Diab Cvd Gene

Therapeutic effect of MG-132 on diabetic cardiomyopathy is associated withits suppression of proteasomal activities: roles of Nrf2 and NF-B

Yuehui Wang,1 Weixia Sun,2,3 Bing Du,2 Xiao Miao,1,3 Yang Bai,2,3 Ying Xin,3,4 Yi Tan,3,5Wenpeng Cui,1,3 Bin Liu,1 Taixing Cui,5,6 Paul N. Epstein,3,7 Yaowen Fu,2 and Lu Cai3,5,71The Second Hospital, Jilin University, Jilin, China; 2The First Hospital, Jilin University, Jilin, China; 3Kosair Childrens HospitalResearch Institute, Department of Pediatrics, University of Louisville, Louisville, Kentucky; 4Norman Bethune Medical College,Jilin University, Jilin, China; 5Chinese-American Research Institute for Diabetic Complications, Wenzhou Medical College,Wenzhou, China; 6Department of Cell Biology and Anatomy, University of South Carolina School of Medicine, Columbia,South Carolina; and 7Departments of Pharmacology and Toxicology, University of Louisville, Louisville, KentuckySubmitted 31 August 2012; accepted in final form 4 December 2012

Wang Y, Sun W, Du B, Miao X, Bai Y, Xin Y, Tan Y, Cui W,Liu B, Cui T, Epstein PN, Fu Y, Cai L. Therapeutic effect ofMG-132 on diabetic cardiomyopathy is associated with its suppres-sion of proteasomal activities: roles of Nrf2 and NF-B. Am J PhysiolHeart Circ Physiol 304: H567H578, 2013. First published December7, 2012; doi:10.1152/ajpheart.00650.2012.MG-132, a proteasomeinhibitor, can upregulate nuclear factor (NF) erythroid 2-related factor2 (Nrf2)-mediated antioxidative function and downregulate NF-B-mediated inflammation. The present study investigated whetherthrough the above two mechanisms MG-132 could provide a thera-peutic effect on diabetic cardiomyopathy in the OVE26 type 1 dia-betic mouse model. OVE26 mice develop hyperglycemia at 23 wkafter birth and exhibit albuminuria and cardiac dysfunction at 3 mo ofage. Therefore, 3-mo-old OVE26 diabetic and age-matched controlmice were intraperitoneally treated with MG-132 at 10 g/kg daily for3 mo. Before and after MG-132 treatment, cardiac function wasmeasured by echocardiography, and cardiac tissues were then sub-jected to pathological and biochemical examination. Diabetic miceshowed significant cardiac dysfunction, including increased left ven-tricular systolic diameter and wall thickness and decreased left ven-tricular ejection fraction with an increase of the heart weight-to-tibialength ratio. Diabetic hearts exhibited structural derangement andremodeling (fibrosis and hypertrophy). In diabetic mice, there wasalso increased systemic and cardiac oxidative damage and inflamma-tion. All of these pathogenic changes were reversed by MG-132treatment. MG-132 treatment significantly increased the cardiac ex-pression of Nrf2 and its downstream antioxidant genes with a signif-icant increase of total antioxidant capacity and also significantlydecreased the expression of IB and the nuclear accumulation andDNA-binding activity of NF-B in the heart. These results suggestthat MG-132 has a therapeutic effect on diabetic cardiomyopathy inOVE26 diabetic mice, possibly through the upregulation of Nrf2-dependent antioxidative function and downregulation of NF-B-me-diated inflammation.

diabetic cardiomyopathy; nuclear factor erythroid 2-related factor 2;nuclear factor-B; proteasome inhibitor; MG132; therapeutic effect

THE UBIQUITIN-PROTEASOME SYSTEM (UPS) represents the majornonlysosomal pathway for the degradation of ubiquitinatedproteins (26). In addition to the removal of misfolded ordamaged proteins, the UPS also plays a crucial role in theregulation of many cellular processes, such as the cell cycle,apoptosis, transcriptional control, and responses to stress. Re-cent studies (26, 36) have indicated that alterations in the UPS

contribute to the pathogenesis and progression of several car-diac diseases, and inhibition of proteasome activity has becomean interesting approach to prevent various diseases, includingcardiac diseases.

Bortezomib (velcadew) was the first proteasome inhibitorapproved by the Federal Drug Administration in 2003 (8).However, side effects of Bortezomib and the development ofresistance in some patients with tumors prompted the study ofnew proteasome inhibitors (8). The cell-permeable MG-132tripeptide (Z-Leu-Leu-Leu-aldehyde) is a peptide aldehydeproteasome inhibitor that also inhibits other proteases, includ-ing calpains and cathepsins. Reportedly nontoxic concentra-tions of MG-132 inhibit Nrf2 proteasomal degradation andstimulate nuclear factor (NF) erythroid 2-related factor 2(Nrf2) translocation into the nucleus (5, 28). By blocking theproteasome, this tripeptide has been shown to induce theexpression of cell-protective proteins, such as heat shockproteins, in vitro and in vitro. MG-132 was found to play animportant role in cardiovascular protection from variousstresses and pathogeneses (4, 19, 28).

One mechanism responsible for the cardiac protection byMG-132 is its upregulation of antioxidants via activation ofNrf2 (28). Nrf2 is a key transcription factor in the regulation ofmultiple antioxidants, including NADPH-quinone oxidoreduc-tase (NQO)-1, heme oxygenase (HO)-1, catalase (CAT), SOD,glutathione peroxidase, and glutamate-cysteine ligase. Theactin-tethered protein kelch-like ECH-associated protein 1 is acytosolic repressor that binds to and retains Nrf2 in the cyto-plasm, which promotes Nrf2 proteasomal degradation andresults in prevention of Nrf2 transcription (14, 30). Therefore,inhibition of proteasome activity is one method to maintaincytoplasm Nrf2 available to nuclear translocation and activa-tion in response to oxidative stress (15).

Another important mechanism underlying MG-132 cardio-vascular protection is its inactivation of NF-B, a nucleartranscription factor that regulates proinflammatory cytokineexpression. Activation of NF-B has been documented inmyocardial ischemia-reperfusion (13), and specific inhibitionof NF-B is cardioprotective (25). NF-B usually is inhibitedvia IB by forming a complex with NF-B. However, IB isdegraded by proteasome ubiquitination, resulting in the releaseof NF-B and nuclear translocation to turn on inflammatorygenes. Therefore, proteasomal inhibitors such as MG-132 caninhibit proteasome activity to provide anti-inflammation func-tion, leading to cardiac protection from ischemic damage.

Address for reprint requests and other correspondence: L. Cai, Dept. ofPediatrics, Univ. of Louisville, 570 S. Preston St., Baxter I, Suite 321B or304F, Louisville, KY 40202 (e-mail: [email protected]).

Am J Physiol Heart Circ Physiol 304: H567H578, 2013.First published December 7, 2012; doi:10.1152/ajpheart.00650.2012.

0363-6135/13 Copyright 2013 the American Physiological Societyhttp://www.ajpheart.org H567

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Diabetic cardiomyopathy is also associated with early cardiacoxidative stress and inflammation, which initiates the late devel-opment of cardiac remodeling and dysfunction (1, 12, 27, 34). Aprevious report (16) has shown that diabetes increased cardiacproteasome function (11, 22), which was associated with diabeticcomplications. Therefore, we hypothesized that MG-132 could beone of the proteasome inhibitors that may be potentially used forthe prevention of diabetic cardiomyopathy via upregulation ofNrf2 to reduce oxidative stress and downregulation of NF-B-mediated inflammation (16). A few studies (3, 4, 17, 19, 28, 31)have shown cardiac and renal protection by proteasome inhibitorfrom other pathogeneses. Dreger et al. (9) demonstrated theprotective effect of low-dose MG-132 on H2O2-induced oxidativestress and damage in cardiomyocytes and also showed increasedsensitivity in myocytes from Nrf2 knockout mice, suggesting theimportant role of Nrf2 in cardiac protection. Using MG-132,several studies (16, 32) have also shown the preventive effect ofinhibition of proteasome activity on diabetic vascular injury.Results from a pilot study (18) indicated that a proteasomalinhibitor that upregulates Nrf2 activity and inhibits NF-B-mediated inflammation provided a preventive effect on diabetes-induced renal dysfunction.

From a clinical setting, there is an urgent need for an efficientapproach that can provide a therapeutic effect rather than onlyprevention, since it is not easily accepted that diabetic patients willreceive preventive medical treatement once diagnosed as diabetic.So far, however, there is no report regarding the therapeutic effectof MG-132 on diabetic complications; in the present study, there-fore, we tried to address the question of whether MG-132 canprovide a therapeutic effect on diabetic cardiomyopathy by testingif MG-132 could reserve or slow the progression of establishedcardiac dysfunction in the OVE26 mouse model of severe type 1diabetes. In addition, mechanistic experiments were focused onthe upregulation of Nrf2-mediated antioxidants and downregula-tion of NF-B-mediated inflammation.

MATERIALS AND METHODS

Animals

The transgenic type 1 diabetic OVE26 mouse model on a FVB back-ground has been characterized in a previous study (38). All mice werehoused at the University of Louisville Research Resources Center at 22Cwith a 12:12-h light-dark cycle and provided with free access to standardrodent chow and tap water. All animal procedures were approved by theInstitutional Animal Care and Use Committee, which is certified by theAmerican Association for Accreditation of Laboratory Animal Care.

OVE26 mice normally develop severe hyperglycemia before 3 wkof age and develop albuminuria by 3 mo of the age, particularly infemale OVE26 mice (38). Sixteen 3-mo-old female OVE26 mice wererandomly divided into two groups: a diabetic group (DM group; n 10) and a MG-132-treated OVE26 group (DM/MG-132 group; n 6).Sixteen age- and sex-matched nondiabetic FVB mice were randomlydivided into two groups: a control group (n 10) and a MG-132-treated group (MG-132 group; n 6). MG-132 (Sigma-Aldich, St.Louis, MO) was dissolved in DMSO at a concentration of 0.0025g/ml and diluted with saline for injection. For MG-132 and DM/MG-132 mice, MG-132 was given intraperitoneally at a dose of 10g/kg body wt daily for 3 mo, based on a recent study (18), startingat 3 mo old in OVE26 mice when these mice already displayedsignificant renal dysfunction. For control and DM mice, equalamounts of physiological saline solution containing 0.0025 gDMSO/ml were given. MG-132 treatment for 3 mo in OVE26 diabeticmice did not affect their blood glucose levels (7).

Four mice from both control and DM groups were killed at 3 mo ofage, and other mice (n 6) were treated with MG-132 for 3 mo andkilled at 6 mo of age. After heart weight and tibia length had beenmeasured, whole heart tissue was harvested for protein and mRNAanalysis. Histopathological observations were predominantly based onthe left ventricle (LV).

Echocardiography

To assess cardiac function, echocardiography was performed inmice using a Visual Sonics Vevo 770 high-resolution imaging system,as previously described (29, 39). Under sedation with Avertin (2,2,2-tribromoethanol, 240 mg/kg ip), mice were placed in the supineposition on a heating pad to maintain body temperature at 3637C.Heart rates were kept 400550 beats/min. Two-dimensional andM-mode echocardiography were used to assess wall motion, chamberdimensions, and cardiac function. Directly measured indexes includedLV internal dimensions (LVID) at diastole and systole, LV posteriorwall thickness (LVPW) at diastole and systole, and interventricularseptal thickness (IVS) at diastole and systole. LV fractional shortening(FS) was determined as follows: FS [(LVID at diastole LVID atsystole)/LVID at diastole] 100. LV ejection fraction (EF) wasdetermined as follows: EF [(LV volume at diastole LV volumeat systole)/LV volume at diastole] 100.

Western Blot Analysis

Western blot analysis was performed as described in our previousstudies (2, 39). The primary antibodies used included 3-nitrotyrosine(3-NT; 1:1,000 dilution), 4-hydroxy-2-nonenal (4-HNE; 1:1,000 di-lution), TNF- (1:500 dilution), ICAM-1 (1:500 dilution), plasmino-gen activator inhibitor (PAI)-1 (1:1,000 dilution), transforminggrowth factor (TGF)-1 (1:500 dilution), fibronectin (1:500 dilution),Nrf2 (1:500 dilution), NQO-1 (1:500 dilution), HO-1 (1:500 dilution),CAT (1:5,000 dilution), atrial natriuretic peptide (ANP; 1:1,000 dilu-tion), NF-B (1:1,000 dilution), IB- (1:1,000 dilution), -tubulin(1:2,000 dilution), and -actin (1:2,000 dilution), all of which werepurchased from Santa Cruz Biotechnology except for TNF- (Ab-cam), 3-NT (Millipore), NF-B, IB-, and -tubulin (Cell Signal-ing), and 4-HNE (Alpha Diagnostic). All antibodies were polyclonalantibodies except for TNF- and PAI-1 antibodies, which were mono-clonal.

Isolation of RNA and Real-Time RT-PCRIsolation of RNA and real-time RT-PCR were performed as described

in our previous study (39) for Nrf2 (primer: Mm00477784_m1), ANP(primer: Mm01255748_g1), -myosin heavy chain (-MHC; primer:Mm00600555_m1), HO-1 (primer: Mm00516005_m1), NQO-1 (primer:Mm253561_m1), CAT (primer: Mm00437229_m1), and the housekeep-ing gene -actin (primer: Mm00607939_s1). All primers were purchasedfrom Applied Biosystems (Foster City, CA). Total RNA was extractedfrom whole heart tissues using TRIzol reagent (RNA STAT 60 Tel-Test,Ambion, Austin, TX). RNA concentration and purity were quantifiedusing a Nanodrop ND-1000 spectrophotometer (Biolab, Ontario, CA).First-strand cDNA was synthesized from total RNA according to theRNA PCR kit (Promega, Madison, WI) following the manufacturersprotocol.

Cardiac Histopathological Examination andImmunohistochemical Staining

Whole cardiac tissue was fixed overnight in 10% phosphate buff-ered formalin, dehydrated in a graded alcohol series, cleared withxylene, embedded in paraffin, and then sectioned at 5 m thicknessfor pathological and immunohistochemical staining. Paraffin sectionswere dewaxed followed by an incubation in 1 target retrievalsolution (Dako, Carpinteria, CA) for 15 min at 98C for antigen

H568 THERAPEUTIC EFFECT OF MG-132 ON DIABETIC CARDIOMYOPATHY

AJP-Heart Circ Physiol doi:10.1152/ajpheart.00650.2012 www.ajpheart.org

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retrieval. Sections were then treated with 3% H2O2 for 15 min at roomtemperature followed by blocking with 5% BSA for 30 min.

Cardiac sections were stained with hematoxylin and eosin andSirius red, respectively, as previously described (2, 39). For immu-nohistochemical staining, sections were incubated with the primaryantibodies of TGF-1 (1:100 dilution, Santa Cruz Biotechnology) andPAI-1 (1:100 dilution, BD Biosciences) overnight at 4C. After beingwashed with PBS, sections were incubated with horseradish peroxi-dase-conjugated secondary antibody (1:300400 dilutions in PBS) for

2 h at room temperature. For the development of color, sections weretreated with the peroxidase substrate 3,3-diaminobenzidine in thedeveloping system (Vector Laboratories, Burlingame, CA).

Proteasome Activity

The 20S proteasome, the catalytic core of the 26S proteasomecomplex, is responsible for the breakdown of short-lived regulatoryproteins, including Nrf2 and NF-B (6, 9). Since MG-132 mainly

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Fig. 1. Therapeutic effects of MG-132 on diabetes-induced cardiac dysfunction. Three-mo-old female OVE26 mice and FVB control mice were given MG-132(10 g/kg) or an equal volume of physiological saline solution daily for 3 mo. Mice were divided into the following groups: control FVB mice (Ctrl group),control mice with MG-132 treatment (MG-132 group), diabetic OVE26 mice (DM group), and diabetic mice with MG-132 treatment (DM/MG-132 group).Cardiac functional (A) and structural (B) changes were evaluated by echocardiography. LVID;d and LVID;s, left ventricular (LV) internal dimension at diastoleand at systole, respectively; EF%, ejection fraction (in %); FS%, fractional shortening (in %); IVS;d and IVS;s, interventricular septal thickness at diastole andat systole, respectively; LVPW;d and LVPW;s, LV posterior wall thickness at diastole and at systole, respectively; 3m and 6m, 3 mo and 6 mo, respectively.Data are presented as means SD. *P 0.05 vs. the Ctrl group; #P 0.05 vs. the DM group.

H569THERAPEUTIC EFFECT OF MG-132 ON DIABETIC CARDIOMYOPATHY


inhibits proteasome chymotrypsin-like activity (24), we determined20S proteasome activity by quantifying the hydrolysis of the SLLVY-AMC-a fluorogenic substrate for chymotrypsin-like activity. 20Sproteasome activity was measured with the 20S proteasome activityassay kit (Millipore).Isolation of Nuclei

Cardiac nuclei were isolated using the nuclei isolation kit (Sigma-Aldich). Whole cardiac tissue (60 mg) from each mouse was homog-enized for 45 s with 300 l cold lysis buffer containing 1 l DTT and0.1% Triton X-100. After that, 600 l of cold 1.8 mol/l cushionsolution [sucrose cushion solution-sucrose cushion buffer-DTT (900:100:1)] were added to the lysis solution. The mixture was transferredto a new tube preloaded with 300 l of 1.8 mol/l sucrose cushionsolution followed by centrifugation at 13,000 rpm for 45 min. Thesupernatant contained the cytosolic components, and nuclei werevisible as a thin pellet at the bottom of the tube.

Measurements of Serum TNF- and Monocyte ChemoattractantProtein-1 and Cardiac Nuclear NF-B DNA-Binding Activity

Serum levels of TNF- and monocyte chemoattractant protein(MCP)-1 were performed using mouse TNF- and MCP-1 ELISA kits

(Invitrogen, Camarillo, CA), as previously described (21). The lowdetection limits were 3 and 9 pg/ml for serum TNF- and MCP-1,respectively. Nuclear p65 DNA-binding activity was determined byan ELISA-based NF-B activity assay using cardiac tissue from thewhole heart (Cayman Chemical, Ann Arbor, MI), according to man-ufacturer instructions.

Total Antioxidant Capacity Assay

The cardiac total antioxidant capacity (TAC) assay was performedusing a commercially available assay kit (Cell Biolabs, San Diego,CA) according to the manufacturers instructions. Briefly, whole hearttissues were homogenized in cold PBS and then centrifuged at 10,000g at 4C for 10 min to collect the supernatant for the TAC assay.Values were calculated using optical density at 490 nm and expressedas micromoles per gram of protein for TAC.

Statistical Analysis

Data were expressed as means SD. For statistical analysis,one-way ANOVA was used as appropriate. An overall F-test wasperformed to test the significance of the ANOVA models. Thesignificance of the interactions and main effects were taken intoconsideration, and multiple comparisons were then performed with a

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Fig. 2. Therapeutic effect of MG-132 on diabe-tes-induced cardiac hypertrophy. A: at both 3and 6 mo of diabetes, the ratio of heart weight totibia length was measured and calculated aftermice had been euthanized. B: cardiac structuralderangement was examined by morphologicalexamination with hematoxylin and eosin stain-ing. The blue arrow indicates myocardial hyper-trophy; the black arrow indicates myocardialcells degeneration; the brown arrow indicates theproliferation of interstitial collagen fibers. Mag-nification: 400. Bar 50 m. CE: mRNAexpression of the cardiac hypertrophic markersatrial natriuretic peptide (ANP; C) and -myosinheavy chain (-MHC; D) as well as the proteinexpression of ANP (E) were measured by real-time quantitative PCR and Western blot analy-sis. Data are presented as means SD. *P 0.05 vs. the Ctrl group; &P 0.05 vs. the 3-mDM group; #P 0.05 vs. the DM group.



Bonferroni test. P values of 0.05 were considered as statisticallysignificant.

RESULTS

Therapeutic Effect of MG-132 on Diabetes-Induced CardiacDysfunction and Hypertrophy

Transgenic OVE26 type 1 DM mice at 3 mo showed increasedLVID at systole and decreased EF and FS percentages, suggest-ing the exist of cardiac dilation and decreased systolic function(Fig. 1A). Hypertrophic measurements of IVS, LVPW, and LVmass were significantly increased in these mice, with a progres-sive manner from 3 to 6 mo (P 0.05; Fig. 1B). However, whensome of the DM mice at 3 mo of age were treated with low-doseMG-132 for 3 mo (DM/MG-132 group), diabetes-induced cardiacdysfunction was almost completely reversed (P 0.05; Fig. 1).

The therapeutic effects of MG-132 on diabetes-inducedcardiac hypertrophy were also reflected by the ratio of heartweight to tibia length (Fig. 2A), which was significantly in-

creased in an age-dependent manner in the DM group from 3to 6 mo but not in the DM/MG-132 group.

To further confirm cardiac hypertrophy, cardiac morphologyand molecular hypertrophy makers were also examined. He-matoxylin and eosin staining showed that, compared with thecontrol and MG-132 groups, the main pathological changes inthe DM group included 1) myocardial hypertrophy, 2) a fewcardiac cells that showed features of degeneration and 3) pro-liferation of interstitial collagen fibers. Those pathologicalchanges were alleviated or not observed in DM/MG-132 mice(Fig. 2B). Real-time PCR analysis showed that DM induced asignificant increase of the cardiac mRNA expression of ANP(Fig. 2C) and -MHC (Fig. 2D), an effect that was completelyabolished by MG-132 treatment in the DM/MG-132 group.Consistent with the ANP mRNA findings, Western blot anal-ysis showed a significant increase of cardiac ANP proteinexpression at 6 mo of age in DM mice but not in DM/MG-132mice (Fig. 2E). These results suggest that 3-mo treatment withlow-dose MG-132 can significantly or even completely pre-

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Fig. 3. Therapeutic effect of MG-132 on diabe-tes-induced cardiac fibrosis. A: cardiac tissuewas stained with Sirius red for collagen. Arrowsindicate the proliferation of interstitial collagenfibers. Original magnification: 200. Bar 50m. B: semiquantitative analysis was done by acomputer imaging system. C: cardiac expres-sion of transforming growth factor (TGF)-1,fibronectin, and plasminogen activator inhibitor(PAI)-1 was examined by Western blot analysis.D: cardiac expression of TGF-1 and PAI-1 wasalso tested by immunohistochemical staining.Arrows indicate positive staining. Data are pre-sented as means SD. *P 0.05 vs. the Ctrlgroup; #P 0.05 vs. the DM group.



vent the progression of the cardiac hypertrophy induced by dia-betes.

Therapeutic Effect of MG-132 on Diabetes-InducedCardiac Fibrosis

Cardiac tissue was examined with Sirius red staining forcollagen (Fig. 3A) followed by semiquantitative analysis witha computer imaging system (Fig. 3B), which showed a signif-icant increase in collagen accumulation in the DM group butnot in the DM/MG-132 group. Western blot analyses of TGF-1, fibronectin, and PAI-1 as important profibrotic mediatorsconfirmed the Sirius red staining results: there was a significantincrease in cardiac fibrosis in the DM group but not in theDM/MG-132 group (Fig. 3C). Furthermore, the increased ex-pression of TGF-1 and PAI-1 was supported by immunohis-tochemical staining results (Fig. 3D).Therapeutic Effect of MG-132 on Diabetes-Induced CardiacInflammation and Oxidative Stress

Since PAI-1 acts as both a profibrotic and inflammatory medi-ator, its increase in the hearts of DM mice suggests possiblecardiac inflammation. Thus, we next examined the protein expres-sion of TNF- (Fig. 4A) and MCP-1(Fig. 4B) in serum by ELISA

and in the heart by Western blot assay (Fig. 4, C and D). SerumTNF- and MCP-1 levels were progressively elevated from 3 to6 mo in the DM group and were completely prevented byMG-132 treatment in the DM/MG-132 group. Increased cardiacTNF- and MCP-1 contents were also observed only in the DMgroup and not in the DM/MG-132 group.

Considering that inflammation in target organs often causesoxidative stress and that oxidative stress is also able to induceinflammation, we examined oxidative stress by measuring3-NT accumulation as an index of protein nitration (Fig. 5A)and 4-HNE as an index of lipid peroxidation (a measure ofoxidative damage; Fig. 5B). Both 3-NT and 4-HNE contentswere significantly increased in the hearts of DM mice but notDM/MG-132 mice. TAC in heart tissue (Fig. 5C) was slightlydecreased (P 0.05) in the DM group at both 3 and 6 mo butwas not changed in the DM/MG-132 group at 6 mo.

Possible Mechanisms for the Therapeutic Effect of MG-132on Diabetic Cardiomyopathy

Diabetes increases cardiac proteasomal activity, an effectprevented by MG-132. Since tetrahydrobiopterin deficiencyhas been reported to uncouple the enzymatic activity of endo-thelial nitric oxide synthase in DM hearts triggered by DM-

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Fig. 4. Therapeutic effect of MG-132 on diabetes-induced inflammatory cytokines. A and B: TNF-(A) and MCP-1 (B) levels in serum were deter-mined by ELISA. C and D: cardiac tissue wassubjected to Western blots for TNF- (C) andICAM-1 (D). Data are presented as means SD.*P 0.05 vs. the Ctrl group; #P 0.05 vs. theDM group.



increased proteasome-dependent mechanisms (35), we exam-ined whether cardiac proteasome activity was increased indiabetic mice. The results shown in Fig. 6 demonstrate thatMG-132 decreased, and diabetes increased, cardiac 20S pro-

teasomal activity. Compared with the DM group, cardiac 20Sproteasomal activity was significantly reduced, even to controllevels, in the DM/MG-132 group (45% DM, P 0.05 vs. theDM group).

MG-132 inhibits cardiac proteasomal activity, resulting in anupregulation of cardiac Nrf2 and its downstream antioxidants.Proteasomal degradation of Nrf2 has been delineated as themajor of mechanism responsible for Nrf2s negative regulation(14, 30). Our finding that MG-132 completely inhibited diabe-tes-upregulated proteasomal activity implies a possibility forMG-132 treatment to decrease the degradation of Nrf2 protein.Therefore, expression of Nrf2 at mRNA and protein levels wasexamined with real-time PCR and Western blot assays, respec-tively. We found that MG-132 had no impact on, but diabeteshad a significant increase in, the mRNA expression of Nrf2(Fig. 7A). The DM/MG-132 group had a similar expression ofNrf2 mRNA as in the DM group (Fig. 7A). The results shownin Fig. 7B demonstrate that both MG-132 and DM increasedthe cardiac expression of Nrf2 protein, so that Nrf2 proteinexpression was synergistically increased in the DM/MG-132group.

Furthermore, the upregulated Nrf2 transcription activity wasreflected by the increase of several downstream antioxidantgenes (Fig. 7, C and D). MG-132 treatment in control micesignificantly increased the expression of NQO-1, HO-1, and

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Fig. 5. Therapeutic effect of MG-132 on diabetes-induced cardiac oxidative stress. A and B: cardiactissue was subjected to Western blots for 3-nitroty-rosine (3-NT; A) and 4-hydroxy-2-nonenal (4-HNE;B). C: total antioxidant capacity (TAC) in cardiactissue was detected and expressed as micromolesper gram of protein. Data are presented as meansSD. *P 0.05 vs. the Ctrl group; #P 0.05 vs. theDM group.

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Fig. 6. Effect of MG-132 on cardiac activity of the proteasome. Cardiac protea-some activity was measured using a 20S proteasome activity kit. Data arepresented as means SD. *P 0.05 vs. the Ctrl group; #P 0.05 vs. the DMgroup.



CAT at the mRNA and protein levels compared with thecontrol group. Diabetes also increased the expression of theseantioxidants at the mRNA and protein levels. Expression ofthese Nrf2 downstream antioxidant genes in the hearts ofDM/MG-132 mice was synergistically increased at both themRNA and protein levels compared with both MG-132 or DMmice.

MG-132 inhibits cardiac proteasomal activity, resulting in areduction of cardiac inflammation by preventing NF-B nu-clear translocation. Since NF-B is bound by IB- in thecytoplasm, inhibiting NF-B nuclear translocation to transcrip-tionally upregulate inflammatory cytokines, a reduction ofIB- will indirectly upregulate NF-B-mediated inflamma-tion. Western blot analysis showed that the expression ofIB- was significantly decreased in hearts of the DM groupbut not the DM/MG-132 group (Fig. 8A). This suggests thatMG-132 inhibits the proteasomal degradation of IB-. To

define whether preservation of a normal level of IB- is ableto prevent NF-B nuclear translocation, cardiac proteins wereseparated into nuclear and cytoplasmic parts, which showedthat the nuclear accumulation of NF-B was significantlyincreased in the DM group but not in the DM/MG-132 group(Fig. 8B). Furthermore, the increased cardiac nuclear NF-BDNA-binding activity only in the hearts of DM mice at both 3and 6 mo was further confirmed by an ELISA-based NF-kBactivity assay (Fig. 8C).

DISCUSSION

The present study is the first to report the therapeutic effectsof chronic treatment with low-dose MG-132 on diabetes-induced cardiomyopathy using the OVE26 diabetic mousemodel. We demonstrated that the therapeutic effect of MG-132on diabetic cardiomyopathy is associated with its suppression

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Fig. 7. Effect of MG-132 on cardiac expression andfunction of nuclear factor (NF) erythroid 2-relatedfactor 2 (Nrf2). A and B: cardiac expression of Nrf2mRNA (A) and protein (B) levels was examinedwith real-time PCR and Western blot assays, re-spectively. C and D: Nrf2 function was measuredby determining the expression of Nrf2 downstreamgenes [NADPH-quinone oxidoreductase (NQO)-1,heme oxygenase (HO)-1, and catalase (CAT)] atmRNA (C) and protein (D) levels. Data are pre-sented as means SD. *P 0.05 vs. the Ctrlgroup; #P 0.05 vs. the DM group.



of diabetic upregulation of proteasome activity, which mayincrease the proteasomal degradation of IB- and Nrf2.Increased degradation of IB- would release its binding andrestricting NF-B in the cytoplasm, leading to an increase ofNF-B nuclear translocation to generate inflammatory cyto-kines, as shown in Fig. 9. These cytokines then initiate anovergeneration of ROS/reactive nitrogen species, cardiac oxi-dative stress and damage, and remodeling and dysfunction. Inaddition, increased proteasomal degradation of Nrf2 will re-duce the transcription of Nrf2 to generate its downstreamantioxidants, which will exacerbate cardiac pathogenic altera-tions, leading to an acceleration of cardiomyopathy develop-ment, as shown in Fig. 9.

Several in vivo and in vitro studies have provided evidencefor the increase in proteasomes under diabetic conditions. Forexample, exposure of human umbilical vein endothelial cells to

a high level of glucose significantly increased 26S proteasomeactivity (35). Proteasomal activity was also found to be in-creased in the hearts of type 1 diabetic mice (11, 22) and in thegastrocnemius muscles of spontaneous type 2 diabetic (db/db)mice (33). Measurements of proteasomal activity in the kid-neys of diabetic rats showed an increase of 26S proteasomalactivity in streptozotocin-induced diabetic rats (18).

An in vivo pilot study (18) has shown the preventive effectof MG-132 on diabetes-induced renal damage. In that study,shortly after the induction of diabetes, rats were treated withMG-132 at 10 g/kg daily for 3 mo. This regimen producedrenal prevention, as indicated by reductions in proteinuria,basement membrane thickening, and glomerular mesangialexpansion. MG-132 also reduced kidney markers of oxidativestress and increased protein levels of Nrf2 and several antiox-idant enzymes. These experiments demonstrated a potential for

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Fig. 8. Effect of MG-132 on the cardiac expres-sion of NF-B and IB- as well as NF-Bactivity. A and B: IB- protein expression (A)and NF-B protein expression in the nuclearand cytosolic portion (B) were examined with aWestern blot assay. C: nuclear p65 DNA-binding activity was determined by an ELISA-based NF-kB activity assay. Data are presentedas means SD. *P 0.05 vs. the Ctrl group;#P 0.05 vs. the DM group.



MG-132 to prevent the development of diabetic complications.However, whether Nrf2 activators have therapeutic effects onthe heart and/or kidney of diabetic subjects remains unknown.Here, we report, for the first time, the therapeutic effects ofMG-132 on diabetic cardiomyopathy in the transgenic type 1diabetic OVE26 mouse model. When these diabetic mice at 3mo old began to exhibit albuminuria as an index of renaldysfunction (38) and cardiac dysfunction (Fig. 1), some ofthem were treated with MG-132 at a very low dose for 3 mo,which offered a significant therapeutic outcome, as indicatedby the completely prevention of diabetes-induced cardiac in-flammation and oxidative stress and damage, leading to acomplete reverse of cardiac remodeling (fibrosis and hypertro-phy) and dysfunction. Diabetic mice without treatment withMG-132 showed the progressive development of cardiac struc-tural remodeling and functional abnormalities.

In terms of the mechanisms underlying the therapeuticeffects of MG-132 on diabetic cardiomyopathy, we assumedthat they are associated with the significant upregulation ofNrf2 expression and transcriptional increases of its down-stream antioxidants and the complete prevention of diabetes-reduced IB content and diabetes-increased NF-B nuclearaccumulation, as shown in Fig. 9.

Reportedly nontoxic concentrations of MG-132 inhibited theproteasomal degradation of Nrf2 to stimulate Nrf2 transloca-tion into the nucleus (5, 28). An in vitro study (9) has shownthat treatment with 0.5 M MG-132 for 48 h protected neo-natal rat cardiac myocytes against H2O2-mediated oxidativestress. This was correlated with reduced levels of intracellularROS and significant upregulation of superoxide, HO-1, andCAT expression. This demonstrated that nontoxic concentra-tions of MG-132 could upregulate Nrf2-mediated antioxidant

NF-B IB KEAP1 Nrf2

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Fig. 9. Illustration of possible mechanisms by whichMG-132 provides therapeutic effects on diabetic cardio-myopathy. Diabetes increases cardiac proteasomal activ-ity, leading to the degradation of Nrf2 and IB- in thecytoplasm. The increased degradation of Nrf2 results in adecrease of Nrf2 nuclear translocation and transcriptionalupregulation of its downstream antioxidants. The in-creased degradation of IB- results in a release ofNF-B from IB- inhibition and an increase of NF-Bnuclear translocation to transcriptional upregulation ofinflammatory cytokines. All these cause cardiac oxida-tive damage, remodeling, and dysfunction (cardiomyop-athy). MG-132 could prevent Nrf2 degradation andNF-B nuclear translocation by inhibiting the protea-some. KEAP1, kelch-like ECH-associated protein 1.



enzymes to confer cardiomyocyte protection (9). We (10) havedemonstrated the high susceptibility of cardiomyocytes fromNrf2-null mice to high-glucose-induced ROS generation anddamage compared with those from Nrf2 wild-type mice.Therefore, we assumed that the preventive effect of the pro-teasomal inhibitor MG-132 was due to elevated Nrf2 proteincontent, which increased the expression of multiple down-stream antioxidant enzymes.

RT-PCR analysis revealed that diabetes, but not MG-132,significantly increased Nrf2 mRNA expression (Fig. 7A), sug-gesting the existence of a compensatory response of the heartto diabetes-induced oxidative stress by upregulation of Nrf2mRNA expression. However, diabetes also increased 20S pro-teasome activity, which should have reduce Nrf2 protein levelsand resulted in less nuclear translocation; therefore, the finaloutcome of Nrf2 protein expression in the diabetic heartremained at a relatively high level compared with the controlgroup. In contrast to the DM group, DM/MG-132 mice showedincreased Nrf2 mRNA expression, which should be attributedto an effect of diabetes (Fig. 7A), and also synergisticallyincreased Nrf2 protein levels, which should be attributed toboth an effect of diabetes on mRNA expression as well as theinhibitory effect of MG-132 on proteasomal degradation of theNrf2 protein level. Expression profiles of Nrf2 downstreamantioxidants at the mRNA and protein levels (Fig. 7, C and D)were similar to the profile of Nrf2 protein expression amongthe groups (Fig. 7B). These upregulated Nrf2-mediated anti-oxidants seem to be responsible for the therapeutic effects onthe heart, based on a previous study (37) reporting that upregu-lated Nrf2 expression in the heart or kidney provided signifi-cant prevention of diabetes-induced damage.

However, due to the fact that the expression of Nrf2 and itsdownstream antioxidants was not significantly decreased in thehearts of DM mice compared with control mice, we assumedthat the increased expression of Nrf2 and its downstreamantioxidants may be not the sole mechanism underlying thetherapeutic effects of MG-132 on diabetic cardiomyopathy.

The role of an excessive inflammatory response in theinitiation of diabetic cardiomyopathy has been discussed re-cently (27, 34). As a key transcription factor to control inflam-mation, NF-B activation was found to play a pivotal role inthe development of cardiomyopathy (20). Recent studies (19,23) have demonstrated the protection of MG-132 in the otherorgans via inhibition of NF-B. Here, we demonstrated theactivation of NF-B in the hearts of OVE26 diabetic mice,which was significantly inhibited by treatment with MG-132 inthe DM/MG-132 group. This suggests that the effective inhi-bition of proteasome activity by MG-132 may be attributed toits effective inhibition of cardiac inflammation, as we observedin this study. As shown in Fig. 9, NF-B triggers the transcrip-tion of many inflammation cytokines. These inflammatorycytokines stimulate the generation of ROS and/or reactivenitrogen species, which induce cardiac oxidative stress anddamage and remodeling, leading to the development of dia-betic cardiomyopathy. Therefore, inhibition of cardiac inflam-mation activation should provide cardiac protection from dia-betes. We demonstrated that diabetes significantly suppressedthe expression of IB- and increased the nuclear accumula-tion of NF-B and DNA-binding capacity. All these effectswere almost completely abolished by MG-132 treatment,

which was accompanied with a complete reverse of diabetes-induced cardiac inflammation and oxidative damage.

A potential limitation of the present study may be the route(intraperitoneal injection) by which MG-132 was given, whichmay not directly applicable to clinics. In the present study, weused intraperitoneal injection because we wanted to ensure theprecise dose given for the low dose of MG-132. Although wecannot directly expect that what we do in this study will be di-rectly extrapolated to clinics, the eventual oral administration ofthe MG-132 for diabetic patients with the appearance of albumin-uria will be easily established if we confirm the therapeutic effect,safety, and underlying mechanisms. The latter two will be furtherinvestigated in future studies.

In summary, the present study demonstrated that the protea-somal inhibitor MG-132 at a low dose (10 g/kg) can providea therapeutic effect on diabetic cardiomyopathy. Here, we usedthe transgenic type 1 diabetic OVE26 mouse model. Whenthese diabetic mice began to exhibit both albuminuria as anindex of renal dysfunction and cardiac dysfunction by echo-cardiography, some of them were treated with MG-132 at avery low dose for 3 mo, which offered a significant therapeuticoutcome, as indicated by the complete prevention of diabetes-induced cardiac inflammation and oxidative damage, leading toa reversal of cardiac remodeling and dysfunction. In contrast,diabetic mice without treatment with MG-132 showed theprogressive development of cardiac inflammation, structuralremodeling, and dysfunction. These therapeutic changes wereassociated with both significant upregulation of Nrf2 expres-sion and transcriptional increases of its downstream antioxi-dants and significant suppression of NF-B-mediated inflam-mation (Fig. 9). Therefore, MG-132 has great potential as atherapeutic agent for diabetic patients, including those withdiabetic cardiomyopathy.

GRANTSThis work was supported in part by American Diabetes Association Basic

Research Award 1-11-BA-17 (to L. Cai), a Chinese-American Research Institutefor Diabetic Complications from Wenzhou Medical College Starting-Up Fund (toL. Cai and Y. Tan), National Natural Science Foundation of China Grants81070189 (to Y. Wang) and 81273509 (to Y. Tan), and Jilin University BethuneFoundation Grant 2012221 (to Y. Wang).

DISCLOSURESNo conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONSAuthor contributions: Y.W., B.L., T.C., Y.F., and L.C. conception and

design of research; Y.W., W.S., B.D., Y.B., Y.X., Y.T., W.C., and B.L.performed experiments; Y.W., W.S., B.D., X.M., Y.B., Y.X., Y.T., and W.C.analyzed data; Y.W., W.S., X.M., Y.T., B.L., T.C., Y.F., and L.C. interpretedresults of experiments; Y.W., W.S., B.D., and W.C. prepared figures; Y.W.,Y.F., and L.C. drafted manuscript; Y.W., W.S., B.D., X.M., Y.B., Y.X., Y.T.,W.C., B.L., T.C., P.N.E., Y.F., and L.C. approved final version of manuscript;T.C., P.N.E., Y.F., and L.C. edited and revised manuscript.

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