Adiponectin Cardioprotection After Myocardial Ischemia/Reperfusion Involves the Reduction of Oxidative/Nitrative Stress

Bernard L. Lopez, Walter Koch, Lawrence Chan, Barry J. Goldstein and Xin L. MaLing Tao, Erhe Gao, Xiangying Jiao, Yuexing Yuan, Shuzhuang Li, Theodore A. Christopher,

Reduction of Oxidative/Nitrative StressAdiponectin Cardioprotection After Myocardial Ischemia/Reperfusion Involves the

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Adiponectin Cardioprotection After MyocardialIschemia/Reperfusion Involves the Reduction of

Oxidative/Nitrative StressLing Tao, MD, PhD; Erhe Gao, MD, PhD; Xiangying Jiao, MD, PhD; Yuexing Yuan, PhD;

Shuzhuang Li, MD; Theodore A. Christopher, MD; Bernard L. Lopez, MD; Walter Koch, PhD;Lawrence Chan, MBBS, DSc; Barry J. Goldstein, MD, PhD; Xin L. Ma, MD, PhD

Background—Several clinical studies have demonstrated that levels of adiponectin are significantly reduced in patientswith type 2 diabetes and that adiponectin levels are inversely related to the risk of myocardial ischemia. The presentstudy was designed to determine the mechanism by which adiponectin exerts its protective effects against myocardialischemia/reperfusion.

Methods and Results—Adiponectin�/� or wild-type mice were subjected to 30 minutes of myocardial ischemia followedby 3 hours or 24 hours (infarct size and cardiac function) of reperfusion. Myocardial infarct size and apoptosis,production of peroxynitrite, nitric oxide (NO) and superoxide, and inducible NO synthase (iNOS) and gp91phox proteinexpression were compared. Myocardial apoptosis and infarct size were markedly enhanced in adiponectin�/� mice(P�0.01). Formation of NO, superoxide, and their cytotoxic reaction product, peroxynitrite, were all significantly higherin cardiac tissue obtained from adiponectin�/� than from wild-type mice (P�0.01). Moreover, myocardial ischemia/reperfusion–induced iNOS and gp91phox protein expression was further enhanced, but endothelial NOS phosphorylationwas reduced in cardiac tissue from adiponectin�/� mice. Administration of the globular domain of adiponectin 10minutes before reperfusion reduced myocardial ischemia/reperfusion–induced iNOS/gp91phox protein expression,decreased NO/superoxide production, blocked peroxynitrite formation, and reversed proapoptotic and infarct-enlargement effects observed in adiponectin�/� mice.

Conclusion—The present study demonstrates that adiponectin is a natural molecule that protects hearts from ischemia/reperfusion injury by inhibition of iNOS and nicotinamide adenine dinucleotide phosphate-oxidase protein expressionand resultant oxidative/nitrative stress. (Circulation. 2007;115:1408-1416.)

Key Words: apoptosis � diabetes mellitus � myocardial infarction

Cardiovascular disease is the most prevalent cause ofmorbidity and mortality in patients with type 2

diabetes. Experimental results have demonstrated thathyperglycemia and hyperlipidemia not only cause vascularinjury that leads to ischemic heart disease but also have adirect adverse impact on ischemic cardiomyocytes, whichresults in larger infarct size1,2 and more severe heartfailure3 after myocardial ischemia. In recent years, enor-mous efforts have been made to identify the mechanismsresponsible for diabetic vascular injury, and many signal-ing pathways have been reported.4 The molecular basisthat links diabetes with increased vulnerability of diabetichearts to ischemic injury and resultant higher mortality hasnot been established, however.

Adiponectin is an adipocytokine secreted from adiposetissue.5,6 It contains a stalk with 22 collagen repeats and a

Clinical Perspective p 1416highly conserved globular domain (gAd). Adiponectin isnormally present in plasma at concentrations up to 30 �g/mL,but it is markedly downregulated in association with obesity-linked diseases such as coronary artery disease and type 2diabetes.7 Clinical observations have revealed that plasmatotal adiponectin concentrations are inversely correlated withthe risk of myocardial infarction.8,9 Moreover, Shibata et al10

recently reported that myocardial ischemic/reperfusion (I/R)injury is amplified in adiponectin knockout (adiponectin�/�)mice that carry significant metabolic phenotypes such asdelayed clearance of free fatty acid in plasma, decreasedmuscle fatty acid-transport protein-1 expression, and in-creased tumor necrosis factor (TNF)-� concentration.11 Thisresult provided the first direct evidence that adiponectin is an

Received September 26, 2006; accepted January 5, 2007.From the Department of Emergency Medicine (L.T., X.J., Y.Y., S.L., T.A.C., B.L.P., X.L.M.), Center for Translational Medicine (E.G., W.K.), and

Division of Endocrinology, Diabetes, and Metabolic Diseases (B.J.G.), Thomas Jefferson University, Philadelphia, Pa; and Section of Diabetes,Endocrinology, and Metabolism (L.C.), Department of Medicine, Baylor College of Medicine, Houston, Tex.

Correspondence to Dr Xin L. Ma, MD, PhD, Department of Emergency Medicine, 1020 Sansom St, Thompson Bldg, Rm 241, Philadelphia, PA 19107.E-mail [email protected]

© 2007 American Heart Association, Inc.

Circulation is available at http://www.circulationaha.org DOI: 10.1161/CIRCULATIONAHA.106.666941

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essential intrinsic molecule that protects the heart from I/Rinjury.

In human and mouse plasma, adiponectin exists in 3 majoroligomeric forms: trimers, hexamers, and a high-molecular-weight form.12 A proteolytic cleavage product of adiponectin,which includes gAd, may occur in human and mouse plas-ma.13 Recently it has been shown that the cleavage ofadiponectin by leukocyte elastase secreted from activatedmonocytes and/or neutrophils could be responsible for thegeneration of gAd.14 However, the physiological or patho-physiological role of this proteolytic cleavage product re-mains largely unknown, and its relationship to cardiovasculardisease is unclear.15

Considerable evidence indicates that apoptosis, a specialform of cell death that differs from necrosis in many aspects,plays an essential role in cardiomyocyte death induced bymyocardial I/R.16 In a recent study, Lin et al demonstratedthat angiotensin II–induced endothelial cell apoptosis in vitrois significantly inhibited by gAd,17 which suggests that gAdmay exert cardiovascular protective effects by its antiapoptot-ic property. Whether gAd may protect I/R-induced cardio-myocyte apoptosis and thus improve cardiac function hasnever been previously investigated. More important, themechanisms by which gAd exerts its antiapoptotic effectremain unknown.

Therefore, the purposes of the present study were todetermine whether myocardial I/R injury is similarly ampli-fied in a different strain of adiponectin�/� mice that carriesminimal metabolic phenotype18; if so, to investigate whetherthe enhanced I/R injury observed in these animals can berescued by a bolus administration of gAd immediately beforereperfusion; and to delineate mechanisms by which adiponec-tin may confer its cardioprotective effects after myocardialI/R.

MethodsExperimental ProtocolsThe experiments were performed in adherence with the NationalInstitutes of Health Guidelines on the Use of Laboratory Animals andwere approved by the Thomas Jefferson University Committee onAnimal Care. Homozygous (adiponectin�/�) and heterozygous adi-ponectin knockout (adiponectin�/�) mice were described previous-ly.18 Male adult mice (wild-type [WT; Taconic Farms, Inc, German-town, NY], adiponectin�/�, and adiponectin�/�) were anesthetizedwith 2% isoflurane, and myocardial infarction (MI) was produced bytemporarily exteriorizing the heart via a left thoracic incision andplacing a 6-0 silk suture slipknot around the left anterior descendingcoronary artery. After 30 minutes of MI, the slipknot was released,and the myocardium was reperfused for 3 hours or 24 hours (forcardiac function and infarct size determination). Sham-operatedcontrol mice (sham MI/R) underwent the same surgical proceduresexcept that the suture placed under the left coronary artery was nottied.

Determination of Myocardial ApoptosisMyocardial apoptosis was determined by TUNEL staining andcaspase-3 activity assay, which includes the entire I/R area com-monly termed as area at risk as described in our previous study.19

Determination of Cardiac Function andMyocardial Infarct SizeAt the end of the 24-hour reperfusion period, mice were re-anesthe-tized and cardiac function was determined by invasive hemodynamic

evaluation methods. The left ventricular systolic pressure, leftventricular end-diastolic pressure, first derivative of the left ventric-ular pressure (�dP/dtmax and �dP/dtmax) and heart rate were obtainedby use of computer algorithms and an interactive videographicsprogram (Po-Ne-Mah Physiology Platform P3 Plus, Gould Instru-ment Systems, Valley View, Ohio). After completion of functionaldetermination, the ligature around the coronary artery was retied, andMI size was determined by the Evans blue/TTC double stainingmethod as described previously.19

Determination of Total Nitric Oxide andNitrotyrosine Content in Cardiac TissueCardiac tissue (area at risk) was rinsed and homogenized. The tissuenitric oxide (NO) and its metabolic products (NO2 and NO3) in thesupernatant, collectively known as NOx, were determined by use ofa chemiluminescence NO detector (Siever 280i NO Analyzer) asdescribed previously.20 Nitrotyrosine content in the I/R cardiactissue, a footprint of in vivo peroxynitrite formation and an index fornitrative stress,21 was determined by use of an ELISA methoddescribed in our previous publication.22

Quantification of Superoxide ProductionMyocardial superoxide content was determined by lucigenin-enhanced luminescence as described in our previous study.23 In situ

Figure 1. A, Lack of adiponectin increases MI size. Evans blue–stained areas (black) indicate nonischemic/reperfused area;2,3,5-Triphenyl-2H-tetrazolium chloride (TTC) stained areas (redstaining) indicate ischemic but viable tissue; Evans blue/TTCstaining negative areas indicate infarcted myocardium. B, gAdreduces MI size in a dose-dependent fashion. AAR indicatesarea at risk; Adp, adiponectin; PT, pretreatment. N�10 to12/group. *P�0.05, **P�0.01 versus WT mice. ##P�0.01 versusAdp�/� mice. §P�0.05, §§P�0.01 versus 1 �g/g gAd.

Tao et al Adiponectin and Myocardial Reperfusion Injury 1409



superoxide detection was performed with dihydroethidium staining(DHE, Molecular Probes, Carlsbad, Calif) as described previously.24

ImmunoblottingProtein from I/R tissue homogenates was separated on SDS-PAGEgels, transferred to nitrocellulose membranes, and Western blottedwith monoclonal antibody against endothelial NO synthase (eNOS),phosphorylated eNOS, inducible NOS (iNOS; Upstate, Chicago, Ill)or gp91phox (Transduction Laboratories, San Jose, Calif). Nitrocellu-lose membranes were then incubated with HRP-conjugated anti-mouse immunoglobulin G antibody (1:2000, Cell Signaling, Dan-vers, Mass) for 1 hour and the blot was developed with a Supersignalchemiluminescence detection kit (Pierce, Rockford, Ill). The immu-noblotting was visualized with a Kodak Image Station 400 (Roch-ester, NY), and the blot densities were analyzed with Kodak1-dimensional software.

ImmunohistochemistryParaformaldehyde-fixed tissues were cut into semithin sections 4 to5 �m thick, and stained with an antibody against nitrotyrosine(Upstate). Immunostaining was developed with a Vectastain ABC kit(Vector Laboratories, Burlingame, Calif).

Determination of Plasma Adiponectin ConcentrationEndogenous plasma adiponectin level was determined with mouseadiponectin ELISA kit and exogenous gAd was determined withhuman adiponectin ELISA kit (Phoenix Pharmaceuticals, Inc., Bel-mont, Calif) in accordance with the manufacturer’s instructions.

Adult Mouse Cardiomyocyte CultureAdult mouse cardiomyocytes were isolated and subjected to 3 hoursof simulated ischemia (SI) and 6 hours of reperfusion (SI/R) asdescribed in our previous studies.25,26 Effects of adiponectin onSI/R-induced iNOS and gp91phox protein expression weredetermined.

Statistical AnalysisAll values in the text and figures are presented as mean�SEM of nindependent experiments. All data (except Western blot density)were subjected to ANOVA followed by Bonferroni correction forpost hoc t test. Western blot densities were analyzed with theKruskal-Wallis test followed by Dunn post hoc test. P�0.05 wasconsidered statistically significant.

The authors had full access to and take full responsibility for theintegrity of the data. All authors have read and agree to themanuscript as written.

ResultsMyocardial Reperfusion Injury Is MarkedlyIncreased in Adiponectin�/� MiceThirty minutes of ischemia followed by 24 hours of reperfu-sion resulted in significant cardiomyocyte death (�45% ofarea at risk) and impaired cardiac function. Compared withWT mice, MI size was enlarged and cardiac function wasfurther depressed in adiponectin�/� mice whose plasma adi-ponectin was undetectable (P�0.01) (Figures 1 and 2). Toobtain more evidence to support a causative role of adiponec-tin deficiency and increased myocardial reperfusion injury, 2additional experiments were performed. In the first series ofexperiments, heterozygous knockout mice (adiponectin�/�)whose plasma adiponectin concentration was significantlyreduced (4.2�0.18 versus 14.4�1.1 �g/mL in WT, P�0.01)were subjected to MI/R. Adiponectin�/� mice also had anincreased MI size and impaired cardiac function, although toa lesser extent than that seen in adiponectin�/� animals(Figures 1 and 2). In the second series of experiments, humanrecombinant gAd (amino acids 108 to 244) was prepared asdescribed27 and administered to the adiponectin�/� mice 10minutes before reperfusion (1 to 4 �g/g, intraperitoneally).Our preliminary experiments demonstrated that no humanadiponectin was detected before gAd administration by use ofa human adiponectin ELISA kit. Intraperitoneally adminis-tered gAd (2 �g/g) was partially absorbed and humanadiponectin was detected in adiponectin�/� and WT mice at10, 60, and 120 minutes postinjection (adiponectin�/�:9.9�0.7, 23.6�1.2, and 20.1�1.9 ng/mL; WT: 11.2�0.7,21.8�1.7, and 20.1�1.9 ng/mL). Treatment with gAd re-duced MI size in a dose-dependent fashion (Figure 1), andadministration of 2 �g/g gAd either 10 minutes before MI or10 minutes before reperfusion reduced MI size to a level not

Figure 2. Lack of adiponectin impairscardiac functional recovery after MI/R.LVEDP indicates left ventricular enddiastolic pressure; dP/dtmax, maximalpositive values of the instantaneousfirst derivative of left ventricular pres-sure; �dP/dtmax, maximal negative val-ues of the instantaneous first derivativeof left ventricular pressure. N�6 to9/group. Adp indicates adiponectin.*P�0.05, **P�0.01 versus WT mice.##P�0.01 versus Adp�/� mice.

1410 Circulation March 20, 2007



only significantly less than that in the adiponectin�/� groupbut also less than that in the WT mice that were administeredvehicle (Figure 1). Moreover, cardiac dysfunction in adi-ponectin�/� mice was reversed by treatment with gAd (Figure2).

Adiponectin Deficiency Markedly IncreasesI/R-Induced Cardiomyocyte Apoptosis That IsRescued by Administration of gAdApoptosis is the major form of cell death after a short periodof ischemia followed by reperfusion. To investigate whetherthe enlarged MI size in adiponectin�/� animals is associatedwith increased cardiomyocyte apoptosis in these animals,TUNEL staining and caspase-3 activity in I/R cardiac tissuewere determined. As illustrated in Figure 3, I/R-inducedcardiomyocyte apoptosis was significantly increased in adi-ponectin�/� mice, evidenced by increased TUNEL stainingand caspase-3 activity. Consistent with MI size, myocardialapoptosis was also increased in adiponectin�/� mice, althoughto a lesser extent than that seen in adiponectin�/� mice.Treatment with gAd completely blocked additional cardio-myocyte apoptosis in adiponectin�/� animals (Figure 3).

Lack of Adiponectin Increases Total NOProduction via Upregulation of iNOS Expressionin the I/R HeartConsiderable evidence exists that the low concentration ofNO released from eNOS or NO donors is antiapoptotic andcardioprotective,28 and that adiponectin has been shown tostimulate NO production via Akt-mediated eNOS phosphor-ylation.29,30 Therefore, we initially hypothesized that lack ofadiponectin may reduce NO production, and thus rendercardiomyocytes to be more susceptible to I/R injury. To oursurprise, total NO production was markedly increased, ratherthan decreased, in I/R tissue obtained from adiponectin�/�

mice (Figure 4). To identify the molecular sources responsi-ble for this increased NO production, eNOS/iNOS proteinexpression and eNOS phosphorylation were determined.There was no change in eNOS expression in adiponectin�/�

Figure 3. Lack of adiponectinincreases MI/R-induced cardiomyo-cyte apoptosis. N�6 to 8/group forTUNEL assay. N�9 to 11 forcaspase-3 assay. Adp indicates adi-ponectin. *P�0.05, **P�0.01 versusWT mice. ##P�0.01 versus Adp�/�

mice.

Figure 4. Effect of adiponectin on eNOS expression and phos-phorylation (peNOS), iNOS expression, and total NO production.All assays were performed on cardiac tissues obtained fromarea at risk. Inserted gel photographs are representative West-ern blot results from 4 to 5 animals/group. N�7 to 12/group fortotal NO assay. Adp indicates adiponectin. *P�0.05, **P�0.01versus WT mice. ##P�0.01 versus Adp�/� mice.




animals. However, eNOS phosphorylation was reduced incardiac tissues obtained from adiponectin�/� mice, and treat-ment with gAd increased eNOS phosphorylation (Figure 4).These results are consistent with previous reports that adi-ponectin is an upstream molecule that stimulates NO produc-tion via eNOS phosphorylation.30 In contrast to eNOS phos-phorylation, the iNOS expression was further enhanced inadiponectin�/� mice, and treatment with gAd blocked iNOSexpression in these animals (Figure 4).

Lack of Adiponectin Increases · O2� Productionvia Upregulation of gp91phox in the I/R HeartOur previous study demonstrated that oxLDL-induced · O2�production in endothelial cells is inhibited by a NADPHinhibitor as well as gAd, which suggests that adiponectin mayinhibit · O2� production from NADPH oxidase.31 Becausegenes that encode NADPH oxidase and iNOS belong to thesame inflammatory gene family, the aforementioned results(adiponectin inhibits iNOS expression) further suggest thatadiponectin may inhibit NADPH oxidase expression andsubsequent · O2� production. To directly test this hypothesis,· O2� production and gp91phox protein expression in the I/Rcardiac tissue were determined. As illustrated in Figure 5,I/R-induced superoxide overproduction was intensified incardiac tissue obtained from adiponectin�/� animals, andreplenishment with gAd before reperfusion attenuated super-oxide production. Moreover, I/R induced overexpression ofgp91phox, a major component of NADPH oxidase, was furtherincreased in adiponectin�/� mice, and administration of gAdbefore reperfusion blocked this adverse effect.

Lack of Adiponectin Increases TNF-� Productionin the I/R HeartAdiponectin has been reported to protect the heart partially byinhibition of TNF-� production.10 Because the adiponectin�/�

mice18 used in the present study differ in their metabolicphenotype from those used by Shibata et al,10 we thus deter-mined whether TNF-� production is also increased in thisparticular strain of adiponectin�/� mice. In cardiac tissues ob-tained from nonischemic control animals, TNF-� levels wereonly slightly increased in our adiponectin�/� mice comparedwith WT mice (1.18�0.08 versus 1.04�0.11 pg/mg protein,P�0.05). However, I/R-induced TNF-� overproduction wassignificantly enhanced in adiponectin�/� mice (1.86�0.16 ver-sus 1.59�0.09 pg/mg protein in WT, P�0.05). Treatment withgAd markedly reduced I/R-induced TNF-� production in theseanimals (1.39�0.15 pg/mg protein, P�0.01).

Overproduction of Peroxynitrite inAdiponectin�/� AnimalsStudies from other investigators as well as our group havedemonstrated that overproduction of peroxynitrite and resultantnitrative stress play a causative role in postischemic myocardialapoptosis.21,26 After demonstration of production of NO and ·O2�, the 2 free radicals that react at a diffusion-limited rate toform the highly cytotoxic molecule peroxynitrite, we then testeda hypothesis that lack of adiponectin may increase peroxynitriteformation, and thus render cardiomyocytes more susceptible toI/R injury. Consistent with our previous findings, I/R resulted insignificant peroxynitrite production as evidenced by a �2.3-foldincrease in nitrotyrosine content in the I/R cardiac tissue. Mostimportant, peroxynitrite formation was further increased inadiponectin�/� mice, and replenishment with gAd before reper-fusion markedly attenuated nitrotyrosine formation (Figure 6).

Inhibition of Peroxynitrite FormationPreferentially Attenuated Cardiac Injury inAdiponectin�/� AnimalsOur results suggest that adiponectin may exert its cardiopro-tective effects by inhibition of the overproduction of per-

Figure 5. Lack of adiponectin increasesgp91phox protein expression and superoxideproduction. In situ superoxide detectionwas performed with dihydroethidium stain-ing (DHE, N�4 to 5 animals/group) andmyocardial superoxide content was quanti-fied with lucigenin-enhanced luminescence(N�7 to 12/group). Inserted gel photo-graphs are representative Western blotresults from 4 to 5 animals/group. Adp indi-cates adiponectin; RLU, relative light units.**P�0.01 versus WT mice. ##P�0.01 ver-sus Adp�/� mice.




oxynitrite. To obtain more evidence to support this conclu-sion, an additional series of experiments was performed.Male adult adiponectin�/� mice or WT mice were subjected toMI/R as described above and treated with either 1400W (2mg/kg), a selective iNOS inhibitor, or M40401 (0.25 mg/kg),a peroxynitrite scavenger,32 10 minutes before reperfusion.As summarized in Figure 7, treatment with 1400W orM40401 significantly reduced nitrotyrosine content in I/Rtissue, which indicates that blockage of excess NO productionfrom iNOS or enhancement of peroxynitrite decompositioneffectively reduced peroxynitrite-induced protein nitration inMI/R cardiac tissue. These treatments also markedly attenu-ated myocardial apoptosis as determined by caspase-3 activ-ity (Figure 7). It is noteworthy that although nitrotyrosinecontent and caspase-3 activity were markedly higher in I/Rtissue from adiponectin�/� mice administered vehicle thantissue from WT mice (Figure 7), treatment with 1400W orM40401 reduced MI/R-induced nitrotyrosine formation and

caspase-3 activation to comparable levels in adiponectin�/�

mice and WT mice (Figure 7). These results demonstrate thatblocking iNOS activity or scavenging peroxynitrite preferen-tially protected hearts against MI/R-induced cardiomyocyteapoptosis in adiponectin�/� mice, and further suggest thatincreased oxidative and nitrative stress in adiponectin�/� miceplays a causative role in the accelerated MI/R injury observedin these animals.

Treatment With gAd Attenuated SI/R-InducediNOS Expression and gp91phox ExpressionHaving demonstrated that in vivo administration of gAdreduces iNOS and gp91phox expression in the I/R heart, wefurther examined whether gAd may exert this antioxidative/antinitrative effect directly on cardiomyocytes. As illustratedin Figure 8, exposure of adult mouse cardiomyocytes to SI/Rcaused an upregulation of iNOS and gp91phox. Treatment withgAd (2 �g/mL) significantly attenuated SI/R-induced iNOSand gp91phox protein expression (P�0.01). These resultsprovide direct evidence that iNOS and gp91phox proteinexpression is upregulated in I/R cardiomyocytes themselves

Figure 6. Lack of adiponectin increases peroxynitrite produc-tion. Inserted photographs are representative immunohisto-chemistry results from 4 to 5 animals/group. N�8 to 11/groupfor ELISA assay. Adp indicates adiponectin. **P�0.01 versusWT mice. ##P�0.01 versus Adp�/� mice.

Figure 7. Effect of 1400W (a selectiveiNOS inhibitor) or M40401 (a peroxyni-trite scavenger) on myocardial peroxyni-trite formation (measured by nitrotyrosinecontent) and cardiomyocyte apoptosis(measured by caspase-3 activity). Adpindicates adiponectin. **P�0.01 versusMI/R�vehicle (V). N�9 to 11animals/group.

Figure 8. Effect of gAd on SI/R-induced iNOS and gp91phox pro-tein expression in adult mouse cardiomyocytes. Inserts: repre-sentative Western blots. Bar graphs: summary of densitometricanalysis. N�5 to 7 hearts/group. *P�0.05, **P�0.01 versussham SI/R, ##P�0.01 versus SI/R�vehicle (V).




and that gAd is capable of inhibition of their expression inthis particular cell type.

DiscussionWe have made several important observations in the presentstudy. First, with an adiponectin�/� mouse strain that exhibitsminimal metabolic phenotype,18 we have provided additionalevidence that adiponectin possesses cardioprotective properties.Second, we have demonstrated for the first time that acuteadministration of gAd shortly before reperfusion significantlyattenuates MI/R injury. Most important, we have obtained thefirst direct evidence that adiponectin protects against MI/Rinjury, at least in part by a novel antioxidative and antinitrativemechanism that has never been previously reported.

Compared with other forms of adiponectin, the proteolyticallycleaved product of adiponectin (ie, gAd) presents in the circu-lation at an extremely lower concentration, and its biologicalsignificance therefore remains uncertain. Interestingly, it hasbeen recently reported that gAd exhibits much more extensive(�20-fold) biological activity than the full-length form.33,34

Moreover, Fruebis and colleagues reported that gAd is not onlymuch more potent in the stimulation of fatty acid oxidation inskeletal muscles than full-length adiponectin but also has muchmore rapid action than full-length adiponectin after a single invivo dose.13 These authors thus speculated that adiponectincirculates in plasma as an inactive precursor of a regulatoryprotein. In a more recent study, Pajvani and colleagues12

demonstrated that formation of the oligomer form of adiponectindepends on disulfide bond formation mediated by Cys-39.Mutation of Cys-39 results in trimers that are subject to proteo-lytic cleavage in the collagenous domain. The proteolytic cleav-age products are significantly more bioactive than the higherorder oligomeric forms of the protein (�30-fold). On the basis ofthese results and results published by others, the authors pro-posed that high-molecular-weight adiponectin complexes thatcirculate in serum represent a precursor pool that can beactivated by a serum reductase. Reduction of high-molecular-weight adiponectin results in its dissociation and the transientappearance of the bioactive trimer. Once reduced to the basictrimer, adiponectin may be subject to proteolysis by membrane-bound proteases found on the cell surface of target cells, whichleads to the formation of more biologically active gAd. There-fore, although the level of gAd is extremely low in circulation,gAd may represent the final active ligand of adiponectin at itstarget cells. In the present study, we have provided the first directevidence that administration of recombinant gAd as a bolus 10minutes before reperfusion reversed the adverse effects ofadiponectin knockout on MI/R injury. These results suggest thatgAd may have broad clinical applications in a variety ofcardiovascular diseases where apoptotic cell death is increased,such as heart failure, atherosclerosis, and hypertension. How-ever, the most potent oligomeric form of adiponectin for cardio-protection remains unknown and warrants further investigation.

Reactive oxygen species (ROS) have long been recognized tocause oxidative stress and act as the major mediators of I/Rinjury. We have previously demonstrated that treatment withgAd significantly inhibited basal · O2� release by bovine aorticendothelial cells, and abolished the oxidized low-density li-poprotein–induced increase in · O2� production.31 Moreover, our

most recent study demonstrated that adiponectin blocked highglucose–induced endothelial · O2� generation in vascular endo-thelial cells, which indicates that the · O2� suppression effect ofadiponectin is a general property of this novel molecule, ratherthan a specific effect on oxidized LDL–induced oxidativeinjury.35 In the present study, we have demonstrated thatI/R-induced gp91phox protein expression and resultant · O2�production were markedly increased in the adiponectin�/� ani-mals. We have also shown that administration of gAd reversedgp91phox overexpression and · O2� production observed inadiponectin�/� animals. These results provide the first directevidence to demonstrate that adiponectin is a natural moleculethat inhibits gp91phox overexpression in I/R cardiac tissue, andthus attenuates oxidative stress-induced tissue injury.

The most interesting finding of the present study is thatadiponectin differentially regulates NO production from eNOSand iNOS. Several previous studies have demonstrated thatadiponectin increases NO production by activation of theAMPK-Akt-eNOS phosphorylation pathway.29,30 Consistentwith these reports, our present study demonstrated that eNOSphosphorylation was reduced in I/R cardiac tissue obtained fromadiponectin�/� mice. Surprisingly, total NO production in the I/Rheart in adiponectin�/� mice was markedly increased, rather thandecreased. This paradoxical result strongly suggests that otherforms of NOS might be increased in these animals. Indeed, wehave provided direct evidence that I/R-induced iNOS expressionwas further increased in adiponectin�/� mice and that treatmentwith gAd significantly reduced iNOS expression in I/R cardiactissue. Taken together with previous studies, our present studysuggests that adiponectin increases NO production from eNOSunder physiological conditions, and thus contributes to adi-ponectin’s vasodilatory/cardioprotective effects. In contrast, un-der pathological conditions where iNOS expression is stimu-lated, adiponectin inhibits NO overproduction by inhibition ofiNOS expression and thus protects tissues from nitrative stress.

NO itself is not toxic and does not produce significant tissueinjury even at a very high concentration.36 However, the NO/ ·O2�reaction and the subsequent production of peroxynitritereverses its biological protective properties and results in oxida-tive/nitrative tissue injury.37 In the present study, we havedemonstrated that I/R-induced peroxynitrite formation wasmarkedly potentiated in adiponectin�/� mice and that treatmentwith gAd attenuated peroxynitrite production. Numerous studieshave demonstrated that peroxynitrite is the most importantnitrative species that causes severe nitrative stress and tissueinjury.37–39 The present study has provided the first evidence thatadiponectin is an endogenous molecule that inhibits the forma-tion of peroxynitrite by inhibition of NADPH oxidase-derived ·O2� and iNOS-derived NO.

Physiological or pharmacological concentrations of NO pro-duced from eNOS or NO donors exert significant cardioprotec-tive effects, whereas the reaction product between NO and · O2�,peroxynitrite, is extremely cytotoxic.36,37 Theoretically, a signal-ing system that possesses dual actions (ie, stimulation of NOproduction and inhibition of · O2� production) would providethe most protection against I/R injury. Unfortunately, most, ifnot all cytokines studied to date stimulate both NO and ·O2�production simultaneously, and result in peroxynitrite pro-duction and subsequent tissue injury. To our knowledge, adi-




ponectin is the first cytokine that has been shown to increasephysiological NO production (via eNOS phosphorylation), in-hibit · O2� production, and block peroxynitrite formation. Thesenovel findings suggest that reduced adiponectin concentration,like that observed in type 2 diabetes, may render cardiomyocytesmore susceptible to I/R injury by deregulation of expression ofinflammatory genes and enhancement of ROS and reactivenitrogen species production.

Limitations and Future DirectionA study by Shibata et al demonstrated that adiponectinconfers its cardioprotective effects through 2 distinct path-ways: the AMPK signaling pathway and the COX-2 signalingpathway.10 The present study demonstrated that adiponectincardioprotection after MI/R involves the reduction of oxida-tive/nitrative stress. However, the signaling hierarchy be-tween activation of AMPK/COX-2 reported by Shibata et al10

and the reduction of ROS/reactive nitrogen species observedin the present study remains unclear. Treatment with met-formin has been reported to inhibit hyperglycemia-inducedsuperoxide production in endothelial cells, which is blockedby the dominant negative form of AMPK transfection.40

Activation of AMPK by insulin-sensitizing drugs markedlyinhibits endotoxin-induced iNOS expression and NO over-production in muscle and adipose tissues, which is blocked byAMPK small interfering RNA.41 It is therefore possible thatreduction of oxidative/nitrative stress functions as a down-stream mechanism by which adiponectin-activated AMPKexerts its cardioprotective effects. On the other hand,adenovirus-mediated transfer of COX-2 blocks sanguinarine-induced NO/superoxide overproduction in LNCaP cells andreduces apoptosis,42 which suggests that COX-2 activationmay be an upstream mechanism by which adiponectin re-duces oxidative/nitrative stress. In addition, we have recentlydemonstrated that gAd suppresses high glucose–inducedROS production in vascular endothelial cells by a cAMP-mediated signaling pathway.35 Taken together, current exper-imental evidence suggests that adiponectin may reduce ROS/reactive nitrogen species overproduction by multipleupstream signaling pathways, and that reduction of oxidative/nitrative stress observed in our present study may be a finalcommon pathway by which adiponectin exerts its cardiopro-tective effects. Moreover, it is possible that different form ofadiponectin may block ROS/reactive nitrogen species pro-duction induced by different stimulation in different cell typeby different signaling pathway. Clarification of the individualsignaling mechanism is of scientific and clinical importanceand thus warrants future investigations.

Sources of FundingThis research was supported by the following grants: NationalInstitutes of Health (NIH) 2R01HL-63828, American DiabetesAssociation 7-05-RA-83, and Commonwealth of Pennsylvania De-partment of Health (to Dr Ma); American Diabetes Association7-6-JF59 (to Dr Tao); NIH DK63018, NIH DK71360, and AmericanDiabetes Association (to Dr Goldstein); and NIH HL51586 and NIHDK68037 (to L. Chan). Dr Christopher has received a grant from theAmerican Heart Association; Dr Koch has received a grant fromNIH.

DisclosuresNone.

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CLINICAL PERSPECTIVECardiovascular disease is the major cause of morbidity and mortality in patients with diabetes mellitus. The molecular linksbetween diabetes and cardiovascular disease have not been fully clarified. Adiponectin is an adipocytokine secreted byadipose tissue that is normally present in plasma, but is reduced with obesity-linked diseases such as coronary artery diseaseand type 2 diabetes. Clinical observations have revealed that plasma total adiponectin concentrations are inverselycorrelated with the risk of myocardial infarction, which suggests that reduced adiponectin plays a pathogenic role in thedevelopment of cardiovascular disease. The present study demonstrates that cardiac injury associated with myocardialischemia/reperfusion is markedly enhanced in adiponectin knockout animals. Acute administration of the globular domainof adiponectin shortly before reperfusion attenuated myocardial injury by a reduction of oxidative/nitrative stress andsubsequent apoptotic cell death. The reduced adiponectin found in patients with type 2 diabetes may increase theirsusceptibility to ischemia/reperfusion injury by deregulation of expression of inflammatory genes and enhancement ofproduction of reactive oxygen/nitrogen species. Restoration of the production of endogenous adiponectin or supplemen-tation with exogenous adiponectin may have therapeutic value in the reduction of myocardial ischemia/reperfusion injuryin patients with type 2 diabetes.




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Adiponectin Cardioprotection After Myocardial Ischemia/Reperfusion Involves the Reduction of Oxidative/Nitrative Stress