Download Therapeutic effect of MG132 on diabetic cardiomyopathy is

Articles in PresS. Am J Physiol Heart Circ Physiol (December 7, 2012). doi:10.1152/ajpheart.00650.2012
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Therapeutic effect of MG132 on diabetic cardiomyopathy is associated with its
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suppression of proteasomal activities: Roles of Nrf2 and NF-κB
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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,
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Wenpeng Cui 1,3, Bin Liu 1, Taixing Cui 5,6, Paul N. Epstein 3,7 , Yaowen Fu 2,*, Lu Cai 3,5,7,*
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1,
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2,
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3,
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5,
The First Hospital of the Jilin University, Jilin, China
KCHRI at the Department of Pediatrics, University of Louisville, KY, USA,
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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
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Department of Cell Biology and Anatomy, University of South Carolina School of Medicine,
Columbia, SC, USA
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Departments of Pharmacology & Toxicology, the University of Louisville, KY, USA
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Running head: Therapeutic effect of MG132 on diabetic cardiomyopathy
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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]
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Copyright © 2012 by the American Physiological Society.
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Abstract
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MG132, a proteasome inhibitor, can up-regulate nuclear factor erythroid 2-related factor 2
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(Nrf2)-mediated anti-oxidative function and down-regulate nuclear factor-(NF-)κB-mediated
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inflammation. The present study investigated whether through above two mechanisms MG132 could
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provide a therapeutic effect on diabetic cardiomyopathy in OVE26 type 1 diabetic mouse model.
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OVE26 mice develop hyperglycemia at 2 – 3 weeks after birth and exhibit albuminuria and cardiac
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dysfunction at 3 months of age. Therefore, 3-month old OVE26 diabetic and age-matched control
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mice were intraperitoneally treated with MG132 at 10 μg/kg daily for 3 months. Before and after
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MG132 treatment, cardiac function was measured by echocardiography and then cardiac tissues were
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subjected to pathological and biochemical examination. Diabetic mice showed significant cardiac
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dysfunction, including increased left ventricular systolic diameter and wall thickness and a decreased
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left ventricular ejection fraction with an increase of heart weight/tibia length ratio. Diabetic hearts
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exhibited structural derangement and remodeling (fibrosis and hypertrophy). In diabetic mice, there
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was also increased systemic and cardiac oxidative damage and inflammation. All of these pathogenic
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changes were reversed by MG132 treatment. MG132 treatment significantly increased cardiac
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expression of Nrf2 and its down-stream antioxidant genes with a significant increase of total
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antioxidant capacity and also significantly decreased the expression of Iκ-B and the nuclear
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accumulation and DNA binding activity of NF-κB in the heart. These results suggest that MG132 has
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a therapeutic effect on diabetic cardiomyopathy in OVE26 diabetic mice possibly through the
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up-regulation of Nrf2-dependent anti-oxidative function and the down-regulation of NF-κB-mediated
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inflammation.
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Key words: Diabetic cardiomyopathy, Nrf2, proteasome inhibitor, MG132, therapeutic effect
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INTRODUCTION
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The ubiquitin-proteasome system (UPS) represents the major non-lysosomal pathway for
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degradation of ubiquitinated proteins (26). In addition to the removal of misfolded or damaged
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proteins, the UPS also plays a crucial role in the regulation of many cellular processes such as the
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cell cycle, apoptosis, transcriptional control, and responses to stress. Recent studies indicate that
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alterations in the UPS contribute to the pathogenesis and progression of several cardiac diseases,
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and inhibition of proteasome activity has become an interesting approach to prevent various
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diseases, including cardiac diseases (26, 36).
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Bortezomib (velcadew) was the first proteasome inhibitor approved by the FDA in 2003 (8).
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However, side effects of Bortezomib and the development of resistance in some tumor patients
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prompted the study of new proteasome inhibitors (8). The cell-permeable MG132 tripeptide
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(Z-Leu-Leu-Leu-aldehyde) is a peptide aldehyde proteasome inhibitor that also inhibits other
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proteases, including calpains and cathepsins. Reportedly non-toxic concentrations of MG132 inhibit
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Nrf2 proteasomal degradation and stimulate Nrf2 translocation into the nucleus (5, 28). By blocking
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the proteasome, this tripeptide has been shown to induce the expression of cell-protective proteins
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such as heat shock proteins in vitro and in vitro. MG132 was found to play an important role in
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cardiovascular protection from various stresses and pathogeneses (4, 19, 28).
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One mechanism responsible for the cardiac protection by MG132 is its up-regulation of
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antioxidants via activation of a nuclear factor-erythroid 2-related factor 2 (Nrf2) (28). Nrf2 is a key
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transcription factor in regulating multiple antioxidants including NADPH-quinone oxidoreductase
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(NQO1), heme oxygenase (HO-1), catalase (CAT), superoxide dismutase, glutathione peroxidase,
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and glutamatecysteine ligase. Actin-tethered protein Keap1 is a cytosolic repressor that binds to and
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retains Nrf2 in the cytoplasm, which promotes Nrf2 proteasomal degradation and results in the
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prevention of Nrf2 transcription (14, 30). Therefore, inhibition of proteasome activity is one method
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to maintain cytoplasm Nrf2 available to nuclear translocation and activation in response to
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oxidative stress (15).
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Another important mechanism underlying MG132 cardiovascular protection is its inactivation
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of NF-κB, a nuclear transcription factor that regulates pro-inflammatory cytokine expression.
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Activation of NF-κB has been documented in myocardial ischemia/reperfusion (13), and specific
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inhibition of NF-κB is cardioprotective (25). Since NF-κB usually is inhibited by IκB via forming a
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complex with NF-κB. However, IκB is degraded by proteasome ubiquitination, resulting in the
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release of NF-κB and nuclear translocation to turn on inflammatory genes. Therefore, proteasomal
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inhibitors such as MG132 can inhibit the proteasome activity to provide anti-inflammation function,
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leading to the cardiac prevention from ischemic damage.
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Diabetic cardiomyopathy is also associated with early cardiac oxidative stress and
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inflammation, which initiates the late development of cardiac remodeling and dysfunction (1, 12, 27,
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34). There was a report, showing that diabetes increased cardiac proteasome function (11, 22),
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which was associated with diabetic complications (16). Therefore, we hypothesized that MG132 as
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one of the proteasome inhibitors may be potentially used for the prevention of diabetic
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cardiomyopathy via up-regulation of Nrf2 to reduce oxidative stress and down-regulation of
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NF-κB-mediated inflammation (16). There were a few studies that have shown the cardiac and renal
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protection by proteasome inhibitor from other pathogeneses (3, 4, 17, 19, 28, 31). Dreger et al. have
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demonstrated the protective effect of low-dose MG132 on hydrogen peroxide-induced oxidative
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stress and damage in cardiomyocytes, and also showed the increased sensitivity in myocytes from
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Nrf2 knockout mice, suggesting the important role of Nrf2 in the cardiac protection (9). Using
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MG132 several studies have also shown the preventive effect of inhibiting proteasome activity on
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diabetic vascular injury (16, 32). A pilot study indicates that proteasomal inhibitor that up-regulates
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Nrf2 activity and inhibits NF-κB-mediated inflammation provided a preventive effect on
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diabetes-induced renal dysfunction (18).
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From clinical setting, there is an urgent need for an efficient approach that can provide a
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therapeutic effect, rather than only prevention, since it is not easily accepted that the diabetic
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patients will give preventive medicine once diagnosed as diabetes. So far, however, there was no
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report regarding the therapeutic effect of MG132 on diabetic complications; in the present study,
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therefore, we have tried to address the question whether MG132 can provide a therapeutic effect on
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diabetic cardiomyopathy by testing if MG132 could reserve or slow the progression of established
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cardiac dysfunction in the OVE26 mouse model of severe Type 1 diabetes. In addition, mechanistic
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studies were focused on the up-regulation of Nrf2-mediated antioxidants and down-regulation of
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NF-κB-mediated inflammation.
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MATERIALS AND METHODS
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Animals. The transgenic type 1 diabetic OVE26 mouse model with FVB background has been
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characterized in previous studies (38). All mice were housed in the University of Louisville
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Research Resources Center at 22 °C with a 12-h light/dark cycle and provided with free access to
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standard rodent chow and tap water. All animal procedures were approved by the Institutional
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Animal Care and Use Committee, which is certified by the American Association for Accreditation
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of Laboratory Animal Care.
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OVE26 mice normally develop severe hyperglycemia before 3 weeks of age and develop
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albuminuria by 3 months of the age, particularly in female OVE26 mice (38). Sixteen 3-month-old
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female OVE26 mice were randomly divided into two groups: diabetic group (DM, n=10) and
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MG132-treated OVE26 group (DM/MG132, n=6). Sixteen age and sex matched non-diabetic FVB
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mice were randomly divided into two groups: control group (n=10) and MG132 group (MG132,
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n=6). MG132 (Sigma-Aldich, St. Louis, MO) was dissolved in dimethyl sulfoxide (DMSO) at a
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concentration of 0.0025 μg/ml and diluted with saline for injection. For MG132 and DM/MG132
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mice, MG132 was given intraperitoneally at a dose of 10 μg/kg body-weight daily for 3 months,
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based on a recent study (18), starting at 3 month old OVE26 mice when these mice already display
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significant renal dysfunction. For control and DM mice equal amounts of physiological saline
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containing 0.0025 μg DMSO/ml were given. MG132 treatment for 3 months for OVE26 diabetic
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mice did not affect their blood glucose levels (7).
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Four mice from both control and diabetic groups were sacrificed at 3 months of age and other
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mice (n=6) were treated with MG132 for 3 months and sacrificed at 6 months of age. After heart
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weight and tibia length was measured, whole heart tissue was harvested for protein and mRNA
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analysis. Histopathological observation was predominantly based on left ventricle.
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Echocardiography. To assess cardiac function, echocardiography was performed for these mice
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using a Visual Sonics Vevo 770 high-resolution imaging system, as described before (29, 39). Under
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sedation with Avertin (2,2,2-tribromoethanol; 240 mg/kg IP), mice were placed in a supine position
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on a heating pad to maintain body temperature at 36 – 37 °C. Animal’s heart rates were kept 400 –
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550 beats per min. Two-dimensional and M-mode echocardiography were used to assess wall
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motion, chamber dimensions and cardiac function. Indices directly measured included left
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ventricular (LV) cavity dimensions in diastole (LVID,d) and systole (LVID,s), LV posterior wall
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thickness in diastole (LVPW,d) and systole (LVPW,s), and interventricle septum thickness in
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diastole (IVS,d) and systole (IVS,s). LV fractional shortening (FS) = [(LVIDd − LVIDs)/LVIDd] ×
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100; LV ejection fraction (EF) = (LV Vol,d − LV Vol,s)/LV Vol,d] × 100.
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Western blot analysis. Western blot was performed as described in our previous studies (2, 39). The
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primary antibodies used include: 3-nitrotyrosine (3-NT, 1:1000 dilution), 4-hydroxy-2-nonenal
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(4-HNE, 1:1000 dilution), TNF-α (1:500 dilution), intercellular adhesion molecule 1 (ICAM-1,
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1:500 dilution), plasminogen activator inhibitor 1 (PAI-1, 1:1000 dilution), TGF-β1 (1:500 dilution),
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fibronectin (1:500 dilution), Nrf2 (1:500 dilution), NQO-1 (1:500 dilution), HO-1 (1:500 dilution),
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CAT (1:5000 dilution), atrial natriuretic peptide (ANP, 1:1000 dilution), NF-κB (1:1000 dilution),
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IκB-α (1:1000 dilution), α-tubulin (1:2000 dilution), and β-actin (1:2000 dilution), all of which
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were purchased from Santa Cruz Biotech. Inc. except for TNF-α from Abcam, 3-NT from Millipore,
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NF-κB, IκB-α, and α-tubulin from cell signaling, and 4-HNE from Alpha Diagnostic International.
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All these antibodies are polyclonal antibodies except for TNF-α and PAI-1 antibodies that are
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monoclonal.
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RNA isolation and real-time RT-PCR. RNA isolation and real-time RT-PCR were performed, as
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described in our previous studies (39), for Nrf2 (primer: Mm00477784_m1), ANP
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(Mm01255748_g1), β-myosin heavy chain (β-MHC) (Mm00600555_m1), Hmox1 (HO-1,
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Mm00516005_m1), Nqo1 (Mm253561_m1), CAT (Mm00437229_m1), and the housekeeping gene
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β-actin (Mm00607939_s1). All these primers were purchased from Applied Biosystems (Foster City,
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CA). Total RNA was extracted from whole heart tissues using Trizol reagent (RNA STAT 60
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Tel-Test, Ambion, Austin, TX). RNA concentration and purity were quantified using a Nanodrop
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ND-1000 spectrophotometer (Biolab, Ontario, CA). First-strand complimentary DNA (cDNA) was
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synthesized from total RNA according to the RNA PCR kit (Promega, Madison, WI, USA),
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following the manufacturer’s protocol.
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Cardiac histopathological examination and immunohistochemical staining. Whole cardiac tissue
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was fixed overnight in 10% phosphate buffered formalin and then dehydrated in a graded alcohol
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series, cleared with xylene, embedded in paraffin, and sectioned at 5 μm thickness for pathological
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and immunohistochemical staining. Paraffin sections were dewaxed, followed by incubation with
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1X target retrieval solution (Dako, Carpinteria, CA) for 15 min at 98°C for antigen retrieval, and
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then treated with 3% hydrogen peroxide for 15 min at room temperature, followed by blocking with
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5% BSA for 30 min.
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Cardiac sections were stained with hematoxylin and eosin (H&E) and Sirius-red, respectively,
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as before (2, 39). For immunohistochemical staining sections were incubated with the primary
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antibodies of TGF-β1 (1:100 dilution, Santa Cruz Biotech. Inc.) and PAI-1 (1:100 dilution, BD
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Biosciences. Inc.) for overnight at 4°C. After washing with PBS, these sections were incubated with
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horseradish peroxidase conjugated secondary antibody (1:300-400 dilutions in PBS) for 2 h at room
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temperature. For the development of color, sections were treated with peroxidase substrate
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3,3-Diaminobenzidine in the developing system (Vector Laboratories, Inc. Burlingame, CA).
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Proteasome activity. The 20S proteasome, catalytic core of the 26S proteasome complex, is
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responsible for the breakdown of short-lived regulatory proteins, including Nrf2 and NF-κB (6, 9).
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Since MG132 mainly inhibits the proteasome chymotrypsin (ChT)-like activity (24), we determined
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20S proteasome activity by quantifying the hydrolysis of SLLVY-AMC-a fluorogenic substrate for
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the ChT-like activity. 20S proteasome activity was measured with the 20S Proteasome Activity
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Assay Kit (Millipore).
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Nuclei isolation. The cardiac nuclei were isolate using nuclei isolation kit (Sigma-Aldich). Whole
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cardiac tissue (60 mg) from each mouse was homogenized for 45 sec. within 300 μl cold lysis
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buffer containing 1 μl dithiothreitol (DTT) and 0.1 % Triton X-100. After that, 600 μl cold 1.8
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mol/L Cushion Solution (Sucrose Cushion Solution: Sucrose Cushion Buffer: DTT= 900:100:1)
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was added to the lysis solution. The mixture was transferred to a new tube pre-loaded with 300 μl
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1.8 mol/ L Sucrose Cushion Solution followed by centrifuge at 13,000 × rpm for 45 min. The
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supernatant contains cytosolic component and nuclei were visible as thin pellet at the bottom of
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tube.
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Measurements of serum TNF-α and MCP-1, and cardiac nuclear NF-κB DNA binding activity.
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Serum levels of TNF-α and MCP-1 were performed using mouse TNF-α and MCP-1 ELISA kits
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(Invitrogen, Camarillo, CA), as described previously (21). The low detection limit was 3 pg/mL and
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9 pg/mL for serum TNF-α and MCP-1, respectively. Nuclear p65 DNA binding activity was
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determined by ELISA-based NF-κB activity assay using cardiac tissue from whole heart (Cayman
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Chemical, Ann Arbor, MI), according to manufacturer instructions.
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Total antioxidant capacity (TAC) assay .The cardiac TAC assay was performed using a
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commercially available assay kit (Cell Biolabs, San Diego, CA), according to the manufacturer's
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instructions. Briefly, whole heart tissues were homogenized in cold PBS and then centrifuged at
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10,000×g at 4°C for 10 min to collect the supernatant for the TAC assay. The values were calculated
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using optical density at 490 nm and expressed as umol/g protein for TAC.
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Statistical analysis. Data were expressed as mean ±SD. For statistical analysis, one-way analysis of
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variance (ANOVA) was used as appropriate. The overall F-test was performed to test the
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significance of the ANOVA models. The significance of the interactions and main effects were
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taken into consideration and then multiple comparisons were performed by the Bonferroni test. The
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significance level was considered at p<0.05.
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RESULTS
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Therapeutic effect of MG132 on diabetes-induced cardiac dysfunction and hypertrophy
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Transgenic OVE26 type 1 diabetic (DM) mice at 3 months showed the increased LVID at systole
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and the decreased EF% and FS%, suggesting the exist of cardiac dilation and decreased systolic
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function (Fig. 1A). Hypertrophic measurements of IVS, LVPW and LV mass were significantly
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increased in these mice with a progressive manner from 3 to 6 months (P<0.05, Fig. 1B). However,
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when some of DM mice at 3 months of age were treated with low-dose MG132 for 3 months,
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diabetes-induced cardiac dysfunction was almost completely reversed in DM/MG132 group
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(P<0.05, Fig. 1).
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Therapeutic effects of MG132 on diabetes-induced cardiac hypertrophy were also reflected by
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the ratio of heart to tibia length (Fig. 2A) that was significantly increased in an age-dependent
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manner in DM group from 3 to 6 months, but not in DM/MG132 group.
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To further confirm cardiac hypertrophy, cardiac morphology and molecular hypertrophy
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makers were also examined. H&E staining showed that compared with control and MG132 groups,
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main pathological changes in DM group include: (1) myocardial hypertrophy; (2) a few cardiac
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cells that showed degeneration features; (3) proliferation of interstitial collagen fibers. Those
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pathological changes were alleviated or not observed in the DM/MG132 mice (Fig. 2B). Real-time
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PCR analysis showed that DM induced a significant increase of cardiac mRNA expression of ANP
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(Fig. 2C) and β-MHC (Fig. 2D), an effect that was completely abolished by MG132 treatment in
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DM/MG132 group. Consistent with ANP mRNA finding, Western blot showed a significant
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increase of cardiac ANP protein expression at 6 months of age in DM mice, but not in DM/MG132
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mice (Fig. 2E). These results suggest that 3-month treatment with low-dose MG132 can
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significantly or even completely prevent the progression of the cardiac hypertrophy induced by
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diabetes.
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Therapeutic effect of MG132 on diabetes-induced cardiac fibrosis
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Cardiac tissue was examined with Sirius-red staining for collagen (Fig. 3A), followed by
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semi-quantitative analysis with computer imaging system (Fig. 3B), which showed a significant
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increase of the collagen accumulation in DM group, but not in DM/MG132 group. Western blots of
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TGF-β1, fibronectin, and PAI-1 as important pro-fibrotic mediators confirmed the Sirius-red
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staining result: a significant increase in cardiac fibrosis in DM group, but not in DM/MG132 group
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(Fig. 3C). Furthermore, the increased expression of TGF-β1 and PAI-1 was supported by
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immunohistochemical stain (Fig. 3D).
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Therapeutic effect of MG132 on diabetes-induced cardiac inflammation and oxidative stress
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Since PAI-1 acts as both pro-fibrotic and inflammatory mediators, its increase in the heart of DM
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mice suggests the possible cardiac inflammation. Next we thus examined the protein expression of
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TNF-α (Fig. 4A) and MCP-1(Fig. 4B) in serum by ELISA and (Figs. 4C,D) in the heart by Western
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blot assay. Serum TNF-α and MCP-1 levels were progressively elevated from 3 to 6 months in DM
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group, which were completely prevented by MG132 treatment in DM/MG132 group. Increased
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cardiac TNF-α and MCP-1 contents were also observed only in DM group, not in DM/MG132
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group.
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Considering that inflammation in the target organs often causes oxidative stress while
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oxidative stress is also able to induce inflammation, the next study examined the oxidative stress by
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measuring 3-NT accumulation as an index of protein nitration (Fig. 5A) and 4-HNE as lipid
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peroxidation (one of oxidative damage) (Fig. 5B). Both 3-NT and 4-HNE contents were
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significantly increased in the heart of DM mice, but not DM/MG132 mice. The TAC in heart tissue
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(Fig. 5C) was slightly decreased (p<0.05) in DM group at both 3 and 6 months, but not changed in
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DM/MG132 group at 6 months.
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Possible mechanisms for the therapeutic effect of MG132 on the diabetic cardiomyopathy
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Diabetes increases cardiac proteasomal activity, an effect prevented by MG132
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Since tetrahydrobiopterin deficiency that was reported to uncouple the enzymatic activity of
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endothelial nitric oxide synthase in DM hearts is triggered by DM-increased proteasome-dependent
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mechanisms (35), we examined here whether cardiac proteasome activity is increased in diabetic
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mice. Fig. 6 shows that MG132 decreases, and diabetes increases, the cardiac 20S proteasomal
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activity. Compared to DM group, the cardiac 20S proteasomal activity was significantly reduced,
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even to control level, in DM/MG132 group (45% DM, P<0.05 vs DM).
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MG132 inhibits cardiac proteasomal activity, resulting in an up-regulation of cardiac Nrf2 and its
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downstream antioxidants
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Proteasomal degradation of Nrf2 has been appreciated as the major of mechanism responsible for
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Nrf2’s negative regulation (14, 30). Our finding that MG132 completely inhibited
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diabetes-up-regulated proteasomal activity implies a possibility for MG132 treatment to decrease
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degradation of Nrf2 protein. Therefore the expression of Nrf2 at mRNA and protein levels were
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examined with real-time PCR and Western blot assays, respectively. We found that MG132 had no
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impact on, but diabetes had a significant increase in, the Nrf2 mRNA expression (Fig. 7A).
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DM/MG132 had a similar expression of Nrf2 mRNA to those observed in DM group (Fig. 7A). Fig.
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7B shows that both MG132 and DM increased cardiac expression of Nrf2 protein, so that Nrf2
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protein expression was synergistically increased in DM/MG132 group.
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Furthermore, up-regulated Nrf2 transcription activity was reflected by the increase of several
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downstream antioxidant genes (Fig. 7C,D). MG132 treatment in control mice significantly
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increased the expression of NQO1, HO-1, and CAT at mRNA and protein levels, compared to
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Control. Diabetes also increased the expression of these antioxidants at mRNA and protein levels.
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The expression of these Nrf2 downstream antioxidant genes in the heart of DM/MG132 mice was
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synergistically increased at both mRNA and protein levels compared to MG132 or DM alone.
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MG132 inhibits cardiac proteasomal activity, resulting in a reduction of cardiac inflammation by
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preventing NF-κB nuclear translocation
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Since NF-κB is bound by IκB-α in cytoplasm that inhibits NF-κB nuclear translocation to
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transcriptionally up-regulate inflammatory cytokines, reduction of IκB-α will indirectly up-regulate
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NF-κB-mediated inflammation. Western blot showed that the expression of IκB-α was significantly
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decreased in the heart of DM group, but not DM/MG132 group (Fig. 8A). This suggests that
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MG132 inhibits proteasomal degradation of IκB-α. To define whether preservation of normal level
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of IκB-α is able to prevention of NF-κB nuclear translocation, cardiac proteins were separated into
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nuclear and cytoplasm parts, which showed that nuclear accumulation of NF-κB was significantly
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increased in DM group, but not in DM/MG132 group (Fig. 8B). Furthermore, the increased cardiac
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nuclear NF-κB DNA binding activity only in the heart of DM mice at both 3 and 6 months was
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further confirmed by ELISA-based NF-kB activity assay (Fig. 8C).
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DISCUSSION
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The present study is the first one to report the therapeutic effect of chronic treatment with low-dose
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MG132 on diabetes-induced cardiomyopathy using OVE26 diabetic mouse model. We
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demonstrated that the therapeutic effect of MG132 on diabetic cardiomyopathy is associated with
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its suppression of diabetic up-regulation of proteasome activity that may increase the proteasomal
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degradation of IκB-α and Nrf2. Increased degradation of IκB-α would release its binding and
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restricting NF-κB in cytoplasm, leading to the increase of NF-κB nuclear translocation to generate
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inflammatory cytokines, as illustrated in Fig. 9. These cytokines will initiate the over-generation of
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ROS/RNS, cardiac oxidative stress and damage, remodeling and dysfunction. In addition, increased
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proteasomal degradation of Nrf2 would reduce Nrf2’s transcription to generate its downstream
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antioxidants, which will exacerbate cardiac pathogenic alterations, leading to an acceleration of
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cardiomyopathy development, as illustrated in Fig. 9.
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Several in vivo and in vitro studies have provided evidence for the increase in proteasomes
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under diabetic conditions. For example, exposure of human umbilical vein endothelial cells to high
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level of glucose significantly increased the 26S proteasome activity (35). Proteasomal activity was
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also found to be increased in the heart of type 1 diabetes (11, 22) and in gastrocnemius muscle of
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spontaneous type 2 diabetic mice (db/db) (33). Measurement of proteasomal activity in the kidney
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of diabetic rats showed an increase of 26S proteasomal activity in STZ diabetic rats (18).
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There was a pilot in vivo study showing the preventive effect of MG132 on diabetes-induced
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renal damage (18). In this study, shortly after diabetes induction rats were treated with MG132 at 10
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μg/kg daily for 3 months. This regimen produced renal prevention as indicated by reductions in
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proteinuria, basement membrane thickening and glomerular mesangial expansion. MG132 also
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reduced kidney markers of oxidative stress and increased protein levels of Nrf2 and several
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antioxidant enzymes. These studies demonstrated a potential for MG132 to prevent the
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development of diabetic complications. However, whether Nrf2 activators have therapeutic effects
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on the heart and/or kidney of diabetic subjects remains unknown. Here we report for the first time
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the therapeutic effect of MG132 on diabetic cardiomyopathy in the transgenic type 1 diabetic
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OVE26 mouse model. When these diabetic mice at 3 months old began to exhibit albuminuria as an
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index of renal dysfunction (38) and cardiac dysfunction (Fig. 1), some of them were treated with
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MG132 at a very low dose for three months, which offered a significant therapeutic outcome,
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indicated by completely preventing diabetes-induced cardiac inflammation and oxidative stress and
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damage, leading to a complete reverse of cardiac remodeling (fibrosis and hypertrophy) and
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dysfunction. Diabetic mice without treatment with MG132 showed progressive development of
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cardiac structural remodeling and functional abnormalities.
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In terms of the mechanisms underlying the therapeutic effect of MG132 on diabetic
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cardiomyopathy, we assumed that it is associated with the significant up-regulation of Nrf2
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expression and transcriptional increases of its downstream antioxidants and the complete prevention
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of diabetes-reduced IκB content and -increased NF-κB nuclear accumulation, as outlined in Fig. 9.
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Reportedly non-toxic concentrations of MG132 inhibited the proteasomal degradation of Nrf2
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to stimulate Nrf2 translocation into the nucleus (5, 28). An in vitro study showed that treatment with
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0.5 µM MG132 for 48 h protected neonatal rat cardiac myocytes against H2O2-mediated oxidative
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stress (9). This was correlated with a reduced level of intracellular ROS and the significant
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up-regulation of superoxide, HO-1, and CAT expression. This demonstrated that non-toxic
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concentrations of MG132 could up-regulate Nrf2-mediated antioxidant enzymes to confer
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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. L.C. wrote, edited, and reviewed the article.
427
428
COMPETING INTERESTS:
429
The authors declare that they have no conflict of interest.
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FIGURE LEGENDS
582
Fig.1. Therapeutic effects of MG132 on diabetes-induced cardiac dysfunction. Three month
583
old female OVE26 mice and FVB control mice were given MG132 (10 µg/kg) or an equal
584
volume of physiological saline daily for 3 months. 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)