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Articles in PresS. Am J Physiol Heart Circ Physiol (December 7, 2012). doi:10.1152/ajpheart.00650.2012 1 Therapeutic effect of MG132 on diabetic cardiomyopathy is associated with its 2 suppression of proteasomal activities: Roles of Nrf2 and NF-κB 3 4 Yuehui Wang 1, Weixia Sun 2,3, Bing Du 2, Xiao Miao 1,3, Yang Bai 2,3, Ying Xin 3,4, Yi Tan 3,5, 5 Wenpeng Cui 1,3, Bin Liu 1, Taixing Cui 5,6, Paul N. Epstein 3,7 , Yaowen Fu 2,*, Lu Cai 3,5,7,* 6 1, 7 2, 8 3, 9 5, The First Hospital of the Jilin University, Jilin, China KCHRI at the Department of Pediatrics, University of Louisville, KY, USA, 4, 10 11 The Second Hospital of the Jilin University, Jilin, China Norman Bethune Medical College of the Jilin University, Jilin, China Chinese-American Research Institute for Diabetic Complications at the Wenzhou Medical College, Wenzhou, China 12 6, 13 Department of Cell Biology and Anatomy, University of South Carolina School of Medicine, Columbia, SC, USA 14 7, 15 Departments of Pharmacology & Toxicology, the University of Louisville, KY, USA 16 17 Running head: Therapeutic effect of MG132 on diabetic cardiomyopathy 18 19 20 21 22 23 24 25 26 * Address for correspondence and reprints: Dr. Lu Cai, MD, PhD Department of Pediatrics, University of Louisville, 570 South Preston Street, Baxter I, Suite 321B or 304F, Louisville, KY 40202, Phone/Fax: (502) 852-2214/852-5634, E-mail: [email protected] Dr. Yaowen Fu, MD, PhD Center of Organ Transplantation, the First Hospital of Jilin University, 71 Xinmin Street, Changchun 130021, Phone: +86-13756661667, E-mail: [email protected] 27 28 29 30 31 1 Copyright © 2012 by the American Physiological Society. 32 Abstract 33 MG132, a proteasome inhibitor, can up-regulate nuclear factor erythroid 2-related factor 2 34 (Nrf2)-mediated anti-oxidative function and down-regulate nuclear factor-(NF-)κB-mediated 35 inflammation. The present study investigated whether through above two mechanisms MG132 could 36 provide a therapeutic effect on diabetic cardiomyopathy in OVE26 type 1 diabetic mouse model. 37 OVE26 mice develop hyperglycemia at 2 – 3 weeks after birth and exhibit albuminuria and cardiac 38 dysfunction at 3 months of age. Therefore, 3-month old OVE26 diabetic and age-matched control 39 mice were intraperitoneally treated with MG132 at 10 μg/kg daily for 3 months. Before and after 40 MG132 treatment, cardiac function was measured by echocardiography and then cardiac tissues were 41 subjected to pathological and biochemical examination. Diabetic mice showed significant cardiac 42 dysfunction, including increased left ventricular systolic diameter and wall thickness and a decreased 43 left ventricular ejection fraction with an increase of heart weight/tibia length ratio. Diabetic hearts 44 exhibited structural derangement and remodeling (fibrosis and hypertrophy). In diabetic mice, there 45 was also increased systemic and cardiac oxidative damage and inflammation. All of these pathogenic 46 changes were reversed by MG132 treatment. MG132 treatment significantly increased cardiac 47 expression of Nrf2 and its down-stream antioxidant genes with a significant increase of total 48 antioxidant capacity and also significantly decreased the expression of Iκ-B and the nuclear 49 accumulation and DNA binding activity of NF-κB in the heart. These results suggest that MG132 has 50 a therapeutic effect on diabetic cardiomyopathy in OVE26 diabetic mice possibly through the 51 up-regulation of Nrf2-dependent anti-oxidative function and the down-regulation of NF-κB-mediated 52 inflammation. 53 54 Key words: Diabetic cardiomyopathy, Nrf2, proteasome inhibitor, MG132, therapeutic effect 2 55 INTRODUCTION 56 The ubiquitin-proteasome system (UPS) represents the major non-lysosomal pathway for 57 degradation of ubiquitinated proteins (26). In addition to the removal of misfolded or damaged 58 proteins, the UPS also plays a crucial role in the regulation of many cellular processes such as the 59 cell cycle, apoptosis, transcriptional control, and responses to stress. Recent studies indicate that 60 alterations in the UPS contribute to the pathogenesis and progression of several cardiac diseases, 61 and inhibition of proteasome activity has become an interesting approach to prevent various 62 diseases, including cardiac diseases (26, 36). 63 Bortezomib (velcadew) was the first proteasome inhibitor approved by the FDA in 2003 (8). 64 However, side effects of Bortezomib and the development of resistance in some tumor patients 65 prompted the study of new proteasome inhibitors (8). The cell-permeable MG132 tripeptide 66 (Z-Leu-Leu-Leu-aldehyde) is a peptide aldehyde proteasome inhibitor that also inhibits other 67 proteases, including calpains and cathepsins. Reportedly non-toxic concentrations of MG132 inhibit 68 Nrf2 proteasomal degradation and stimulate Nrf2 translocation into the nucleus (5, 28). By blocking 69 the proteasome, this tripeptide has been shown to induce the expression of cell-protective proteins 70 such as heat shock proteins in vitro and in vitro. MG132 was found to play an important role in 71 cardiovascular protection from various stresses and pathogeneses (4, 19, 28). 72 One mechanism responsible for the cardiac protection by MG132 is its up-regulation of 73 antioxidants via activation of a nuclear factor-erythroid 2-related factor 2 (Nrf2) (28). Nrf2 is a key 74 transcription factor in regulating multiple antioxidants including NADPH-quinone oxidoreductase 75 (NQO1), heme oxygenase (HO-1), catalase (CAT), superoxide dismutase, glutathione peroxidase, 76 and glutamatecysteine ligase. Actin-tethered protein Keap1 is a cytosolic repressor that binds to and 77 retains Nrf2 in the cytoplasm, which promotes Nrf2 proteasomal degradation and results in the 3 78 prevention of Nrf2 transcription (14, 30). Therefore, inhibition of proteasome activity is one method 79 to maintain cytoplasm Nrf2 available to nuclear translocation and activation in response to 80 oxidative stress (15). 81 Another important mechanism underlying MG132 cardiovascular protection is its inactivation 82 of NF-κB, a nuclear transcription factor that regulates pro-inflammatory cytokine expression. 83 Activation of NF-κB has been documented in myocardial ischemia/reperfusion (13), and specific 84 inhibition of NF-κB is cardioprotective (25). Since NF-κB usually is inhibited by IκB via forming a 85 complex with NF-κB. However, IκB is degraded by proteasome ubiquitination, resulting in the 86 release of NF-κB and nuclear translocation to turn on inflammatory genes. Therefore, proteasomal 87 inhibitors such as MG132 can inhibit the proteasome activity to provide anti-inflammation function, 88 leading to the cardiac prevention from ischemic damage. 89 Diabetic cardiomyopathy is also associated with early cardiac oxidative stress and 90 inflammation, which initiates the late development of cardiac remodeling and dysfunction (1, 12, 27, 91 34). There was a report, showing that diabetes increased cardiac proteasome function (11, 22), 92 which was associated with diabetic complications (16). Therefore, we hypothesized that MG132 as 93 one of the proteasome inhibitors may be potentially used for the prevention of diabetic 94 cardiomyopathy via up-regulation of Nrf2 to reduce oxidative stress and down-regulation of 95 NF-κB-mediated inflammation (16). There were a few studies that have shown the cardiac and renal 96 protection by proteasome inhibitor from other pathogeneses (3, 4, 17, 19, 28, 31). Dreger et al. have 97 demonstrated the protective effect of low-dose MG132 on hydrogen peroxide-induced oxidative 98 stress and damage in cardiomyocytes, and also showed the increased sensitivity in myocytes from 99 Nrf2 knockout mice, suggesting the important role of Nrf2 in the cardiac protection (9). Using 100 MG132 several studies have also shown the preventive effect of inhibiting proteasome activity on 4 101 diabetic vascular injury (16, 32). A pilot study indicates that proteasomal inhibitor that up-regulates 102 Nrf2 activity and inhibits NF-κB-mediated inflammation provided a preventive effect on 103 diabetes-induced renal dysfunction (18). 104 From clinical setting, there is an urgent need for an efficient approach that can provide a 105 therapeutic effect, rather than only prevention, since it is not easily accepted that the diabetic 106 patients will give preventive medicine once diagnosed as diabetes. So far, however, there was no 107 report regarding the therapeutic effect of MG132 on diabetic complications; in the present study, 108 therefore, we have tried to address the question whether MG132 can provide a therapeutic effect on 109 diabetic cardiomyopathy by testing if MG132 could reserve or slow the progression of established 110 cardiac dysfunction in the OVE26 mouse model of severe Type 1 diabetes. In addition, mechanistic 111 studies were focused on the up-regulation of Nrf2-mediated antioxidants and down-regulation of 112 NF-κB-mediated inflammation. 113 114 MATERIALS AND METHODS 115 Animals. The transgenic type 1 diabetic OVE26 mouse model with FVB background has been 116 characterized in previous studies (38). All mice were housed in the University of Louisville 117 Research Resources Center at 22 °C with a 12-h light/dark cycle and provided with free access to 118 standard rodent chow and tap water. All animal procedures were approved by the Institutional 119 Animal Care and Use Committee, which is certified by the American Association for Accreditation 120 of Laboratory Animal Care. 121 OVE26 mice normally develop severe hyperglycemia before 3 weeks of age and develop 122 albuminuria by 3 months of the age, particularly in female OVE26 mice (38). Sixteen 3-month-old 123 female OVE26 mice were randomly divided into two groups: diabetic group (DM, n=10) and 5 124 MG132-treated OVE26 group (DM/MG132, n=6). Sixteen age and sex matched non-diabetic FVB 125 mice were randomly divided into two groups: control group (n=10) and MG132 group (MG132, 126 n=6). MG132 (Sigma-Aldich, St. Louis, MO) was dissolved in dimethyl sulfoxide (DMSO) at a 127 concentration of 0.0025 μg/ml and diluted with saline for injection. For MG132 and DM/MG132 128 mice, MG132 was given intraperitoneally at a dose of 10 μg/kg body-weight daily for 3 months, 129 based on a recent study (18), starting at 3 month old OVE26 mice when these mice already display 130 significant renal dysfunction. For control and DM mice equal amounts of physiological saline 131 containing 0.0025 μg DMSO/ml were given. MG132 treatment for 3 months for OVE26 diabetic 132 mice did not affect their blood glucose levels (7). 133 Four mice from both control and diabetic groups were sacrificed at 3 months of age and other 134 mice (n=6) were treated with MG132 for 3 months and sacrificed at 6 months of age. After heart 135 weight and tibia length was measured, whole heart tissue was harvested for protein and mRNA 136 analysis. Histopathological observation was predominantly based on left ventricle. 137 138 Echocardiography. To assess cardiac function, echocardiography was performed for these mice 139 using a Visual Sonics Vevo 770 high-resolution imaging system, as described before (29, 39). Under 140 sedation with Avertin (2,2,2-tribromoethanol; 240 mg/kg IP), mice were placed in a supine position 141 on a heating pad to maintain body temperature at 36 – 37 °C. Animal’s heart rates were kept 400 – 142 550 beats per min. Two-dimensional and M-mode echocardiography were used to assess wall 143 motion, chamber dimensions and cardiac function. Indices directly measured included left 144 ventricular (LV) cavity dimensions in diastole (LVID,d) and systole (LVID,s), LV posterior wall 145 thickness in diastole (LVPW,d) and systole (LVPW,s), and interventricle septum thickness in 146 diastole (IVS,d) and systole (IVS,s). LV fractional shortening (FS) = [(LVIDd − LVIDs)/LVIDd] × 6 147 100; LV ejection fraction (EF) = (LV Vol,d − LV Vol,s)/LV Vol,d] × 100. 148 149 Western blot analysis. Western blot was performed as described in our previous studies (2, 39). The 150 primary antibodies used include: 3-nitrotyrosine (3-NT, 1:1000 dilution), 4-hydroxy-2-nonenal 151 (4-HNE, 1:1000 dilution), TNF-α (1:500 dilution), intercellular adhesion molecule 1 (ICAM-1, 152 1:500 dilution), plasminogen activator inhibitor 1 (PAI-1, 1:1000 dilution), TGF-β1 (1:500 dilution), 153 fibronectin (1:500 dilution), Nrf2 (1:500 dilution), NQO-1 (1:500 dilution), HO-1 (1:500 dilution), 154 CAT (1:5000 dilution), atrial natriuretic peptide (ANP, 1:1000 dilution), NF-κB (1:1000 dilution), 155 IκB-α (1:1000 dilution), α-tubulin (1:2000 dilution), and β-actin (1:2000 dilution), all of which 156 were purchased from Santa Cruz Biotech. Inc. except for TNF-α from Abcam, 3-NT from Millipore, 157 NF-κB, IκB-α, and α-tubulin from cell signaling, and 4-HNE from Alpha Diagnostic International. 158 All these antibodies are polyclonal antibodies except for TNF-α and PAI-1 antibodies that are 159 monoclonal. 160 161 RNA isolation and real-time RT-PCR. RNA isolation and real-time RT-PCR were performed, as 162 described in our previous studies (39), for Nrf2 (primer: Mm00477784_m1), ANP 163 (Mm01255748_g1), β-myosin heavy chain (β-MHC) (Mm00600555_m1), Hmox1 (HO-1, 164 Mm00516005_m1), Nqo1 (Mm253561_m1), CAT (Mm00437229_m1), and the housekeeping gene 165 β-actin (Mm00607939_s1). All these primers were purchased from Applied Biosystems (Foster City, 166 CA). Total RNA was extracted from whole heart tissues using Trizol reagent (RNA STAT 60 167 Tel-Test, Ambion, Austin, TX). RNA concentration and purity were quantified using a Nanodrop 168 ND-1000 spectrophotometer (Biolab, Ontario, CA). First-strand complimentary DNA (cDNA) was 169 synthesized from total RNA according to the RNA PCR kit (Promega, Madison, WI, USA), 7 170 following the manufacturer’s protocol. 171 172 Cardiac histopathological examination and immunohistochemical staining. Whole cardiac tissue 173 was fixed overnight in 10% phosphate buffered formalin and then dehydrated in a graded alcohol 174 series, cleared with xylene, embedded in paraffin, and sectioned at 5 μm thickness for pathological 175 and immunohistochemical staining. Paraffin sections were dewaxed, followed by incubation with 176 1X target retrieval solution (Dako, Carpinteria, CA) for 15 min at 98°C for antigen retrieval, and 177 then treated with 3% hydrogen peroxide for 15 min at room temperature, followed by blocking with 178 5% BSA for 30 min. 179 Cardiac sections were stained with hematoxylin and eosin (H&E) and Sirius-red, respectively, 180 as before (2, 39). For immunohistochemical staining sections were incubated with the primary 181 antibodies of TGF-β1 (1:100 dilution, Santa Cruz Biotech. Inc.) and PAI-1 (1:100 dilution, BD 182 Biosciences. Inc.) for overnight at 4°C. After washing with PBS, these sections were incubated with 183 horseradish peroxidase conjugated secondary antibody (1:300-400 dilutions in PBS) for 2 h at room 184 temperature. For the development of color, sections were treated with peroxidase substrate 185 3,3-Diaminobenzidine in the developing system (Vector Laboratories, Inc. Burlingame, CA). 186 187 Proteasome activity. The 20S proteasome, catalytic core of the 26S proteasome complex, is 188 responsible for the breakdown of short-lived regulatory proteins, including Nrf2 and NF-κB (6, 9). 189 Since MG132 mainly inhibits the proteasome chymotrypsin (ChT)-like activity (24), we determined 190 20S proteasome activity by quantifying the hydrolysis of SLLVY-AMC-a fluorogenic substrate for 191 the ChT-like activity. 20S proteasome activity was measured with the 20S Proteasome Activity 192 Assay Kit (Millipore). 8 193 Nuclei isolation. The cardiac nuclei were isolate using nuclei isolation kit (Sigma-Aldich). Whole 194 cardiac tissue (60 mg) from each mouse was homogenized for 45 sec. within 300 μl cold lysis 195 buffer containing 1 μl dithiothreitol (DTT) and 0.1 % Triton X-100. After that, 600 μl cold 1.8 196 mol/L Cushion Solution (Sucrose Cushion Solution: Sucrose Cushion Buffer: DTT= 900:100:1) 197 was added to the lysis solution. The mixture was transferred to a new tube pre-loaded with 300 μl 198 1.8 mol/ L Sucrose Cushion Solution followed by centrifuge at 13,000 × rpm for 45 min. The 199 supernatant contains cytosolic component and nuclei were visible as thin pellet at the bottom of 200 tube. 201 202 Measurements of serum TNF-α and MCP-1, and cardiac nuclear NF-κB DNA binding activity. 203 Serum levels of TNF-α and MCP-1 were performed using mouse TNF-α and MCP-1 ELISA kits 204 (Invitrogen, Camarillo, CA), as described previously (21). The low detection limit was 3 pg/mL and 205 9 pg/mL for serum TNF-α and MCP-1, respectively. Nuclear p65 DNA binding activity was 206 determined by ELISA-based NF-κB activity assay using cardiac tissue from whole heart (Cayman 207 Chemical, Ann Arbor, MI), according to manufacturer instructions. 208 209 Total antioxidant capacity (TAC) assay .The cardiac TAC assay was performed using a 210 commercially available assay kit (Cell Biolabs, San Diego, CA), according to the manufacturer's 211 instructions. Briefly, whole heart tissues were homogenized in cold PBS and then centrifuged at 212 10,000×g at 4°C for 10 min to collect the supernatant for the TAC assay. The values were calculated 213 using optical density at 490 nm and expressed as umol/g protein for TAC. 214 215 9 216 Statistical analysis. Data were expressed as mean ±SD. For statistical analysis, one-way analysis of 217 variance (ANOVA) was used as appropriate. The overall F-test was performed to test the 218 significance of the ANOVA models. The significance of the interactions and main effects were 219 taken into consideration and then multiple comparisons were performed by the Bonferroni test. The 220 significance level was considered at p<0.05. 221 222 RESULTS 223 Therapeutic effect of MG132 on diabetes-induced cardiac dysfunction and hypertrophy 224 Transgenic OVE26 type 1 diabetic (DM) mice at 3 months showed the increased LVID at systole 225 and the decreased EF% and FS%, suggesting the exist of cardiac dilation and decreased systolic 226 function (Fig. 1A). Hypertrophic measurements of IVS, LVPW and LV mass were significantly 227 increased in these mice with a progressive manner from 3 to 6 months (P<0.05, Fig. 1B). However, 228 when some of DM mice at 3 months of age were treated with low-dose MG132 for 3 months, 229 diabetes-induced cardiac dysfunction was almost completely reversed in DM/MG132 group 230 (P<0.05, Fig. 1). 231 Therapeutic effects of MG132 on diabetes-induced cardiac hypertrophy were also reflected by 232 the ratio of heart to tibia length (Fig. 2A) that was significantly increased in an age-dependent 233 manner in DM group from 3 to 6 months, but not in DM/MG132 group. 234 To further confirm cardiac hypertrophy, cardiac morphology and molecular hypertrophy 235 makers were also examined. H&E staining showed that compared with control and MG132 groups, 236 main pathological changes in DM group include: (1) myocardial hypertrophy; (2) a few cardiac 237 cells that showed degeneration features; (3) proliferation of interstitial collagen fibers. Those 238 pathological changes were alleviated or not observed in the DM/MG132 mice (Fig. 2B). Real-time 10 239 PCR analysis showed that DM induced a significant increase of cardiac mRNA expression of ANP 240 (Fig. 2C) and β-MHC (Fig. 2D), an effect that was completely abolished by MG132 treatment in 241 DM/MG132 group. Consistent with ANP mRNA finding, Western blot showed a significant 242 increase of cardiac ANP protein expression at 6 months of age in DM mice, but not in DM/MG132 243 mice (Fig. 2E). These results suggest that 3-month treatment with low-dose MG132 can 244 significantly or even completely prevent the progression of the cardiac hypertrophy induced by 245 diabetes. 246 247 Therapeutic effect of MG132 on diabetes-induced cardiac fibrosis 248 Cardiac tissue was examined with Sirius-red staining for collagen (Fig. 3A), followed by 249 semi-quantitative analysis with computer imaging system (Fig. 3B), which showed a significant 250 increase of the collagen accumulation in DM group, but not in DM/MG132 group. Western blots of 251 TGF-β1, fibronectin, and PAI-1 as important pro-fibrotic mediators confirmed the Sirius-red 252 staining result: a significant increase in cardiac fibrosis in DM group, but not in DM/MG132 group 253 (Fig. 3C). Furthermore, the increased expression of TGF-β1 and PAI-1 was supported by 254 immunohistochemical stain (Fig. 3D). 255 256 Therapeutic effect of MG132 on diabetes-induced cardiac inflammation and oxidative stress 257 Since PAI-1 acts as both pro-fibrotic and inflammatory mediators, its increase in the heart of DM 258 mice suggests the possible cardiac inflammation. Next we thus examined the protein expression of 259 TNF-α (Fig. 4A) and MCP-1(Fig. 4B) in serum by ELISA and (Figs. 4C,D) in the heart by Western 260 blot assay. Serum TNF-α and MCP-1 levels were progressively elevated from 3 to 6 months in DM 261 group, which were completely prevented by MG132 treatment in DM/MG132 group. Increased 11 262 cardiac TNF-α and MCP-1 contents were also observed only in DM group, not in DM/MG132 263 group. 264 Considering that inflammation in the target organs often causes oxidative stress while 265 oxidative stress is also able to induce inflammation, the next study examined the oxidative stress by 266 measuring 3-NT accumulation as an index of protein nitration (Fig. 5A) and 4-HNE as lipid 267 peroxidation (one of oxidative damage) (Fig. 5B). Both 3-NT and 4-HNE contents were 268 significantly increased in the heart of DM mice, but not DM/MG132 mice. The TAC in heart tissue 269 (Fig. 5C) was slightly decreased (p<0.05) in DM group at both 3 and 6 months, but not changed in 270 DM/MG132 group at 6 months. 271 272 Possible mechanisms for the therapeutic effect of MG132 on the diabetic cardiomyopathy 273 Diabetes increases cardiac proteasomal activity, an effect prevented by MG132 274 Since tetrahydrobiopterin deficiency that was reported to uncouple the enzymatic activity of 275 endothelial nitric oxide synthase in DM hearts is triggered by DM-increased proteasome-dependent 276 mechanisms (35), we examined here whether cardiac proteasome activity is increased in diabetic 277 mice. Fig. 6 shows that MG132 decreases, and diabetes increases, the cardiac 20S proteasomal 278 activity. Compared to DM group, the cardiac 20S proteasomal activity was significantly reduced, 279 even to control level, in DM/MG132 group (45% DM, P<0.05 vs DM). 280 281 MG132 inhibits cardiac proteasomal activity, resulting in an up-regulation of cardiac Nrf2 and its 282 downstream antioxidants 283 Proteasomal degradation of Nrf2 has been appreciated as the major of mechanism responsible for 284 Nrf2’s negative regulation (14, 30). Our finding that MG132 completely inhibited 12 285 diabetes-up-regulated proteasomal activity implies a possibility for MG132 treatment to decrease 286 degradation of Nrf2 protein. Therefore the expression of Nrf2 at mRNA and protein levels were 287 examined with real-time PCR and Western blot assays, respectively. We found that MG132 had no 288 impact on, but diabetes had a significant increase in, the Nrf2 mRNA expression (Fig. 7A). 289 DM/MG132 had a similar expression of Nrf2 mRNA to those observed in DM group (Fig. 7A). Fig. 290 7B shows that both MG132 and DM increased cardiac expression of Nrf2 protein, so that Nrf2 291 protein expression was synergistically increased in DM/MG132 group. 292 Furthermore, up-regulated Nrf2 transcription activity was reflected by the increase of several 293 downstream antioxidant genes (Fig. 7C,D). MG132 treatment in control mice significantly 294 increased the expression of NQO1, HO-1, and CAT at mRNA and protein levels, compared to 295 Control. Diabetes also increased the expression of these antioxidants at mRNA and protein levels. 296 The expression of these Nrf2 downstream antioxidant genes in the heart of DM/MG132 mice was 297 synergistically increased at both mRNA and protein levels compared to MG132 or DM alone. 298 299 MG132 inhibits cardiac proteasomal activity, resulting in a reduction of cardiac inflammation by 300 preventing NF-κB nuclear translocation 301 Since NF-κB is bound by IκB-α in cytoplasm that inhibits NF-κB nuclear translocation to 302 transcriptionally up-regulate inflammatory cytokines, reduction of IκB-α will indirectly up-regulate 303 NF-κB-mediated inflammation. Western blot showed that the expression of IκB-α was significantly 304 decreased in the heart of DM group, but not DM/MG132 group (Fig. 8A). This suggests that 305 MG132 inhibits proteasomal degradation of IκB-α. To define whether preservation of normal level 306 of IκB-α is able to prevention of NF-κB nuclear translocation, cardiac proteins were separated into 307 nuclear and cytoplasm parts, which showed that nuclear accumulation of NF-κB was significantly 13 308 increased in DM group, but not in DM/MG132 group (Fig. 8B). Furthermore, the increased cardiac 309 nuclear NF-κB DNA binding activity only in the heart of DM mice at both 3 and 6 months was 310 further confirmed by ELISA-based NF-kB activity assay (Fig. 8C). 311 312 DISCUSSION 313 The present study is the first one to report the therapeutic effect of chronic treatment with low-dose 314 MG132 on diabetes-induced cardiomyopathy using OVE26 diabetic mouse model. We 315 demonstrated that the therapeutic effect of MG132 on diabetic cardiomyopathy is associated with 316 its suppression of diabetic up-regulation of proteasome activity that may increase the proteasomal 317 degradation of IκB-α and Nrf2. Increased degradation of IκB-α would release its binding and 318 restricting NF-κB in cytoplasm, leading to the increase of NF-κB nuclear translocation to generate 319 inflammatory cytokines, as illustrated in Fig. 9. These cytokines will initiate the over-generation of 320 ROS/RNS, cardiac oxidative stress and damage, remodeling and dysfunction. In addition, increased 321 proteasomal degradation of Nrf2 would reduce Nrf2’s transcription to generate its downstream 322 antioxidants, which will exacerbate cardiac pathogenic alterations, leading to an acceleration of 323 cardiomyopathy development, as illustrated in Fig. 9. 324 Several in vivo and in vitro studies have provided evidence for the increase in proteasomes 325 under diabetic conditions. For example, exposure of human umbilical vein endothelial cells to high 326 level of glucose significantly increased the 26S proteasome activity (35). Proteasomal activity was 327 also found to be increased in the heart of type 1 diabetes (11, 22) and in gastrocnemius muscle of 328 spontaneous type 2 diabetic mice (db/db) (33). Measurement of proteasomal activity in the kidney 329 of diabetic rats showed an increase of 26S proteasomal activity in STZ diabetic rats (18). 330 There was a pilot in vivo study showing the preventive effect of MG132 on diabetes-induced 14 331 renal damage (18). In this study, shortly after diabetes induction rats were treated with MG132 at 10 332 μg/kg daily for 3 months. This regimen produced renal prevention as indicated by reductions in 333 proteinuria, basement membrane thickening and glomerular mesangial expansion. MG132 also 334 reduced kidney markers of oxidative stress and increased protein levels of Nrf2 and several 335 antioxidant enzymes. These studies demonstrated a potential for MG132 to prevent the 336 development of diabetic complications. However, whether Nrf2 activators have therapeutic effects 337 on the heart and/or kidney of diabetic subjects remains unknown. Here we report for the first time 338 the therapeutic effect of MG132 on diabetic cardiomyopathy in the transgenic type 1 diabetic 339 OVE26 mouse model. When these diabetic mice at 3 months old began to exhibit albuminuria as an 340 index of renal dysfunction (38) and cardiac dysfunction (Fig. 1), some of them were treated with 341 MG132 at a very low dose for three months, which offered a significant therapeutic outcome, 342 indicated by completely preventing diabetes-induced cardiac inflammation and oxidative stress and 343 damage, leading to a complete reverse of cardiac remodeling (fibrosis and hypertrophy) and 344 dysfunction. Diabetic mice without treatment with MG132 showed progressive development of 345 cardiac structural remodeling and functional abnormalities. 346 In terms of the mechanisms underlying the therapeutic effect of MG132 on diabetic 347 cardiomyopathy, we assumed that it is associated with the significant up-regulation of Nrf2 348 expression and transcriptional increases of its downstream antioxidants and the complete prevention 349 of diabetes-reduced IκB content and -increased NF-κB nuclear accumulation, as outlined in Fig. 9. 350 Reportedly non-toxic concentrations of MG132 inhibited the proteasomal degradation of Nrf2 351 to stimulate Nrf2 translocation into the nucleus (5, 28). An in vitro study showed that treatment with 352 0.5 µM MG132 for 48 h protected neonatal rat cardiac myocytes against H2O2-mediated oxidative 353 stress (9). This was correlated with a reduced level of intracellular ROS and the significant 15 354 up-regulation of superoxide, HO-1, and CAT expression. This demonstrated that non-toxic 355 concentrations of MG132 could up-regulate Nrf2-mediated antioxidant enzymes to confer 356 cardiomyocyte protection (9). We have demonstrated the high susceptibility of cardiomyocytes 357 from Nrf2-null mice to high glucose-induced ROS generation and damage as compared to those 358 from Nrf2 wild-type mice (10). Therefore, we have assumed that the preventive effect of the 359 proteasomal inhibitor MG132 was due to elevated Nrf2 protein content that increases the expression 360 of multiple downstream antioxidant enzymes. 361 RT-PCR analysis revealed that diabetes, but not MG132, significantly increased Nrf2 mRNA 362 expression (Fig. 7A), suggesting the existence of a compensatory response of the heart to 363 diabetes-induced oxidative stress by up-regulating Nrf2 mRNA expression. However, diabetes also 364 increased the 20S proteasome activity that should reduce Nrf2 protein level and less nuclear 365 translocation; therefore, the final outcome of Nrf2 protein expression in diabetic heart remains at a 366 relatively high level compared to control. In contrast to DM group, DM/MG132 mice showed an 367 increased Nrf2 mRNA expression that should be attributed to diabetic effect (Fig. 7A), and also 368 synergistically increased Nrf2 protein level that should be attributed to both diabetic effect on 369 mRNA expression and also MG132’s inhibitory effect on proteasomal degradation of Nrf2 protein 370 level. Expression profiles of Nrf2 down-stream antioxidants at mRNA and protein levels (Fig. 7C,D) 371 are similar to the profile of Nrf2 protein expression among the groups (Fig. 7B). These up-regulated 372 Nrf2-mediated antioxidants seems responsible for the therapeutic effect on the heart, based on 373 several previous studies reporting that up-regulated Nrf2 expression in the heart or kidney provided 374 significant prevention of diabetes-induced damage (37). 375 376 However, due to the Nrf2 expression and its down-stream antioxidants were not significantly decreased in the heart of DM mice as compared to control, we assumed that the increased 16 377 expression of Nrf2 and its down-stream antioxidants may be not sole mechanism underlying the 378 therapeutic effect of MG132 on diabetic cardiomyopathy. 379 The role of an excessive inflammatory response in the initiation of diabetic cardiomyopathy 380 has been discussed recently (27, 34). As a key transcription factor to control inflammation, NF-κB 381 activation was found to play a pivotal role in the development of cardiomyopathy (20). Recent 382 studies demonstrated the protection of MG132 in the other organs via inhibition of NF-κB (19, 23). 383 Here, we demonstrated the activation of NF-κB in the heart of OVE26 diabetic mice, which was 384 significantly inhibited by treatment with MG132 in DM/MG132 group. This suggests that the 385 effective inhibition of proteasome activity by MG132 may attribute to its effective inhibition of 386 cardiac inflammation, as we observed in this study. As illustrated in Fig. 9, NF-κB triggers the 387 transcription of many inflammation cytokines. These inflammatory cytokines stimulate the 388 generation of ROS and/or RNS that induces cardiac oxidative stress and damage and remodeling, 389 leading to the development of diabetic cardiomyopathy. Therefore, inhibition of cardiac 390 inflammation activation should provide a cardiac protection from diabetes. We demonstrated that 391 diabetes significantly suppressed the expression of IκB-α and increased the nuclear accumulation of 392 NF-κB and DNA binding capacity. All these effects were almost completely abolished by MG132 393 treatment, which was accompanied with a complete reverse of diabetes-induced cardiac 394 inflammation and oxidative damage. 395 A potential limitation of the present study may be the route (I.P. injection) by which MG132 396 was given, which may not directly applicable to clinics. In the current study, we used I.P. injection 397 since we wanted to ensure the precise dose given for the low dose of MG132. Although we can not 398 directly expect that what we do in this study will be directly extrapolated to clinics, the eventual 399 oral administration of the MG-132 for the diabetic patients with the appearance of albuminuria will 17 400 be easily established if we confirm the therapeutic effect, the safety, and the underlying mechanisms. 401 The latter two will be further investigated in the future studies. 402 In summary, the present study has demonstrated that proteasomal inhibitor MG132 at low 403 dose (10 µg/kg) can provide a therapeutic effect on diabetic cardiomyopathy. Here we used the 404 transgenic type 1 diabetic OVE26 mouse model. When these diabetic mice began to exhibit both 405 albuminuria as an index of renal dysfunction and cardiac dysfunction by echocardiography, some of 406 them were treated with MG132 at a very low dose for three months, which offered a significant 407 therapeutic outcome, indicated by a complete prevention of diabetes-induced cardiac inflammation, 408 oxidative damage, leading to a reverse of cardiac remodeling and dysfunction. In contrast, diabetic 409 mice without treatment with MG132 showed progressive development of cardiac inflammation, 410 structural remodeling, and dysfunction. These therapeutic changes were associated with both 411 significant up-regulation of Nrf2 expression and transcriptional increases of its downstream 412 antioxidants and significant suppression of NF-κB-mediated inflammation (Fig. 9). MG132 413 therefore has great potential as a therapeutic agent for diabetic patients including those with diabetic 414 cardiomyopathy. 415 416 ACKNOWLEDGEMENT 417 This study was supported in part by the Basic Research Award from American Diabetes Association 418 (1-11-BA-17, to LC), the Starting-Up Fund for Chinese-American Research Institute for Diabetic 419 Complications from Wenzhou Medical College (to LC & YT), regular grants from the National 420 Natural Science Foundation of China (81070189 to YW) and (81273509 to YT), and the Jilin 421 University Bethune Foundation (2012221, to YW). 422 18 423 AUTHOR CONTRIBUTION 424 Y.W., W.S., B.D., Y.B., X.M., W.C., Y.X. and Y.T. researched data. Y.W., Y.T. B.L., T.C., Y.F. and 425 L.C. reviewed the article. Y.W., Y.T., T.C., P.N.E., Y.F. and L.C. contributed initial discussion of and 426 overseeing the project. 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Cardiac functional (A) and structural 585 (B) changes were evaluated by echocardiography. Data were presented as means ± SD. 586 *,p< 0.05 vs control; #, p< 0.05 vs DM group. (LVID;d=LV end-diastolic diameter; 587 LVID;s=LV end-systolic diameter ; LVPW=LV posterior wall; IVS=interventricular 588 septum; FS=fractional shortening; EF: ejection fraction.) 589 Fig. 2. Therapeutic effect of MG132 on diabetes-induced cardiac hypertrophy. At both 3 and 6 590 months of diabetes, the ratio of the heart weight to tibia length (A) were measured and 591 calculated after mice were sacrificed. Cardiac structural derangement was examined by 592 morphological examination with H&E staining (B). Blue arrow indicates myocardial 593 hypertrophy; black arrow indicates myocardial cells degeneration; blown arrow indicates 594 proliferation of interstitial collagen fibers (400×). Cardiac hypertrophic markers ANP (C) 595 and β-MHC (D) mRNA and ANP protein (E) expression were measured by real time qPCR 596 and Western blot. Data were presented as means± SD. *, p< 0.05 vs control; &, p< 0.05 vs 3 597 m DM group; #, p< 0.05 vs DM group. Bar=50 µM; DM: diabetes; 3m or 6m= 3 months or 598 6 months of diabetes. 599 Fig. 3. Therapeutic effect of MG132 on diabetes-induced cardiac fibrosis. Cardiac tissue was 600 stained with Sirius-red for collagen (A). Arrows indicate proliferation of interstitial collagen 601 fibers (Original magnification x 200). Bars: 50 µm. Semi-quantitative analysis was done by 602 computer imaging system (B). Cardiac expression of TGF-β1 and fibronectin and PAI-1 23 603 were examined by Western blot assay (C). Cardiac expression of TGF-β1 and PAI-1 were 604 also tested by immunohistochemical staining (D). Arrows indicate positive staining. Data 605 were presented as means± SD. *,p< 0.05 vs control; #, p< 0.05 vs DM group. . Bar=50 µM. 606 Fig. 4. Therapeutic effect of MG132 on diabetes-induced inflammatory cytokines. TNF-α (A) 607 and MCP-1(B) levels in serum were determined by ELISA. Cardiac tissue was subjected to 608 Western blots for TNF-α (C) and ICAM-1 (D). Data were presented as means± SD. *, p< 609 0.05 vs control; #, p< 0.05 vs DM group. 610 Fig. 5. Therapeutic effect of MG132 on diabetes-induced cardiac oxidative stress. Cardiac 611 tissue was subjected to Western blots for 3-NT (A) and 4-HNE (B). Total antioxidant 612 capacity (TAC) in cardiac tissue (C) were detected and expressed as umol/g proteins. Data 613 were presented as means± SD. *, p< 0.05 vs control; #, p< 0.05 vs DM group. 614 Fig. 6. Effect of MG132 on cardiac activity of proteasome. Cardiac proteasome activity was 615 measured using 20S proteasome activity kit. Data are presented as mean ± SD. *, P<0.05 vs. 616 control group and #, P<0.05 vs. DM group. 617 Fig. 7. Effect of MG132 on cardiac expression and function of Nrf2. Cardiac expression of Nrf2 618 mRNA (A) and protein (B) levels were examined with real-time PCR and Western blot 619 assays, respectively. Nrf2 function was measured by determining the expression of Nrf2 620 downstream genes (NQO1, HO-1 and CAT) at mRNA (C) and protein (D) levels. Data are 621 presented as mean ± SD. *, P<0.05 vs. control group and #, P<0.05 vs. DM group. 622 Fig. 8. Effect of MG132 on cardiac expression of NF-κB , IκB-α and NF-κB activity assay. 24 623 IκB-α protein expression (A) and NF-κB protein expression in the nuclear and cytosolic 624 portion proteins (B) were examined with Western blot assay. Nuclear p65 DNA binding 625 activity (C) was determined by ELISA-based NF-kB activity assay. Data are presented as 626 mean ± SD. *, P<0.05 vs. control group and #, P<0.05 vs. DM group. 627 Fig. 9. Illustration of possible mechanisms by which MG132 provides the therapeutic effects 628 on diabetic cardiomyopathy. Diabetes increases cardiac proteasomal activity, leading to 629 the degradation of Nrf2 and IκB-α in cytoplasm. The increased degradation of Nrf2 results 630 in the decrease of Nrf2 nuclear translocation and transcriptional upregulation of its 631 down-stream antioxidants; The increased degradation of IκB-α results in a release of NF-κB 632 from IκB-α inhibition and an increase of NF-κB nuclear translocation to transcriptional 633 up-regulation of inflammatory cytokines. All these will cause cardiac oxidative damage, 634 remodeling and dysfunction (cardiomyopathy). MG-132 could prevent Nrf2 degradation and 635 NF-κB nuclear translocation by inhibiting proteasome. 25 Figure 1 LVID;s LVID;d 3.6 3 *# 2.7 1.8 2 90 * * * # 1 # 60 * 60 FS% 4.5 Ctrl MG132 DM DM/MG132 EF% A # * * 3m 6m 40 20 30 0.9 * *# 0.5 3m 0 6m 0.9 * 3m *# 0.6 0.3 0.0 3m 0.0 6m 1.2 LVPW;s IVS;s * 0.4 0.0 3m 6m 1.2 # * * 0.6 0.0 3m 120 6m * *# 80 40 0 6m 1.8 # 0.8 3m 0 6m LV Mass(mg) IVS;d 1.0 0 6m LV Mass corrected(mg) B 3m LVPW;d 0.0 3m 100 * 6m *# 50 0 3m 6m Figure 2 B A 10 *&# * 5 DM 3m 6m D 8 * 6 4 # 2 0 l Ctr G132 M DM G132 /M DM (fold to control) 0 C ANP/E$ctin mRNA (fold to control) MG132 Ctrl EMHC/E$ctin mRNA Heart/Tibia (mg/mm) Saline Ctrl MG132 DM DM/MG132 4 * 3 2 # 1 0 l Ctr G132 M ANP β-Actin ANP/EActin E 2.4 DM G132 /M DM * 1.6 # 0.8 0.0 l Ctr G132 M DM G132 /M DM Figure 3 MG132 B Ctrl C TGF-β1 Fibronectin PAI-1 β-Actin D Ctrl TGF-β1 PAI-1 MG132 Relative protein expression DM 2.4 1.8 * 3 (fold to control) Saline Collgen content A 2 # 1 0 l Ctr G132 DMG132 M /M DM Ctrl MG132 DM DM/MG132 * # 1.2 *# *# 0.6 0.0 1 ctin eta e b n F ro TG Fib DM I-1 PA DM/MG132 Figure 4 A 120 80 Ctrl MG132 DM DM/MG132 * * # 40 0 3m Serum MCP-1(pg/ml) Serum TNF-D(pg/ml) B 6m C 150 100 * * # 50 0 3m 6m D β-Actin β-Actin 1.8 * 1.2 # 0.6 0.0 l Ctr G132 M DM G132 /M DM ICAM-1/EActin ICAM-1 TNF-D/EActin TNF-α 2.0 * 1.5 # 1.0 0.5 0.0 l Ctr G132 M DM G132 /M DM Figure 5 A B β-Actin β-Actin # 1.2 0.6 0.0 C TAC μmol/g protein * 1.8 4HNE/EActin 4HNE 3-NT/EActin 3-NT 8 l Ctr G132 M 6 4 * * # * 2 0 3m 6m * 1.2 # 0.6 0.0 DM G132 /M DM Ctrl MG132 DM DM/MG132 1.8 l Ctr G132 M DM G132 /M DM 20S proteasome activity (fold to control) Figure 6 * 2.4 1.6 0.8 0.0 # * l Ctr G 1 3 2 M D M G132 /M DM Figure 7 A * * 2 Nrf2/EActin 3 Nrf2 β-Actin 1 0 l Ctr G132 M DM G132 /M DM 6 Ctrl MG132 DM DM/MG132 4 # 2 * * 2.7 # * * 1.8 0.9 0.0 l Ctr G132 M DM G132 /M DM # # ** * * 0 O NQ 1 D NQO1 HO-1 CAT β-Actin HO Relative protein levels C 4 Relative mRNA levels Nrf2/E$ctin mRNA (fold to control) B -1 CA T # 4 # 3 2 # ** ** NQO1 HO-1 * * 1 0 CAT Figure 8 INB-D/EActin A IκB-α β-Actin 1.6 1.2 0.8 B NF-κB(c) α-Tubulin β-Actin NF-N%(c)/EActin 1.4 0.7 0.0 l Ctr G132 M 3 2 Ctrl MG132 DM DM/MG132 * 3m DM G132 /M DM * * 1.4 0.7 0.0 * # 1 0 l Ctr G132 M 2.1 DM G132 /M DM (fold to control) C # NF-N% DNA binding activity NF-N%(n)/DTubulin NF-κB(n) * * 0.4 0.0 2.1 # 6m l Ctr G132 M DM G132 /M DM Figure 9 Diabetes Proteasome activity MG132 NF-κB IκBα KEAP1 Nrf2 Cytoplasm Nucleus NF-κB Nrf2 Inflammation cytokines Antioxidant genes Oxidative damage Cardiac Remodeling Cardiomyopathy (Cardiac Dysfunction)