear.

Unsaturated aldehydes such as HNE are strong electrophiles thateact readily with reduced glutathione [6], nucleophilic centers ofroteins [17], DNA [18] and phospholipids [19]. Although severaltudies have shown that during ischemia and reperfusion, proteinsovalently adducted to HNE accumulate in the heart [7], the patho-hysiological significance of protein–HNE adducts formation hasot been assessed. Modified or misfolded proteins in the heart areemoved by proteolysis [20] and excessive accumulation of unde-raded proteins results in ER stress. Indeed, previous studies havehown that myocardial ischemia induces ER stress [21] and triggershe unfolded protein response [22,23].

Proteins are normally folded in the ER with the assistance ofR chaperones such as GRP78, before being transported to cellularocations specified by protein localization signals. Increased pro-ein adducts in the ER or a decrease in the ability to properly foldroteins disrupts ER homeostasis and induces ER stress [24,25]. Toecrease this stress, cells trigger the unfolded protein responseUPR), which is an attempt to restore homeostasis [24]. DuringPR protein synthesis is decreased, expression of ER chaperones is

ncreased in an attempt to refold proteins, and misfolded proteinsre remove by proteolysis [24,26,27]. Unresolved ER stress, how-ver, triggers apoptosis [24,27]. In view of the evidence showingxcessive accumulation of HNE-adducted proteins and ER stress inhe ischemic heart, we tested the hypothesis that removal of HNEy AR prevents ischemic injury by diminishing ER stress. Our resultshow that HNE is a potent agent of ER stress and that overexpres-ion of AR in the heart decreases ischemia-induced ER stress. Thesebservations support the notion that ER stress in the ischemic hearts in part mediated by the accumulation of proteins modified byldehydic products of lipid peroxidation.

. Materials and methods

.1. Chemicals and reagents

Reagents HNE, DHN, POVPC and PHVPC were synthesized asescribed previously [10,28]. Unless stated otherwise, all chemicalsere purchased from Sigma–Aldrich (St. Louis, MO, USA). Supplies

or electrophoresis and Western blot analysis were purchased fromioRad (Hercules, CA, USA). Primary antibodies against eIF2-� and

NK as well as antibodies recognizing the phosphorylated proteins

nd secondary HRP-conjugated antibodies were purchased fromell Signaling Technology (Danvers, MA, USA). Antibodies againstTF3 and the phosphorylated and non-phosphorylated form ofERK were obtained from Santa Cruz Biotechnology, Inc. (Santaruz, CA, USA). Antibodies recognizing actin were purchased from

Dwla3

teractions 178 (2009) 242–249 243

igma–Aldrich (St. Louis, MO, USA) and antibodies against AR wereaised in house as described before.

.2. Animals

Wild-type (WT) C57BL/6 mice were purchased from Jacksonaboratories and cardio-specific AR overexpressing transgenic (TG)

ice were raised under standard conditions within our animalacility. The transgene was constructed by inserting a 0.95-kb rat ARDNA next to the mouse �-myosin heavy chain (�-MHC) promotern the multiple cloning site of the Myo vector. These mice were bredn to a C57 background to have only cardiac specific overexpressionf AR. These animals breed and grow normally. The care and use ofll animals was approved by the University of Louisville IACUC.

.3. Cell culture and treatment of VSMC and HUVEC

Vascular smooth muscle cells (VSMC) obtained fromprague–Dawley rat aortic explants were cultured in Dulbecco’sodified Eagle Medium (DMEM) supplemented with 10% fetal

alf serum (FCS) and 1% penicillin/streptomycin purchased fromnvitrogen (Carlsbad, CA, USA). Human umbilical vein endothelialells (HUVEC), obtained from PromoCell (Heidelberg, Germany),ere cultured in endothelial basal medium (EBM, Clonetics/Lonza,alkersville, MD, USA) containing human endothelial growth

actor (hEGF), hydrocortisone, gentamycin/amphotericin B (GA),ovine brain extract (BBE) and 2% fetal bovine serum (FBS, EGMingleQuots®, Clonetics/Lonza, Walkersville, MD, USA). Cells wereultured under standard cell culture conditions (37 ◦C, 5% CO2).

For indicated treatments, the cells were grown to a confluencef 90–95%. Serum-starved VSMC or HUVEC (20 h, 0.1% EBM-media)ere incubated as indicated with HNE (25 or 50 �M), DHN (25 �M),

OVPC (25 or 50 �M), PHVPC (25 �M) or acrolein (50 �M). Tovoid reaction between the aldehydes and constituents of the cul-ure medium, the cells were incubated with aldehydes for 30 or0 min in Hanks’ balanced salt solution (HBSS) containing 20 mMEPES, 135 mM NaCl, 5.4 mM KCl, 1.0 mM MgCl2, 2.0 mM CaCl2,.0 mM NaH2PO4, 5.5 mM glucose, pH 7.4. Long-term incubation6 h total) required to measure changes in protein expression ofTF3, HBSS was replaced by complete media after 2 h and the cellsere incubated for an additional 4 h in the complete medium. Cells

reated with thapsigargin (1 �M Th) were used as positive control.fter treatment, the cells were washed three times with ice-coldBS (Invitrogen, Carlsbad, CA, USA) and processed for Westernnalysis.

.4. Isolation and treatment of cardiomyocytes

Cardiomyocytes were isolated from mice (10–12 weeks) asescribed previously [29]. Briefly, the hearts were excised fromice, cannulated and perfused with oxygenated (95% O2 and 5%

O2) Tyrode solution containing 126 mM NaCl, 4.4 mM KCl, 1 mMgCl2·6H2O, 18 mM NaHCO3, 11 mM glucose, 4 mM HEPES, 20 mM

,3-butanedione monoxime (BDM), and 30 mM taurine, pH 7.4. Thextracellular matrix was digested by perfusing the heart for 12 minith Tyrode solution containing 0.6 units/ml Liberase IV (Roche,asel, Switzerland). The heart was minced in Tyrode solution sup-lemented with BSA and Liberase IV and cells were allowed to settle

MEM (Invitrogen, Carlsbad, CA, USA) containing 20 mM BDM, cellsere incubated in fresh media and allowed to attach overnight on

aminin coated dishes. For treatment, the medium was removednd cells were incubated for 30 min with 25 or 50 �M HNE or00 nM thapsigargin in HBSS.

2 ical In

2

aT

2

KccK3f(gtf

2

bbTi

2

oEwplaT(

drdB

uc

2

lnwpT3swbdm

2

og

3

3

acIoAoiHw

FefAa

44 R.J. Keith et al. / Chemico-Biolog

.5. Echocardiography

Cardiovascular function of WT and AR overexpressing mice wasnalyzed by baseline two-dimensional echocardiography (Toshiba380 Powervision) performed as previously described [30].

.6. Global myocardial ischemia–reperfusion ex vivo

Murine hearts were excised, cannulated and perfused withrebs Henseleit buffer by a standard Langendorf retrograde pro-edure as described previously [31]. Briefly, the hearts wereannulated and perfused at a constant pressure of 80 mm Hg withHB supplemented with glucose (11 mM) and CaCl2 (2.5 mM) at7 ◦C oxygenated with 95% O2 and 5% CO2. Hearts were equilibratedor 10 min and subjected to three treatment groups: perfusion alone15 or 30 min), no-flow global ischemia (I, 15 or 30 min), no-flowlobal ischemia followed by reperfusion (I/R 15/15 or 30 min). Afterreatment the hearts were removed from cannulation and snaprozen in liquid N2.

.7. In situ ischemia

The murine model of myocardial ischemia and reperfusion haseen described in detail [32]. Myocardial infarction was producedy a 15-min coronary occlusion followed by 15 min of reperfusion.he ischemic zone (IZ) was identified and segmented from the non-schemic zone (NIZ) for analysis.

.8. Western analysis

Protein lysates were prepared from pulverized hearts, cardiomy-cytes, HUVEC, or VSMC using a lysis buffer (25 mM HEPES, 1 mMDTA, 1 mM EGTA, 1% NP-40 and 0.1% SDS, pH 7.0) supplementedith protease inhibitor cocktail (1:100 Sigma–Aldrich) and phos-hatase inhibitor cocktail (1:100, Pierce, Rockland, IL, USA). After

ysis and sonication, the lysates were centrifuged for 15 min at 4 ◦Ct 4000 × g (for myocardial tissue) or 12,000 × g (for cell extracts).otal protein concentration was measured using a commercial kitBradford, Bio-Rad, Hercules, CA, USA).

SDS/PAGE and Western blotting were performed as previouslyescribed [33]. Western blots were developed using ECL® pluseagent (Amersham Biosciences, Piscataway, NJ, USA) and wereetected with a Typhoon 9400 variable mode imager (Amershamiosciences, Piscataway, NJ, USA). Band intensity was quantified

waptr

ig. 1. Aldehydes derived from lipid peroxidation induce ER stress. (A) Representative Weither 25 or 50 �M HNE or thapsigargin for 30 min. The blots were developed with antibrom lysates of VSMC incubated with 50 �M POVPC, 50 �M HNE or 50 �M acrolein. The blfter band quantification, the extent of phosphorylation was calculated from the ratio of ts mean ± S.E.M. *P < 0.01 (n = 3–4).

teractions 178 (2009) 242–249

sing the Image Quant TL software (Amersham Biosciences, Pis-ataway, NJ, USA).

.9. AR activity and sorbitol measurements

AR activity was determined as previously described [31]. Brieflyeft ventricular tissue was homogenized, centrifuged and the super-atant was collected for activity measurements. Enzyme activityas measured at 25 ◦C, 1–1.5 mg of protein, 100 mM potassiumhosphate (pH 6.0), 15 mM NADPH, and 10 mM glyceraldehyde.he reaction was monitored by the rate of oxidation of NADPH at40 nm for 3 min. For sorbitol measurements, left ventricular tis-ue 0.1–0.2 g pulverized, homogenized then centrifuged was mixedith the protein-free supernatant (1.0 ml), and 0.5 ml of glycine

uffer (pH 9.4) containing 3.6 mM NAD+ and 6 units of sorbitolehydrogenase. The sorbitol levels were measured spectrofluoro-etrically, as described previously [34].

.10. Statistical analysis

Data are presented as mean ± S.E.M. and paired student t-test orne way ANOVA (L.S.D., post hoc) was used for comparison betweenroups.

. Results

.1. Aldehydes derived from lipid peroxidation induce ER stress

The ER stress triggered UPR is characterized by an alarm and andaptive phase involving the activation of multiple signaling cas-ades to halt protein synthesis and increase protein folding [27].n this response, phosphorylation of PERK represents a major limbf the alarm phase. This leads to the phosphorylation of eIF2-�.ccordingly, we investigated whether aldehydes generated fromxidized lipids increase phosphorylation of these kinases. For thissolated mouse cardiac myocytes were treated with 25 or 50 �MNE for 30 min and changes in PERK and eIF2-� phosphorylationere measured by Western analysis. As shown in Fig. 1, treatment

ith HNE led to an increase in the phosphorylation of both PERK

nd eIF2-�. The extent of increase in phosphorylation of theseroteins with 50 �M HNE was comparable to that obtained withhapsigargin, indicating that the HNE-induced response was asobust as thapsigargin in stimulating the PERK pathway of UPR.

stern blot of lysates prepared from isolated mouse cardiomyocytes incubated withodies raised against phoshpo-PERK, PERK, and phospho-eIF2-�. (B) Western blotsots were developed using antibodies that recognize phospho-PERK and total PERK.he band intensities of the phospho-PERK normalized to total PERK. Data are shown

ical Interactions 178 (2009) 242–249 245

aPps[dElPatUc

3

odaortca[iwJab

stect�tHmaciwlwioofial

3

lioaign

Fig. 2. Reduction decreases the ability of HNE to induce stress responses. (A) Repre-sentative Western blots of lysates from HUVECs incubated with POVPC (25 �M) orPHVPC (25 �M) developed using antibodies directed against phospho-JNK or totalJNK. Quantification performed by densitometric analysis of phoshpo-JNK normal-ized to total JNK is shown in lower panel. (B) Western blots of lysates from HUVECsincubated with 25 �M HNE or DHN. The levels of ATF3 and phospho-eIF2-� weremeasured as indicated after 6 h or 30 and 60 min, respectively. (C) Stimulation ofPERK phosphorylation in isolated cardiomyocytes. Western blots were performedwith lysates of cells treated with 25 �M HNE or 25 �M DHN for 30 min and devel-oped using an anti-phospho-PERK antibody. Data are shown as mean ± S.E.M. In

R.J. Keith et al. / Chemico-Biolog

To examine whether lipid-derived aldehydes other than HNElso trigger UPR, we examined the effects of POVPC and acrolein.OVPC is one of the most abundant oxidation product of 1-almitoyl-2-arachidonyl-phosphatidylcholine (PAPC) and has beenhown to increase endothelial adhesion and cytokine production35]. Acrolein is also generated by oxidized lipids. It is also pro-uced by myeloperoxidase-catalyzed oxidation of threonine [36].xposure of VSMC to POVPC (25 �M) or acrolein (25 �M) for 30 mined an increase in PERK phosphorylation. The extent of increase inERK phosphorylation was most robust with acrolein, while HNEnd POVPC were equally effective. Based on these data we concludehat the major aldehydes generated by lipid peroxidation triggerPR in both cardiac myocytes as well as vascular smooth muscleells.

.2. Reduction decreases the ability of aldehydes to trigger UPR

Our previous work shows that AR catalyzes the reductionf POVPC [28], HNE [37] and acrolein [38]. Because reductionecreases carbonyl reactivity, we assumed it represents a mech-nism for detoxification, which diminish the biological activitiesf the parent aldehyde. To test this assumption, we synthesizededuced metabolites of HNE and POVPC i.e. DHN and PHVPC. Toest the relative efficacies of POVPC and PHVPC, we examinedhanges in JNK. Previous studies show that induction of ER stressnd activation of the UPR results in increased JNK phosphorylation27]. As shown in Fig. 2A treatment of HUVECs with 25 �M POVPCncreased phosphorylation of p46, although the levels of total JNK

ere not affected. In total an approximately four-fold increase inNK phosphorylation was observed. Cells treated with PHVPC showminimal increase in phospho-JNK, indicating clearly that POVPC,ut not its reduced analog PHVPC, stimulates JNK phosphorylation.

To examine the relative efficacies of HNE and DHN, we mea-ured changes in ATF3 and phospho-eIF2-�. Serum-starved HUVECreated with HNE for 6 h showed a significant increase in ATF3xpression, which was nearly undetectable in untreated cells. Inontrast to HNE treatment, no increase in ATF3 was observed in cellsreated with DHN. Because ATF3 is downstream to the PERK–eIF2-

pathway, we also examined the efficacy of DHN in stimulatinghe phosphorylation of eIF2-�. As shown in Fig. 2B, treatment withNE led to robust phosphorylation of eIF2-� within 30 min of treat-ent. This increase in phosphorylation was still evident 60 min

fter treatment. No increase in phospho-eIF2-� was observed inells treated with DHN either 30 or 60 min of treatment. To exam-ne how DHN affects UPR in cardiac myocytes, the cells were treated

ith equal concentrations of either HNE or DHN. Western blots ofysates prepared from these cells showed that although treatment

ith HNE for 30 min led to a strong increase in phospho-PERK, noncrease was observed in cells treated with DHN. Based on thesebservations we conclude that aldehydes generated from lipid per-xidation, but not their corresponding alcohols trigger UPR. Thisnding is consistent with the view that reduction by AR decreasesldehyde toxicity and therefore the catalytic activity of AR in situ isikely to prevent the induction of stress responses.

.3. Myocyte-specific AR-transgenic mice

To study the role of AR we had previously used pharmaco-ogical inhibitors; however, the non-specific effects of chemicalnhibitors could not be rigorously ruled out. Hence to minimize such

ff-target effects, we designed gain-of-function experiments tossess whether an increase in AR activity would prevent ischemia-nduced ER stress. As described under Section 2, AR-TG mice wereenerated on a C57 background. These mice were phenotypicallyormal and bred as well as their WT littermates. Cardiac extracts

panel (A), *P < 0.01; control (untreated cells) versus POVPC and # versus PHVPC(n = 4). In panel (B), *P < 0.01 HNE-treated versus untreated cells and # versus DHN(n = 3).

246 R.J. Keith et al. / Chemico-Biological Interactions 178 (2009) 242–249

Fig. 3. Cardiac-myocyte specific overexpression of aldose reductase in mouse heart.The AR-transgenic mice were generated in our laboratory as described under Sec-tion 2 using the �-myosin heavy chain (�-MHC) promoter to drive the expressionof the transgene. (A) Representative Western blots of myocardial lysates from wildtype (WT) and two TG lines—S 137 and S 138 developed with anti-AR antibody.The additional band in TG hearts with the slightly higher molecular weight (due toHis-Tag) was ascribed to the transgene. For quantification, the intensities of bothbands were summed and normalized to GAPDH. (B) AR activity measured in wholehearts lysates from WT and TG mice. Enzyme activity was measured using 10 mMgc(

fBgiaioataouohifwn

Table 1Cardiac parameters in AR-TG and WT mice.

M-mode echocardiogram WT AR-TG

Body weight 35 ± 0.76 31.28 ± 1.4HR (beats/min) 527 ± 24 537 ± 14Heart/body weight 5.0 ± 0.18 5.8 ± 0.30LEFR

oa

3s

mfilz(ditifhIwodi

4

eri(tia

rSsdl�tHtusltstress associated with vasculitis [42], myocardial hypertrophy [43],

lyceraldehdye and 0.1 mM NADPH. Sorbitol content was determined enzymati-ally by using sorbitol dehydrogenase. Data are presented as mean ± S.E.M. *P < 0.01n = 3–4).

rom these mice showed a large increase in AR protein (Fig. 3A).ecause it was attached to a His-tag, the product of the trans-ene could be readily distinguished from the native protein. Thencrease in AR protein was accompanied by an increase in enzymectivity. Both lines showed a 2–4-fold increase in enzyme activ-ty determined in cardiac lysates with saturating concentrationsf substrates. In contrast, there was a <2-fold increase in sorbitolccumulation, indicating that despite high levels of expression,he overall activity of the enzyme was not drastically increasednd there was no osmotic overload imposed by overexpressionf AR. Because the S 137 strain showed maximal activity, it wassed for all further experiments. Table 1 summarizes parametersf cardiac contractility. No changes in body weight, heart rate,eart/body weight, or LV/body weight ratios were observed. Signif-

cantly, echocardiographic measurements of ejection fraction andractional shortening in the TG mice were similar to those obtainedith their WT littermates, indicating that overexpression of AR didot affect cardiac contractility or induce cardiac hypertrophy. These

hwNa

V/body weight 3.6 ± 0.14 4.08 ± 0.27jection fraction 0.67 ± 0.01 0.68 ± 0.01ractional shortening 0.71 ± 0.01 0.68 ± 0.01WT 0.34 ± 0.01 0.33 ± 0.01

bservations suggest that transgenic overexpression of AR does notffect basal function in mouse hearts.

.4. Overexpression of AR prevents ischemic activation of ERtress in the heart

To examine ischemia-induced changes hearts from WT and TGice were perfused and subjected to global ischemia and reper-

usion ex vivo. As shown in Fig. 4A, hearts subjected to 30 min ofschemia displayed an increase in PERK phosphorylation. A simi-ar increase in phospho-PERK was also observed in the ischemicone of hearts subjected to 30 min of coronary occlusion in situFig. 4B), indicating that the increase in PERK phosphorylation wasependent on ex vivo perfusion, but was also induced in situ. Global

schemia in isolated hearts was also associated with an increase inhe phosphorylation of eIF2-�. The phosphorylation of eIF2-� wasncreased further upon reperfusion (Fig. 4A). After 30 min of reper-usion, the increase in phospho-eIF2-� was more than two-foldigher than hearts that were perfused with the aerobic buffer alone.

n contrast, no increases in the phospho-PERK or phospho-eIF2-�ere observed in transgenic heart subjected to 30 min of ischemia

r 30 min of ischemia followed by 30 min of reperfusion. From theseata we conclude that an increase in AR activity prevents ER stress

nduced in hearts subjected to ischemia–reperfusion.

. Discussion

The major findings of this paper are that aldehydes gen-rated from lipid peroxidation trigger the unfolded proteinesponse and that increased metabolism of these aldehydes dur-ng myocardial ischemia and reperfusion via aldose reductaseAR) prevents ischemia-induced ER stress. These findings supporthe view that ER stress is a significant component of myocardialschemia–reperfusion injury and that it is mediated in part by theldehyde products of lipid peroxidation.

Aldehydes are the major end products of lipid peroxidationeactions that take place in membrane proteins and lipoproteins.everal aldehydes are generated in oxidized lipids. Aldehydesuch as POVPC are formed due to fragmentation of bis-allylicouble bonds of unsaturated fatty acids esterified to a phospho-

ipid backbone. Free aldehydes such as HNE are generated from-6 polyunsaturated fatty acids and are one of the most reac-

ive products of lipid peroxidation [39]. Under some conditions,NE represents 95% of the total unsaturated aldehyde produc-

ion. Normal cellular levels of HNE range from 0.8 to 2.8 �M butp to 10–100 mM HNE has been measured in oxidizing micro-omes [40,41]. Several conditions associated with oxidative stresseads to increased accumulation of proteins covalently adductedo lipid-derived aldehydes. In particular, cardiovascular oxidative

eart failure [44], and ischemic hearts [7,45–47] are associatedith an increase in the accumulation of protein–aldehyde adducts.onetheless, in most cases, the functional significance of suchdduct formation is unknown. Hence we tested the hypothesis that

R.J. Keith et al. / Chemico-Biological In

Fig. 4. Overexpression of AR decrease ischemia-induced ER stress in the heart. (A)Representative Western blots of lysates from perfused hearts which were eithersubjected to perfusion (P) alone, 30 min of ischemia (I), or 30 min ischemia followedby 30 min of reperfusion (I/R) ex vivo. The blots were developed using anti-phospho-PERK, anti-phospho-eIF2-� or anti-eIF2-� antibodies. Quantification for WT and TGhearts are shown in a bar diagram. (B). Wild type mouse hearts were subjected to1app

aEs

adrksdfgcUgg

1a�sobGfob

pkbEa[vwJpiEDltoE

haesreoiAomso[mwAphtlE

aietCaep

5 min coronary ligation and 15 min reperfusion in vivo. Whole hearts were lysednd representative Western blots developed with anti-phospho-PERK compared thehosphorylation of PERK in ischemic (IZ) and non-ischemic (NIZ) zones. Data areresented as mean ± S.E.M. *P < 0.01 (n = 3–4), #P < 0.01 vs. WT (n = 3–4).

ccumulation of aldehyde-modified proteins in the heart inducesR stress and triggers the unfolded protein response, which is aignificant cause of myocardial ischemia–reperfusion injury.

Our results show that both free (HNE) and esterified (POVPC)ldehydes are potent UPR triggers. Treatment of isolated car-iac myocytes, vascular smooth muscle cells or endothelial cellsesulted in increased phosphorylation of PERK and eIF2-�. Bothinases play a central role in orchestrating UPR in response to ERtress. The mammalian UPR is mediated by three distinct pathwaysue to IRE-1, PERK and ATF6 activation following their dissociationrom GRP78 (Bip) [27]. Not all forms and inducers of ER stress trig-

er identical UPR which is not an all-or-none response. In neonatalardiac myocytes, simulated ischemia induces all three arms ofPR (PERK, ATF6, and IRE-1) [22,48,49] and their downstream tar-ets (CHOP, procaspase-12 cleavage) [50], however, in a model oflobal brain ischemia, PERK–eIF2-� were activated, but ATF6, XBP-

iiUcn

teractions 178 (2009) 242–249 247

, CHOP, and ATF4 were not [51]. Hence our studies showing thatldehydes stimulate the phosphorylation of both PERK and eIF2-

provide strong evidence that lipid-derived aldehydes cause ERtress. This notion is further supported by the robust activationf ATF3 by HNE in endothelial cells. Although it has been shownefore that HNE upregulates ER stress genes such as ATF3, HERP andadd34 [52], the significance of these changes is not clear. There-

ore, our observation that ATF3 is induced by HNE after activationf PERK and eIF2-� supports the idea that induction of ATF3 maye in response to ER stress.

Treatment with POVPC was also found to increase JNK phos-horylation. While JNK is considered to a classical stress-activatedinase, recent studies suggest that its phosphorylation is enhancedy UPR signaling activated by ER stress [27]. Activation of JNK byR stress has been shown to be downstream of IRE1 activation [53],nd phosphorylation of JNK is also impaired in PERK-null fibroblasts54]. Although mechanisms by which aldehydes-induced JNK acti-ation were not examined, phosphorylation of this stress kinaseith ER stress markers suggests that as described in other cells,

NK activation may be related to ER stress. More importantly, phos-horylation of JNK was stimulated by POVPC but not by PHVPC,

ndicating that unlike the parent aldehydes, alcohols cannot induceR stress. A similar conclusion is supported by the observations thatHN was unable to induce eIF2-� phosphorylation in endothe-

ial cells and PERK phosphorylation in cardiac myocytes. Takenogether, these observations support the concept that metabolismf aldehydes to alcohols in the heart abolishes their ability to induceR stress and to induce stress-kinase signaling.

Our previous work shows that reduction of aldehydes in theeart is catalyzed by AR. Extensive studies show that AR cat-lyzes the reduction of structurally diverse range of aldehydes. Thenzyme is particularly efficient in reducing medium to long-chainaturated and unsaturated aldehydes [55]. AR also catalyzes theeduction of aldehyde–glutathione conjugates; in many cases withfficiency higher than that of the parent aldehyde [56]. In addition,ur studies show that reduction of HNE and GS–HNE is preventedn hearts pre-treated with AR inhibitors [10] and that inhibition ofR results in the accumulation of HNE in the heart [15]. Inhibitionf AR has also been shown to increase HNE during vascular inflam-ation [42]. AR is upregulated upon exposure to oxidative stress

pecifically in the areas of high HNE formation [42]. Furthermore,ur recent studies show that AR is activated in the ischemic heart31] and that pharmacological inhibition of the enzyme increasesyocardial ischemia–reperfusion injury [16]. In extension of thisork, data presented here show that transgenic overexpression ofR in cardiac myocytes prevented ischemia–reperfusion-inducedhosphorylation of eIF2-�. These data are consistent with ourypothesis (Scheme 1) that AR protects the heart by preventinghe accumulation of lipid peroxidation-derived aldehydes, which ifeft unmetabolized could contribute to ischemic injury by inducingR stress.

Results from several recent studies suggest that ER stress may ben important consequence of ischemia and a significant mediator ofschemia–reperfusion injury. It has been shown that brief ischemicpisodes increase the expression of GRP94, GRP78 [22,48,57] andhat prolonged ischemia leads to the activation of UPR componentsHOP and caspase-12 that can trigger cell death. Induction of GRP78nd GRP94 has also been reported in mouse hearts subjected to I/Rx vivo [57] and elevated levels of GRP78 have been reported in theeri-infarct zone of the heart [22]. That UPR triggered by ER stress

s a significant cause of myocardial I/R injury is suggested by stud-es reporting that overexpression of GRP94 [25] or pre-induction ofPR prevents hypoxic cardiac myocyte cell death [58]. Conversely,ardiomyocyte death due to simulated I/R is increased by dominantegative XBP-1 [22] and anti-sense GRP78 could abolish the protec-

248 R.J. Keith et al. / Chemico-Biological Interactions 178 (2009) 242–249

Scheme 1. ER stress in the ischemic heart. Myocardial ischemia leads to an increase in ROS generation which results in an increase in the formation of aldehydes derived fromoxidized lipids. These aldehydes are reduced by AR to alcohols, which are inactive. Aldehydes that escape AR reduction form covalent adducts with proteins which induceE ERK po tion oa chapep id per

triitlioipAh

C

A

H

R

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

R stress. Modified proteins induce a dissociation of GRP78 from PERK leading to Pf protein translation. Although not studied here, ER stress also results in the activas genes encoding ER chaperones and ERAD components. The transcription of ERrevented in hearts in which overexpression of AR prevents the accumulation of lip

ive effects of early pre-conditioning [59]. Activated AFT6 has beenecently reported to protect the heart from I/R injury [57] indicat-ng that enhancing the expression of ER chaperones could preventschemic injury. In agreement with these observations we foundhat ischemia lead to an increase in eIF2-� and PERK phosphory-ation. Similar results were obtained in hearts subjected to globalschemia–reperfusion ex vivo and in hearts subjected to coronarycclusion and reperfusion in situ, indicating model-independentnduction of ER stress in the ischemic heart. Significantly, eIF2-�hosphorylation was attenuated in AR-TG hearts, suggesting thatR is a significant regulator of ischemia-induced ER stress in theeart.

onflict of interest

No conflicts of interest.

cknowledgements

This work was supported in part by NIH grants ES11860,L55477, HL59378, and HL78825.

eferences

[1] F. Levi, F. Lucchini, E. Negri, C. La Vecchia, Trends in mortality from cardiovascu-lar and cerebrovascular diseases in Europe and other areas of the world, Heart88 (2002) 119–124.

[2] W. Rosamond, K. Flegal, K. Furie, A. Go, K. Greenlund, N. Haase, S.M. Hailpern,M. Ho, V. Howard, B. Kissela, S. Kittner, D. Lloyd-Jones, M. McDermott, J. Meigs,C. Moy, G. Nichol, C. O’Donnell, V. Roger, P. Sorlie, J. Steinberger, T. Thom, M.Wilson, Y. Hong, Heart disease and stroke statistics-2008 update—A reportfrom the American Heart Association Statistics Committee and Stroke StatisticsSubcommittee, Circulation 117 (2008) E25–E146.

[3] J.M. Downey, Free radicals and their involvement during long-term myocardialischemia and reperfusion, Annu. Rev. Physiol. 52 (1990) 487–504.

[4] R. Bolli, The late phase of preconditioning, Circ. Res. 87 (11) (2000) 972–983.[5] L.B. Becker, New concepts in reactive oxygen species and cardiovascular reper-

fusion physiology, Cardiovasc. Res. 61 (3) (2004) 461–470.[6] H. Esterbauer, R.J. Schaur, H. Zollner, Chemistry and biochemistry of 4-

hydroxynonenal, malonaldehyde and related aldehydes, Free Radic. Biol. Med.11 (1) (1991) 81–128.

[7] P. Eaton, J.M. Li, D.J. Hearse, M.J. Shattock, Formation of 4-hydroxy-2-nonenal-modified proteins in ischemic rat heart, Am. J. Physiol. 276 (1999) H935–H943.

[8] A. Renner, M.R. Sagstetter, H. Harms, V. Lange, M.E. Gotz, O. Elert, Formationof 4-hydroxy-2-nonenal protein adducts in the ischemic rat heart after trans-plantation, J. Heart Lung Transplant. 24 (6) (2005) 730–736.

[9] A. Bhatnagar, Electrophysiological effects of 4-hydroxynonenal, an aldehydic

product of lipid peroxidation, on isolated rat ventricular myocytes, Circ. Res. 76(2) (1995) 293–304.

10] S. Srivastava, A. Chandra, L.F. Wang, W.E. Seifert Jr., B.B. DaGue, N.H. Ansari,S.K. Srivastava, A. Bhatnagar, Metabolism of the lipid peroxidation product,4-hydroxy-trans-2-nonenal, in isolated perfused rat heart, J. Biol. Chem. 273(1998) 10893–10900.

[

[

hosphorylation, which in turn phosphorylates eIF2-� resulting in the suppressionf IRE-1�- XBP-1 pathway inducing the transcription of inflammatory genes as wellrones is enhanced to increase the protein capacity of the cell. These changes areoxidation-derived aldehydes.

11] S. Srivastava, A. Chandra, N.H. Ansari, S.K. Srivastava, A. Bhatnagar, Iden-tification of cardiac oxidoreductase(s) involved in the metabolism of thelipid peroxidation-derived aldehyde-4-hydroxynonenal, Biochem. J. 329 (1998)469–475.

12] S. Choudhary, T. Xiao, S. Srivastava, W. Zhang, L.L. Chan, L.A. Vergara, F.J.G.M.Van Kuijk, N.H. Ansari, Toxicity and detoxification of lipid-derived aldehydesin cultured retinal pigmented epithelial cells, Toxicol. Appl. Pharmacol. 204(2005) 122–134.

13] Y.B. Li, Z.X. Cao, H. Zhu, M.A. Trush, Differential roles of 3H-1,2-dithiole-3-thione-induced glutathione, glutathione S-transferase and aldose reductase inprotecting against 4-hydroxy-2-nonenal toxicity in cultured cardiomyocytes,Arch. Biochem. Biophys. 439 (2005) 80–90.

14] A. Fukuda, Y. Nakamura, H. Ohigashi, T. Osawa, K. Uchida, Cellular responseto the redox active lipid peroxidation products: induction of glutathione S-transferase P by 4-hydroxy-2-nonenal, Biochem. Biophys. Res. Commun. 236(1997) 505–509.

15] K. Shinmura, R. Bolli, S.Q. Liu, X.L. Tang, E. Kodani, Y.T. Xuan, S. Srivastava, A.Bhatnagar, Aldose reductase is an obligatory mediator of the late phase ofischemic preconditioning, Circ. Res. 91 (2002) 240–246.

16] K. Kaiserova, X.L. Tang, S. Srivastava, A. Bhatnagar, Role of nitric oxide in regulat-ing aldose reductase activation in the ischemic heart, J. Biol. Chem. 283 (2008)9101–9112.

17] G. Poli, F. Biasi, G. Leonarduzzi, 4-Hydroxynonenal-protein adducts: a reliablebiomarker of lipid oxidation in liver diseases, Mol. Aspects Med. 29 (2008)67–71.

18] M.P. Stone, Y.J. Cho, H. Huang, H.Y. Kim, I.D. Kozekov, A. Kozekova, H. Wang, I.G.Minko, R.S. Lloyd, T.M. Harris, C.J. Rizzo, Interstrand DNA cross-links inducedby alpha,beta-unsaturated aldehydes derived from lipid peroxidation and envi-ronmental sources, Acc. Chem. Res. 41 (2008) 793–804.

19] K.A. Zemski Berry, R.C. Murphy, Characterization of acrolein-glycerophosphoethanolamine lipid adducts using electrospray massspectrometry, Chem. Res. Toxicol. (1920) 1342–1351.

20] T. Grune, K.J. Davies, The proteasomal system and HNE-modified proteins, Mol.Aspects Med. 24 (4–5) (2003) 195–204.

21] A. Azfer, J. Niu, L.M. Rogers, F.M. Adamski, P.E. Kolattukudy, Activationof endoplasmic reticulum stress response during the development ofischemic heart disease, Am. J. Physiol. Heart Circ. Physiol. 291 (3) (2006)H1411–H1420.

22] D.J. Thuerauf, M. Marcinko, N. Gude, M. Rubio, M.A. Sussman, C.C. Glem-botski, Activation of the unfolded protein response in infarcted mouseheart and hypoxic cultured cardiac myocytes, Circ. Res. 99 (3) (2006)275–282.

23] C.C. Glembotski, The role of the unfolded protein response in the heart, J. Mol.Cell. Cardiol. 44 (2008) 453–459.

24] M. Boyce, J. Yuan, Cellular response to endoplasmic reticulum stress: a matterof life or death, Cell Death Differ. 13 (3) (2006) 363–373.

25] C.C. Glembotski, Endoplasmic reticulum stress in the heart, Circ. Res. 101 (2007)975–984.

26] M. Schroder, R.J. Kaufman, The mammalian unfolded protein response, Annu.Rev. Biochem. 74 (2005) 739–789.

27] C. Xu, B. Bailly-Maitre, J.C. Reed, Endoplasmic reticulum stress: cell life anddeath decisions, J. Clin. Invest. 115 (10) (2005) 2656–2664.

28] S. Srivastava, M. Spite, J.O. Trent, M.B. West, Y. Ahmed, A. Bhatnagar, Aldosereductase-catalyzed reduction of aldehyde phospholipids, J. Biol. Chem. 279

(2004) 53395–53406.

29] T.D. O’Connell, M.C. Rodrigo, P.C. Simpson, Isolation and culture of adult mousecardiac myocytes, Methods Mol. Biol. 357 (2007) 271–296.

30] Y. Guo, W.J. Wu, Y. Qiu, X.L. Tang, Z. Yang, R. Bolli, Demonstration of an earlyand a late phase of ischemic preconditioning in mice, Am. J. Physiol. 275 (1998)H1375–H1387.

ical In

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

R.J. Keith et al. / Chemico-Biolog

31] K. Kaiserova, S. Srivastava, J.D. Hoetker, S.O. Awe, X.L. Tang, J. Cai, A. Bhatnagar,Redox activation of aldose reductase in the ischemic heart, J. Biol. Chem. 281(22) (2006) 15110–15120.

32] Q. Li, Y. Guo, Y.T. Xuan, C.J. Lowenstein, S.C. Stevenson, S.D. Prabhu, W.J. Wu, Y.Zhu, R. Bolli, Gene therapy with inducible nitric oxide synthase protects againstmyocardial infarction via a cyclooxygenase-2-dependent mechanism, Circ. Res.92 (7) (2003) 741–748.

33] M.B. West, B.G. Hill, Y.T. Xuan, A. Bhatnagar, Protein glutathiolation by nitricoxide: an intracellular mechanism regulating redox protein modification,FASEB J. 20 (10) (2006) 1715–1717.

34] J.I. Malone, G. Knox, S. Benford, T.A. Tedesco, Red cell sorbitol: an indicator ofdiabetic control, Diabetes 29 (11) (1980) 861–864.

35] J.A. Berliner, N.M. Gharavi, Endothelial cell regulation by phospholipid oxida-tion products, Free Radic. Biol. Med. 45 (2) (2008) 119–123.

36] J.F. Stevens, C.S. Maier, Acrolein: sources, metabolism, and biomolecular inter-actions relevant to human health and disease, Mol. Nutr. Food Res. 52 (1) (2008)7–25.

37] S. Srivastava, A. Chandra, A. Bhatnagar, S.K. Srivastava, N.H. Ansari, Lipid per-oxidation product, 4-hydroxynonenal and its conjugate with GSH are excellentsubstrates of bovine lens aldose reductase, Biochem. Biophys. Res. Commun.217 (1995) 741–746.

38] K.V. Ramana, B.L. Dixit, S. Srivastava, A. Bhatnagar, G.K. Balendiran, S.J.Watowich, J.M. Petrash, S.K. Srivastava, Characterization of the glutathionebinding site of aldose reductase, Chem. Biol. Interact. 130–132 (2001)537–548.

39] A. Benedetti, M. Comporti, H. Esterbauer, Identification of 4-hydroxynonenalas a cytotoxic product originating from the peroxidation of liver microsomallipids, Biochim. Biophys. Acta 620 (2) (1980) 281–296.

40] A. Benedetti, R. Fulceri, M. Ferrali, L. Ciccoli, H. Esterbauer, M. Comporti,Detection of carbonyl functions in phospholipids of liver-microsomes in Ccl4-poisoned and Brccl3-poisoned rats, Biochim. Biophys. Acta 712 (3) (1982)628–638.

41] A. Benedetti, M. Comporti, R. Fulceri, H. Esterbauer, Cyto-toxic aldehydes orig-inating from the peroxidation of liver microsomal lipids—Identification of4,5-dihydroxydecenal, Biochim. Biophys. Acta 792 (2) (1984) 172–181.

42] H.L. Rittner, V. Hafner, P.A. Klimiuk, L.I. Szweda, J.J. Goronzy, C.M. Weyand,Aldose reductase functions as a detoxification system for lipid peroxidationproducts in vasculitis, J. Clin. Invest. 103 (7) (1999) 1007–1013.

43] M. Benderdour, G. Charron, D. DeBlois, B. Comte, R.C. Des, Cardiac mitochondrialNADP+-isocitrate dehydrogenase is inactivated through 4-hydroxynonenaladduct formation: an event that precedes hypertrophy development, J. Biol.Chem. 278 (46) (2003) 45154–45159.

44] S. Srivastava, B. Chandrasekar, A. Bhatnagar, S.D. Prabhu, Lipid peroxidation-derived aldehydes and oxidative stress in the failing heart: role of aldosereductase, Am. J. Physiol. Heart Circ. Physiol. 283 (2002) H2612–H2619.

45] D.T. Lucas, L.I. Szweda, Declines in mitochondrial respiration during cardiacreperfusion: age-dependent inactivation of alpha-ketoglutarate dehydroge-nase, Proc. Nat. Acad. Sci. U.S.A. 96 (12) (1999) 6689–6693.

[

[

teractions 178 (2009) 242–249 249

46] A. Musatov, C.A. Carroll, Y.C. Liu, G.I. Henderson, S.T. Weintraub, N.C. Robinson,Identification of bovine heart cytochrome c oxidase subunits modified by thelipid peroxidation product 4-hydroxy-2-nonenal, Biochemistry 41 (25) (2002)8212–8220.

47] M. Veronneau, B. Comte, C. Des Rosiers, Quantitative gaschromatographic–mass spectrometric assay of 4-hydroxynonenal boundto thiol proteins in ischemic/reperfused rat hearts, Free Radic. Biol. Med. 33(10) (2002) 1380–1388.

48] E. Szegezdi, A. Duffy, M.E. O’Mahoney, S.E. Logue, L.A. Mylotte, T. O’Brien, A.Samali, ER stress contributes to ischemia-induced cardiomyocyte apoptosis,Biochem. Biophys. Res. Commun. 349 (4) (2006) 1406–1411.

49] D.J. Thuerauf, H. Hoover, J. Meller, J. Hernandez, L. Su, C. Andrews, W.H.Dillmann, P.M. McDonough, C.C. Glembotski, Sarco/endoplasmic reticulumcalcium ATPase-2 expression is regulated by ATF6 during the endoplasmicreticulum stress response: intracellular signaling of calcium stress in a cardiacmyocyte model system, J. Biol. Chem. 276 (51) (2001) 48309–48317.

50] G. Mouw, J.L. Zechel, J. Gamboa, W.D. Lust, W.R. Selman, R.A. Ratcheson,Activation of caspase-12, an endoplasmic reticulum resident caspase, afterpermanent focal ischemia in rat, Neuroreport 14 (2) (2003) 183–186.

51] R. Kumar, G.S. Krause, H. Yoshida, K. Mori, D.J. DeGracia, Dysfunction of theunfolded protein response during global brain ischemia and reperfusion, J.Cereb. Blood Flow Metab. 23 (2003) 462–471.

52] J.D. West, L.J. Marnett, Alterations in gene expression induced by the lipid per-oxidation product, 4-hydroxy-2-nonenal, Chem. Res. Toxicol. 18 (11) (2005)1642–1653.

53] F. Urano, X.Z. Wang, A. Bertolotti, Y.H. Zhang, P. Chung, H.P. Harding, D. Ron, Cou-pling of stress in the ER to activation of JNK protein kinases by transmembraneprotein kinase IRE1, Science 287 (5453) (2000) 664–666.

54] S.H. Liang, W. Zhang, B.C. McGrath, P. Zhang, D.R. Cavener, PERK (eIF2alphakinase) is required to activate the stress-activated MAPKs and inducethe expression of immediate-early genes upon disruption of ER calciumhomoeostasis, Biochem. J. 393 (2006) 201–209.

55] S. Srivastava, S.J. Watowich, J.M. Petrash, S.K. Srivastava, A. Bhatnagar, Structuraland kinetic determinants of aldehyde reduction by aldose reductase, Biochem-istry 38 (1999) 42–54.

56] K.V. Ramana, B.L. Dixit, S. Srivastava, G.K. Balendiran, S.K. Srivastava, A. Bhat-nagar, Selective recognition of glutathiolated aldehydes by aldose reductase,Biochemistry 39 (2000) 12172–12180.

57] J.J. Martindale, R. Fernandez, D. Thuerauf, R. Whittaker, N. Gude, M.A. Sussman,C.C. Glembotski, Endoplasmic reticulum stress gene induction and protec-tion from ischemia/reperfusion injury in the hearts of transgenic mice witha tamoxifen-regulated form of ATF6, Circ. Res. 98 (9) (2006) 1186–1193.

58] P.L. Zhang, M. Lun, J. Teng, J. Huang, T.M. Blasick, L. Yin, G.A. Herrera, J.Y. Che-ung, Preinduced molecular chaperones in the endoplasmic reticulum protectcardiomyocytes from lethal injury, Ann. Clin. Lab. Sci. 34 (4) (2004) 449–457.

59] K. Shintani-Ishida, M. Nakajima, K. Uemura, K. Yoshida, Ischemic precondi-tioning protects cardiomyocytes against ischemic injury by inducing GRP78,Biochem. Biophys. Res. Commun. 345 (4) (2006) 1600–1605.