Prevention by sulforaphane of diabetic cardiomyopathy is associated with up-regulation of Nrf2 expression and transcription activation

Journal of Molecular and Cellular Cardiology 57 (2013) 82–95

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Original article

Prevention by sulforaphane of diabetic cardiomyopathy is associated with up-regulation of Nrf2 expression and transcription activation Yang Bai a, b, c, Wenpeng Cui b, d, Ying Xin b, e, Xiao Miao b, d, Michelle T. Barati f, Chi Zhang b, Qiang Chen b, g, Yi Tan b, Taixing Cui h, Yang Zheng a,⁎, Lu Cai a, b, f,⁎⁎ a

The Cardiovascular Center, The First Hospital of Jilin University, Changchun, China The Department of Pediatrics, University of Louisville, Louisville, KY, USA The Jilin Province People's Hospital, Changchun, China d The Second Hospital of Jilin University, Changchun, China e The Department of Pathology, Bethune Medical College of Jilin University, Changchun, China f The Department of Medicine, University of Louisville, Louisville, KY, USA g The School of Public Health of Jilin University, Changchun, China h The Department of Cell Biology and Anatomy, University of South Carolina School of Medicine, Columbia, SC, USA b c

a r t i c l e

i n f o

Article history: Received 26 June 2012 Received in revised form 18 December 2012 Accepted 8 January 2013 Available online 23 January 2013 Keywords: Sulforaphane Nrf2 Cardiomyopathy Oxidative damage Cardiac dysfunction

a b s t r a c t This study was to investigate whether sulforaphane (SFN) can prevent diabetic cardiomyopathy. Type 1 diabetes was induced in FVB mice by multiple intraperitoneal injections with low-dose streptozotocin. Hyperglycemic and age-matched control mice were treated with or without SFN at 0.5 mg/kg daily in five days of each week for 3 months and then kept until 6 months. At 3 and 6 months of diabetes, blood pressure and cardiac function were assessed. Cardiac fibrosis, inflammation, and oxidative damage were assessed by Western blot, real-time qPCR, and histopathological examination. SFN significantly prevented diabetes-induced high blood pressure and cardiac dysfunction at both 3 and 6 months, and also prevented diabetes-induced cardiac hypertrophy (increased the ratio of heart weight to tibia length and the expression of atrial natriuretic peptide mRNA and protein) and fibrosis (increased the accumulation of collagen and expression of connective tissue growth factor and tissue growth factor-β). SFN also almost completely prevented diabetes-induced cardiac oxidative damage (increased accumulation of 3-nitrotyrosine and 4-hydroxynonenal) and inflammation (increased tumor necrotic factor-α and plasminogen activator inhibitor 1 expression). SFN up-regulated NFE2-related factor 2 (Nrf2) expression and transcription activity that was reflected by increased Nrf2 nuclear accumulation and phosphorylation as well as the mRNA and protein expression of Nrf2 downstream antioxidants. Furthermore, in cultured H9c2 cardiac cells silencing Nrf2 gene with its siRNA abolished the SFN's prevention of high glucose-induced fibrotic response. These results suggest that diabetes-induced cardiomyopathy can be prevented by SFN, which was associated with the up-regulated Nrf2 expression and transcription function. © 2013 Elsevier Ltd. All rights reserved.

1. Introduction Diabetic cardiovascular complications include both macrovascular and microvascular diseases. Changes in the heart independent of macrovascular disease that may exist at the same time are recognized as diabetic cardiomyopathy. Diabetic cardiomyopathy is the most common complications of diabetes and also a main cause of the mortality for diabetic patients [1]. Accumulating evidence already indicates that to ⁎ Correspondence to: Y. Zheng, Cardiovascular Center at the First Hospital of Jilin University, 71 Xinmin Street, Changchun, 130021, China. Tel.: + 86 13756661288. ⁎⁎ Correspondence to: L. Cai, Department of Pediatrics, University of Louisville, 570 South Preston Street, Baxter I, Suite 304F, Louisville, KY 40202, USA. Tel.: + 1 502 852 2214; fax: + 1 502 852 5634. E-mail addresses: [email protected] (Y. Zheng), [email protected] (L. Cai). 0022-2828/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.yjmcc.2013.01.008

prevent the development and progression of cardiomyopathy in the patients with diabetes could not be done by controlling glucose level or blood pressure, lowing lipid level, and blockading the renin–angiotensin system [1]. Therefore, an effective approach to prevent or delay the development and progression of these lethal complications for diabetic patients are urgently needed. As three key metabolic abnormalities, hyperglycemia, hyperlipidemia, and inflammation all stimulate the generation of reactive oxygen or nitrogen species (ROS or RNS). Extra production of these species is a causative of the development of diabetic complications, including cardiomyopathy [1–3]. Accordingly, antioxidant prevention or therapy of diabetic complications has been explored, but, to date, there was no exogenous antioxidant that efficiently prevents diabetic cardiomyopathy in clinics. Therefore, the activation of tissue's endogenous antioxidant components has been proposed as an attractive strategy [4].

Y. Bai et al. / Journal of Molecular and Cellular Cardiology 57 (2013) 82–95

The transcription factor NFE2-related factor 2 (Nrf2) as one member of the cap ‘n’ collar family is a master regulator of cellular detoxification responses and redox status [5]. Under physiological conditions Nrf2 locates in the cytoplasm and binds to its inhibitor kelch-like ECHassociated protein 1 (KEAP1) [6]. KEAP1 could mediate a rapid ubiquitination and subsequent degradation of Nrf2 by the proteasome [6]. Upon exposure of cells to oxidative stress or electrophilic compounds, Nrf2 is free from KEAP1 and translocates into the nucleus to bind to antioxidant-responsive elements in the promoters of gene encoding antioxidant enzymes such as NADPH quinone oxidoreductase (NQO1), heme oxygenase-1 (HO-1), glutathione S-transferase, superoxide dismutase (SOD), catalase (CAT), and γ-glutamylcysteine synthetase, to increase the expression of these antioxidants against oxidative stress and associated inflammation and damage [6,7]. Sulforaphane (SFN) is an organosulfur compound and obtained from cruciferous vegetables such as broccoli, brussels sprouts or cabbages [8]. Emerging evidence indicates the association of the increased consumption of cruciferous vegetables with a decreased risk of several degenerative and chronic diseases, including cardiovascular disease under diabetic and non-diabetic conditions. SFN has garnered particular interest as an indirect antioxidant due to its extraordinary ability to induce the expression of several enzymes via the KEAP1/Nrf2 pathway [8]. Therefore the present study investigated whether the chronic use of SFN can prevent the development of diabetic cardiomyopathy. To this end, we have used a type 1 diabetic mouse model induced with multiple low-dose streptozotocin (MLD-STZ). Diabetic and control mice were treated with SFN for 3 months and then kept for another 3 month without SFN treatment. SFN could almost completely prevent the development of diabetic cardiomyopathy along with an up-regulation of Nrf2 expression and transcription function in the heart. Furthermore, we found that the exposure of the cultured cardiac H9c2 cells to high glucose (HG) increased fibrotic effect, which could be completely prevented by pre-treatment with SFN that also significantly increased Nrf2 expression and transcription. Silencing Nrf2 expression completely abolished the prevention by SFN of HG-induced fibrotic effect, suggesting the important role of Nrf2 in the cardiac protection by SFN against HG in vitro or diabetes in vivo. 2. Materials and methods 2.1. Animals FVB male mice, 8–10 weeks of age, were purchased from the Jackson Laboratory (Bar Harbor, Maine) and housed in the University of Louisville Research Resources Center at 22 °C with a 12-h light/dark cycle with free access to standard rodent chow and tap water. All experimental procedures for these animals were approved by the Institutional Animal Care and Use Committee of the University of Louisville, which is compliant with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). For induction of the type 1 diabetes, mice were injected intraperitoneally with MLD-STZ [Sigma-Aldich, St. Louis, MO, dissolved in 0.1 M sodium citrate (pH 4.5)] at 50 mg/kg body weight daily for 5 consecutive days while age-matched control mice were received multiple injections of the same volume of sodium citrate buffer. Five days after the last injection of STZ, mice with hyperglycemia (blood glucose levels≥250 mg/dl) were defined as diabetic, as before [9]. SFN (Sigma-Aldich) was subcutaneously given at 0.5 mg/kg for five days in each week for 3 months. At the end of 3-month SFN treatment, some mice were sacrificed for experimental measurements, and the rest of these SFN-treated control and diabetic mice were kept for additional 3 months without SFN treatment and then sacrificed for experimental measurements. Dose of SFN used was based on the previous study [10]. Mice were randomly allocated into four groups (n =6 at least per group): Control, SFN, diabetes (DM)

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and DM plus SFN (DM/SFN). Since SFN was dissolved in 1% dimethyl sulfoxide (DMSO) and diluted in PBS, mice serving as vehicle controls were given the same volume of PBS containing 1% DMSO. 2.2. Non-invasive blood pressure Blood pressure (BP) was measured by tail-cuff manometry using a CODATM non-invasive BP monitoring system (Kent Scientific Corporation, Torrington, CT). Mice were kept in warm on the heating pad to ensure sufficient blood flow to the tail. Mice were restrained in a plastic tube restrainer. Occlusion and volume-pressure recording cuffs were placed over the tail. Each mouse was allowed to adapt to the restrainer for 5 min prior to BP measurement. The BP was measured for 10 acclimation cycles followed by 20 measurement cycles. After three days of training for the BP measurement, formal measurements for the unanesthetized BP and heart rate (HR) were collected (Table 1), as described previously [11]. 2.3. Echocardiography Transthoracic echocardiography (Echo) was performed for Avertin anesthetized mice at rest using a high-resolution imaging system for small animals (Vevo 770, VisualSonics, Canada), equipped with a high-frequent ultrasound probe (RMV-707B). All hair was removed from the chest using a chemical hair remover and the aquasonic clear ultrasound gel (Parker Laboratories, Fairfield, NJ) without bubble was applied to the thorax surface to optimize the visibility of the cardiac chambers. Parasternal long-axis and short-axis views were acquired. Left ventricular (LV) dimensions and wall thicknesses were determined from parasternal short axis M-mode images. The anesthetized HR was collected. Meanwhile, ejection fraction (EF), fractional shortening (FS), and LV mass were calculated by Vevo770 software (Table 2). The final data represent averaged values of 10 cardiac cycles [12]. 2.4. Heart pathology, immunohistochemical and immunofluorescent staining After anesthesia, hearts were isolated and fixed in 10% buffered formalin and then dehydrated in graded alcohol series, cleared with xylene, embedded in paraffin, and sectioned at 5 μm thickness. Tissue sections were dewaxed and then incubated with 1 × target retrieval solution (Dako, Carpinteria, CA) in a microwave oven for 15 min at

Table 1 Effect of SFN on diabetes-induced unanesthetized blood pressure change and heart rate. Control 3M HR (beats/min) Diastolic BP (mm Hg) Systolic BP (mm Hg) Mean BP (mm Hg) 6M HR (beats/min) Diastolic BP (mmHg) Systolic BP (mm Hg) Mean BP (mm Hg)

SFN

DM

DM/SFN

657 ± 35 76.7 ± 1.37

644 ± 5 74.09 ± 2.85

626 ± 17 85.83 ± 1.11

627 ± 10 78.45 ± 1.22

105.02 ± 1.19

100.29 ± 2.35

120.59 ± 1.47

106.53 ± 5.63

86.14 ± 0.53

82.82 ± 3.31

97.42 ± 1.63

87.81 ± 3.93

637 ± 26 78.68 ± 2.64

644 ± 9 76.57 ± 2.57

626 ± 12 92.42 ± 3.04⁎

609 ± 13 80.91 ± 1.28#

107.45 ± 1.96

101.07 ± 0.81

125.75 ± 3.85⁎

109.97 ± 3.55#

88.27 ± 2.26

84.73 ± 1.75

103.53 ± 3.13⁎

90.60 ± 1.94#

Notes: Data were presented as means ± SEM. HR = heart rate; BP = blood pressure. ⁎ p b 0.05 vs. control. # p b 0.05 vs. DM group.

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Table 2 Protective effect of SFN on diabetes-induced cardiac dysfunction and anesthetized heart rate. Control 3M HR (beats/min) LVID;d (mm) LVID;s (mm) IVS;d (mm) IVS;s (mm) LVPW;d (mm) LVPW;s (mm) %EF (%) % FS (%) LV mass (mg) LV mass corrected (mg)

SFN

DM

Cardiac fibrosis was examined with 0.1% Sirius-red F3BA and 0.25% Fast green FCF for the collagen accumulation, as described in our previous study [13].

DM/SFN

2.5. Cell culture 495 ± 19 3.56 ± 0.11 1.39 ± 0.04 0.71 ± 0.03 1.07 ± 0.03 0.82 ± 0.01 1.8 ± 0.12 90.36 ± 0.79 60.94 ± 1.29 91.15 ± 1.17 72.92 ± 2.93

449 ± 23 449 ± 6 3.59 ± 0.02 3.45 ± 0.06 1.56 ± 0.06 2.17 ± 0.01⁎ 0.71 ± 0.01 0.75 ± 0.01⁎ 1.12 ± 0.02 0.9 ± 0.01 0.88 ± 0.01 1.02 ± 0.03⁎ 1.67 ± 0.01 1.26 ± 0.03⁎ 87.46 ± 0.94 68.78 ± 1.31⁎ 56.78 ± 1.33 39.12 ± 0.28⁎ 95.8 ± 2.82 106.77 ± 2.12⁎ 77.44 ± 2.25 85.42 ± 1.7⁎

463 ± 14 3.49 ± 0.19 1.77 ± 0.1# 0.72 ± 0.01# 0.99 ± 0.05 0.9 ± 0.01# 1.47 ± 0.02# 81.58 ± 1.5# 49.41 ± 1.58# 99.32 ± 1.76# 79.46 ± 2.81#

H9c2 cells were purchased from ATCC and, as suggested by ATCC, maintained in DMEM (Dulbecco's Modification of Eagle's Medium) with 4.5 g/L glucose and L-glutamine & sodium pyruvate (Mediatech, Manassas, VA), supplemented with 10% bovine calf serum and 1% Penicillin–Streptomycin Solution at 37 °C with 5% CO2. When experiments were performed these cells were exposed to either HG (22 mM or 4 g/L) or control glucose (5.5 mM or 1 g/L) in DMEM containing 10% bovine calf serum and 1% Penicillin–Streptomycin Solution. 2.6. Immunocytochemistry

6M HR (beats/min) 479 ± 7 458 ± 21 454 ± 13 452 ± 7 LVID;d (mm) 3.57 ± 0.09 3.59 ± 0.10 3.94 ± 0.01⁎ 3.64 ± 0.04# ⁎ LVID;s (mm) 1.65 ± 0.11 1.74 ± 0.03 2.55 ± 0.03 1.81 ± 0.07# IVS;d (mm) 0.78 ± 0.03 0.74 ± 0.01 0.93 ± 0.02⁎ 0.81 ± 0.01# IVS;s (mm) 1.09 ± 0.03 1.02 ± 0.04 0.93 ± 0.01⁎ 1.09 ± 0.02# LVPW;d (mm) 0.88 ± 0.04 0.93 ± 0.02 1.01 ± 0.01⁎ 0.91 ± 0.02# LVPW;s (mm) 1.73 ± 0.03 1.77 ± 0.06 1.14 ± 0.03⁎ 1.39 ± 0.12# %EF (%) 84.45 ± 0.74 84.14 ± 0.52 65.05 ± 0.25⁎ 81.75 ± 1.31# %FS (%) 52.83 ± 0.87 52.38 ± 0.71 35.14 ± 0.61⁎ 49.72 ± 1.41# LV mass (mg) 112.58 ± 2.28 108.13 ± 2.6 149.98 ± 2.08⁎ 108.43 ± 1.05# LV mass corrected 90.06 ± 1.66 86.5 ± 2.1 119.99 ± 1.82⁎ 86.74 ± 0.84# (mg) Notes: Data were presented as means ± SEM. LVID;d = LV end-diastolic diameter; LVID;s = LV end-systolic diameter; LVPW = LV posterior wall; IVS = interventricular septum; FS = fractional shortening; EF = ejection fraction. ⁎ p b 0.05 vs. control. # p b 0.05 vs. DM group.

98 °C for antigen retrieval, followed by treatments with 3% hydrogen peroxide for 15 min at room temperature and with 5% bovine serum albumin (Sigma-Aldich) for 30 min, respectively. These sections were incubated with primary antibodies overnight at 4 °C. Primary antibodies are those against 3-nitrotyrosine (3-NT, Millipore, Billerica, MA) at 1:400 dilution, 4-hydroxy-2-nonenal (4-HNE, Alpha Diagnostic International, San Antonio, TX) at 1:400 dilution, plasminogen activator inhibitor-1 (PAI-1, BD Bioscience, San Jose, CA) at 1:200 dilution, tumor necrosis factor-alpha (TNF-α, Abcam, Cambridge, MA) at 1:100 dilution, Nrf2 (Abcam) at 1:50 dilution, and phosphorylated Nrf2 at Ser40 (p-Nrf2) at 1: 200 dilution. For immunohistochemical staining, the above sections were then washed with PBS and incubated with horseradish peroxidase conjugated secondary antibodies (1:300–400 dilutions with PBS) for 2 h in room temperature. To develop the color, sections were treated with peroxidase substrate DAB (3,3-Diaminobenzidine, Vector Laboratories, Burlingame, CA) and counterstained with hematoxylin. For immunofluorescent staining, the above sections that have been incubated with primary antibody were washed with PBS and then incubated with secondary antibodies (Cy3-conjugated donkey anti-rabbit antibody at 1:200 dilution) (Jackson ImmunoReserch Laboratories, West Grove, PA), followed by counterstaining with 4, 6-diamidino-2phenylindole dihydrochloride (DAPI, Sigma-Aldich). Confocal images were acquired using an Olympus Fluoview FV-1000 confocal scanner coupled to an Olympus 1×81 inverted microscope, a PlanApoN 60× objective, and FV-10 ASW 2.1 software. Multi channel scanning configuration with sequential line scanning was setup for acquisition of Cy3 and DAPI staining, using 543 nm HeNe and 405 laser diode, at a speed of 4 μs/pixel. Optimal brightness setting for each channel was configured by determining the HV setting yielding maximal intensity without saturation. Images of single planes are presented in the corresponding figures.

H9c2 cells were cultured in a glass culture chamber (Nalge Nunc International, Rochester, NY) and exposed to HG for 24 h with or without SFN pretreatment for 1 h (10 μM). Acetone-fixed cells were blocked with 5% bovine serum albumin (Sigma-Aldich) for 1 h at room temperature and then incubated with rabbit anti-Nrf2 at 1:50 (Abcam) overnight at 4 °C. After PBS washing, cells were incubated with secondary antibody (Cy3-conjugated donkey anti-rabbit antibody at 1:200 dilution), counterstained with DAPI, and mounted, observed under confocal microscope. 2.7. Real-time qPCR Collected heart was snap frozen in liquid nitrogen and kept at − 80 °C. Total RNA was extracted using the TRIzol Reagent (Invitrogen, Carlsbad, CA). RNA concentrations and purities were quantified using a Nanodrop ND-1000 spectrophotometer. First-strand complimentary DNA (cDNA) was synthesized from total RNA according to manufacturer's protocol from the RNA PCR kit (Promega, Madison, WI). Reverse transcription was performed using 1 μg of total RNA in 12.5 μL of the solution containing 4 μL 25 mM MgCl2, 4 μL AMV reverse transcriptase 5× buffer, 2 μL dNTP, 0.5 μL RNase inhibitor, 1 μL of AMV reverse transcriptase, and 1 μL of oligo dT primer, which were added with nuclease-free water to make a final volume of 20 μL. Reaction system was run at 42 °C for 50 min and 95 °C for 5 min. Primers [ANP: Mm01255748_ g1; Hmox1: Mm00516005_ m1; NqO1: Mm01253561_ m1; Zn/Cu-SOD (Sod1): Mm01344233_ g1; Mn-SOD (Sod2): Mm01313000_ m1; CAT: Mm00437229_ m1; MT1: Mm00496660_ g1; CTGF: Mm01192933_ g1; β-actin: Mm00607939_ s1] and [Hmox1: Rn 3160, NqO1: Rn 11234, Sod1: Rn 6059, Sod2: Rn 10488, CAT: Rn 3001, CTGF: Rn 00573960_ g1, β-actin: Rn00667869_m1] for PCR were purchased from Applied Biosystems (Carlsbad, CA). Real-time qPCR was carried out in a 20 μL reaction buffer that included a 10 μL of TaqMan Universal PCR Master Mix, 1 μL of primer, and 9 μL of cDNA with the ABI 7300 Real-Time qPCR system. Fluorescent intensity of each sample was measured at each temperature to monitor the amplification of the target gene. Comparative cycle time (CT) was used to determine fold differences between samples [14]. 2.8. siRNA transfection For knocking down Nrf2 expression, rattus norvegicus nontargeting Stealth RNAi™ siRNA (RSS343558 and 343559) along with corresponding negative control (Invitrogen) were transfected into H9c2 cells for 36 h by Lipofectamine™ 2000 transfection reagent (Invitrogen), followed by SFN (10 μM) and glucose (22 mM) treatment 24 h. The sense and antisense sequences of rat Nrf2 were as follows: 5′-GGAAACCUUACUCUCCCAGUGAGUA-3′ and 5′-UACUCACUGGGAGA GUAAGGUUUCC-3′ (RSS343558); 5′-ACGCAGGAGAGGGAAGAAUAAA

Y. Bai et al. / Journal of Molecular and Cellular Cardiology 57 (2013) 82–95

A

85

B

Control

SFN

DM

DM/SFN Heart/Tibia(mg/mm)

3M 6M

*#

*#

10

5

0 3M

3M C

6M

6M

D Control SFN DM DM/SFN

* 8

* 4

ANP

17KD

Actin

42KD

*# *

#

4

ANP/Actin

ANP mRNA expression

12

*

* *

2

*#

#

0 3M

6M 0 3M

6M

Fig. 1. SFN prevention from diabetes-induced cardiac hypertrophy in vivo. Diabetic and age-matched mice were treated with SFN at 0.5 mg/kg daily for five days in each week for 3 months and then kept until 6 months. At both 3 and 6 months of diabetes, heart size (A) and the ratio of the heart weight to tibia length (B) were measured and calculated after mice were sacrificed. Cardiac hypertrophic markers ANP mRNA (C) and protein (D) expression were measured by real-time qPCR and Western blot. Data were presented as means ± SD (n = 6 at least). *, p b 0.05 vs. control; #, p b 0.05 vs. DM. DM: diabetes; 3 M or 6 M = 3 months or 6 months of diabetes.

GUU-3′ and 5′-AACUUUAUUCUUCCCUCUCCUGCGU-3′ (RSS343559). The Nrf2 siRNA at 40 nmol/L was transiently transfected into differentiated H9c2 cells with Lipofectamine 2000 according to the manufacturer's instructions (Invitrogen) for 36 h. Transfection efficiency was assessed by Western blot analysis for Nrf2 protein expression. Effect of Nrf2 siRNA knockdown on the expression of CTGF and PAI-1 in HG-treated cells were assessed by Western blot analysis [15].

(1:2000), CAT (1:100000), and β-actin (1:1000), 3-NT (1:1000), 4-HNE (1:1000), PAI-1 (1:2000), and TNF-α (1:500). After three washes with Tris-buffered saline (pH 7.2) containing 0.05% Tween 20, membranes were incubated with appropriate secondary antibodies for 1 h at room temperature. Antigen–antibody complexes were then visualized using an enhanced chemiluminescence kit (Thermo scientific, Rockford, IL) [13].

2.9. Western blot assay 2.10. Statistical analysis Heart tissues were homogenized and cardiomyocytes were sonicated in RIPA buffer (Santa Cruz). Total proteins were extracted and separated on 10% SDS-PAGE gels followed by transferring to a nitrocellulose membrane (Bio-Rad, Hercules, CA). The membrane was blocked with a 5% non-fat dried milk for 1 h and then incubated overnight at 4 °C with the following antibodies: Nrf2 (1:500), NQO1 (1:500), HO-1 (1:500), and ANP (1:500), connective tissue growth factor (CTGF, Santa Cruz Biotechnology, Santa, CA, 1:1000), transforming growth factor β1 (TGF-β1, Santa Cruz Biotechnology, 1:1000), SOD1 (1:1000), SOD2

Data were collected from several animals (n= 6 at least per group) and presented as means ±SD or SEM as indicated. We used Image Pro Plus 6.0 software to identify the positive staining and Image Quant 5.2 to analyze Western blot. Comparisons were performed by one or two-way ANOVA for the different groups, followed by post hoc pairwise repetitive comparisons using Tukey's test with Origin 7.5 Lab data analysis and graphing software. Statistical significance was considered as p b 0.05.

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the body weight among groups from 2 to 24 weeks after MLD-STZ treatment (Supplemental Fig. 1A). After MLD-STZ, however, blood glucose levels in DM and DM/SFN groups significantly increased without difference (Supplemental Fig. 1B).

3. Results 3.1. General effects of SFN on diabetic and control mice Diabetic and age-matched control mice were subcutaneously given SFN at 0.5 mg/kg daily for five days in each week for 3 months. Some mice were sacrificed for experimental measurements and the rest of SFN-treated control and diabetic mice were kept for another 3 months without SFN and then sacrificed for experimental measurements. During and after SFN treatment, body weights and non-fasting blood glucose levels were monitored. There was no significant change for

3.2. SFN prevented diabetes-induced cardiac dysfunction and hypertrophy Unanesthetized BP and HR of these mice were examined by tail-cuff manometry, which showed that both diastolic and systolic BPs were progressively increased from 3 to 6 months in DM group (Table 1), an effect that was almost completely attenuated by SFN treatment.

A

SFN

DM

DM/SFN

6M

3M

Control

*#

B

*

CTGF mRNA expression

Collagen Content

2

#

1

Control SFN DM DM/SFN

0 3M

6M

*

6

*

4

#

#

2

0 3M

6M

C

3M

6M

10

8

*

*

4

TGF-β1 Actin

38KD

*#

*#

6

4

12.5KD

TGF-β1/Actin

CTGF

CTGF/Actin

3

*

*

2

#

#

1

2

42KD 0 3M

6M

0 3M

6M

Fig. 2. SFN prevention from diabetes-induced cardiac fibrosis in vivo. Cardiac sections were subject to Sirius-red staining with 0.1% Sirius-red F3BA and 0.25% Fast green FCF for collagen accumulation (A), to real-time qPCR analysis of CTGF mRNA expression (B), and also Western blot for CTGF and PAI-1 protein expression (C). Data were presented as means ± SD (n = 6 at least). *, p b 0.05 vs. control; #, p b 0.05 vs. DM group. Bar = 100 μM.

Y. Bai et al. / Journal of Molecular and Cellular Cardiology 57 (2013) 82–95

There was no significant difference for the HR among groups (Table 1). Anesthetized HRs of these mice, detected by Echo examination (Table 2), were lower than unanesthetized (Table 1), but not different among groups. Echo examination for cardiac function revealed the progressive increases in IVS and LVPW and the progressive decreases in EF and FS from 3 to 6 months (Table 2). SFN treatment of diabetic mice almost completely prevented these cardiac dysfunctions. From Table 2, it was also clear that diabetes induced a progressive increase of LV mass from 3 to 6 months, suggesting the possible induction of cardiac hypertrophy. This was confirmed by the increased size of the heart (Fig. 1A) and the increased ratio of the heart weight to tibia length (Fig. 1B) in DM groups from 3 to 6 months. Furthermore, molecular hypertrophy marker ANP was also progressively increased at

Control

SFN

DM

both mRNA (Fig. 1C) and protein expression (Fig. 1D) levels. However, all these hypertrophic changes were significantly prevented by the 3-month SFN treatment (Table 1, Figs. 1A–D). 3.3. SFN prevented diabetes-induced cardiac fibrosis, inflammation, and oxidative stress We examined the fibrotic effect of diabetes on the heart by Sirius-red staining for collagen (Fig. 2A). DM induced a significant collagen accumulation, predominantly in interstitial, but also include perivascular area, which was increased with the disease process from 3 months to 6 months. SFN treatment completely prevented the collagen accumulation. DM-induced cardiac fibrosis was further confirmed by increased CTGF expression at both mRNA (Fig. 2B) and protein (Fig. 2C) levels.

DM/SFN

*

1.8

PAI-1 expression

PAI-1

6M

3M

A

87

* 1.2

#

#

0.6

0.0 3M

6M

2.4

SFN

DM

DM/SFN TNF-α expression

Control TNF- α

6M

3M

B

*

*

1.6

#

# 0.8

0.0 3M

3M

6M

47KD

TNF-α

17KD

Actin

42KD

PAI-1/Actin

4

PAI-1

Control SFN 6 DM DM/SFN

* * *#

#

*

2

0 3M

6M

TNF- α/Actin

C

4

6M

*

* #

#

2

0 3M

6M

Fig. 3. SFN protection from diabetes-induced inflammation in vivo. Both immunohistochemical staining, followed by a semi-quantitative analysis of positive staining (A,B) and Western blot (C) were done to measure PAI-1 and TNF-α protein expression. Data were presented as means ± SD (n = 6 at least). *, p b 0.05 vs. control; #, p b 0.05 vs. DM group. Bar = 100 μM.

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Y. Bai et al. / Journal of Molecular and Cellular Cardiology 57 (2013) 82–95

A

SFN

Control

DM/SFN

DM

2.4

3-NT expression

3-NT

*

1.8

* #

# 1.2

0.6

0.0 3M

DM

SFN

Control

DM/SFN

2.4

4-HNE expression

B

6M

4- HNE

*

1.8

*

#

#

1.2

0.6

0.0 3M

C

D

6M

3M

6M

6M

3M

98KD

3-NT

98KD

4-HNE 16KD

16KD 42KD

3-NT/Actin

3

2

*

Control SFN DM DM/SFN

* #

#

Actin

1

42KD 3

4-HNE/Actin

Actin

2

* #

*

#

1

0

0 3M

6M

3M

6M

Fig. 4. SFN protection from diabetes-induced oxidative stress in vivo. Both immunohistochemical staining, followed by a semi-quantitative analysis of positive staining (A,B) and Western blot (C,D) were performed to measure oxidative damage by 3-NT (A,C) and 4-HNE (B,D). Data were presented as means±SD (n=6 at least). *, pb 0.05 vs. control; #, pb 0.05 vs. DM group. Bar=100 μM.

Cardiac TGF-β1 protein expression was also significantly increased in DM group (Fig. 2C). There was no significant increase of CTGF or TGF-β1 expression in DM/SFN group (Figs. 2B,C).

Since inflammation and oxidative stress have been suggested to play an important role in DM-induced cardiac pathogenesis [1,16–18], by immunohistochemical staining and Western blot assay we first examined

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the expression of inflammatory factors, PAI-1 (Figs. 3A,C) and TNF-α (Figs. 3B,C), which were significantly increased in DM group, but not in DM/SFN group.

The next study with immunohistochemical staining and Western blot showed the significant increase in cardiac oxidative damage, by examining 3-NT accumulation as an index of nitrosative damage

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Fig. 5. SFN induced Nrf2 expression in vivo. Nrf2 expression was detected by Western blot (A), confocal observation with immunofluorescent staining (B), and immunohistochemical staining for its phosphorylation at Ser40 (p-Nrf2, C). Arrows indicate the nuclear accumulation of Nrf2 (400× magnification). The percentage of p-Nrf2 positive staining nuclear in the total nuclear was counted (D). Data were presented as means ± SD (n = 6 at least). *, pb 0.05 vs. control; #, p b 0.05 vs. DM group.

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Fig. 6. SFN induced the expression of Nrf2 downstream genes in vivo. Nrf2 function was measured by determining the expression of Nrf2 downstream genes (HO-1, NQO1, MT, CAT, SOD1 and SOD2) at mRNA and protein levels with real-time qPCR (A) and Western blot (B). Data were presented as means ± SD (n = 6 at least). *, p b 0.05 vs. control; #, p b 0.05 vs. DM group.

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(Figs. 4A,C) and 4-HNE accumulation as an index of lipid peroxidation (Figs. 4B,D), an effect prevented significantly by SFN treatment. 3.4. Mechanistic studies: SFN cardiac protection from diabetes was associated with up-regulation of Nrf2 expression and transcription function 3.4.1. SFN up-regulated the expression of Nrf2 and its downstream genes in vivo Above results showed that SFN can protect diabetic induction of cardiac hypertrophy, fibrosis, inflammation and oxidative damage. SFN is an Nrf2 activator; therefore, whether SFN protects the heart from diabetes by activating Nrf2 was examined first by measuring Nrf2 expression and its transcription function in the diabetic heart. By Western blot (Fig. 5A) Nrf2 expression was found to be significantly increased in the heart of SFN-treated control mice both at 3 months (the end of 3-month SFN treatment) and 6 months (3 months after 3-month SFN treatment). Nrf2 expression in the heart of DM mice was increased at 3 months, but significantly decreased at 6 months of diabetes. There was no significantly further increase in the cardiac Nrf2 expression in DM/SFN group, compared to SFN group or DM group at the end of 3-month SFN treatment, but there was a significant increase of cardiac Nrf2 expression in DM/SFN group compared to DM group at 6 months of diabetes, i.e.: at 3 months after the end of 3-month SFN treatment (Fig. 5A). Immunofluorescent staining also showed that SFN can stimulate the translocation of Nrf2 into nucleus (Fig. 5B), suggesting the activation of Nrf2 function. Since Nrf2 phosphorylation at Ser40 is an indication of its activity, we have performed the immunohistochemical staining for the p-Nrf2 with hematoxylin counterstaining (Figs. 5C,D), which showed the significant increase in p-Nrf2 expression in the nucleus of the heart in SFN and DM/SFN groups, and slightly in DM group. In the next study, we further examine the Nrf2 transcription function by detecting the expression of its downstream anti-oxidative genes. Fig. 6 showed that mRNA expression of HO-1, NQO1, MT, CAT, SOD1, and SOD2 in the heart of SFN-treated control mice significantly increased both at 3 months (the end of 3-month SFN treatment) and 6 months (3 months after the end of 3-month SFN treatment). DM significantly increased the cardiac mRNA expression of these genes at 3 months, but decreased it at 6 months of diabetes. Cardiac mRNA expression of these genes was significantly higher in the heart of DM/SFN mice than that of DM mice at either 3 months or 6 months of diabetes. The change of these Nrf2 downstream genes (Fig. 6) is consistent with p-Nrf2 expression in the nucleus (Figs. 5C,D). 3.4.2. SFN protected high glucose (HG)-induced inflammatory and fibrotic responses via up-regulating Nrf2 expression and function in vitro The above in vivo finding clearly suggests the possible contribution of SFN-up-regulated Nrf2 expression and function to the protection against diabetes-induced cardiomyopathy. To further explore this, we treated H9c2 cells with HG (22 mM) or control glucose (LG, 5.5 mM) for 24 h to model diabetic condition. Some LG- and HG-treated H9c2 cells were given SFN at 10 μM for 1 h before and 24 h during HG treatment. Real time qPCR analysis (Fig. 7A) demonstrated that CTGF mRNA expression was increased in HG-treated cells compared to control, but not in HG/SFN-treated cells, which were also confirmed by its protein expression with Western blot (Fig. 7B). Fig. 7B also shows a significant increase of PAI-1 expression in HG group, but not in HG/SFN group (Fig. 7B). Nrf2 protein expression, examined by both Western blot (Fig. 7C) and immunofluorescent staining (Fig. 7D), was increased in SFN or HG groups, and synergistically increased in HG/SFN group with

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an increased nuclear accumulation (Fig. 7D). To confirm the activation of Nrf2 transcription function, the expression of its downstream antioxidants (HO-1, NQO1, CAT, SOD1 and SOD2) was examined at both mRNA (Fig. 7A) and protein level (Fig. 7C). Treatment with SFN significantly increased the expression of these antioxidant genes at both mRNA and protein levels while HG treatment significantly increase HO-1, CAT mRNA and protein expression and significantly decreased SOD1 and SOD2 mRNA and protein expression. However, all these antioxidant expression levels are higher in HG/SFN group than that in HG group (Figs. 7A,C). The above in vitro findings are in agreement with the in vivo observation (Fig. 6), confirming the association of SFN cardiac protection against from DM in vivo and HG in vitro with the up-regulated Nrf2 expression and function. To define the direct role of Nrf2 in the prevention of DM-and HG-induced fibrotic effect, we have applied Nrf2 siRNA to silence Nrf2 mRNA expression. Results showed that Nrf2 specific siRNA RSS343558 could almost completely silenced Nrf2 expression and its downstream target gene HO-1 express at the protein level after cells were exposed to siRNA for 36 h (Fig. 8A). In the cells that Nrf2 expression was silenced with RSS343558 Nrf2 siRNA, SFN prevention of HG-induced up-regulation of CTGF and PAI-1 expression was abolished (Fig. 8B). 4. Discussion We have provided the first experimental evidence to show the importance of Nrf2 in the cardiac protection against diabetes-induced damage with cardiomyocytes from Nrf2-KO mice [15]; however, whether the up-regulation of cardiac Nrf2 expression and transcription function can prevent diabetes-induced cardiac damage, particularly for the development of diabetic cardiomyopathy, in a chronic animal model remains undefined yet. The present study provided new evidence to show the significant prevention of the development of diabetic cardiomyopathy with Nrf2 activator SFN, which was associated with the up-regulation of cardiac Nrf2 expression and transcription function. Using MLD-STZ-induced type 1 diabetic mouse model here the diabetic cardiomyopathy has been successfully established, reflected by significantly progressive increases of cardiac dysfunction, including the progressive increases of diastolic and systolic BPs and LV mass, the progressive decrease of LV ejection fraction and increase of cardiac remodeling (fibrosis), along with the significant increases of cardiac inflammation and oxidative damage (Table 1, Figs. 1–4). Interestingly, we showed that cardiac Nrf2 expression (Fig. 5A) in DM group was increased at 3 months, but significantly decreased at 6 months of diabetes. The increased and decreased expression of Nrf2 at the early and late stages of diabetes was also accompanied with the increase and decrease of its transcription function, mirrored by its nuclear translocation (Fig. 5B) and phosphorylation (Figs. 5C,D) along with the expression of its down-stream antioxidants (Fig. 6), except for MT gene expression. It is known that Nrf2 expression and transcription in the cells in vitro and tissues in vivo are increased in response to oxidative stress [19–21]. We have shown in our early study that treatment with glucose at 20 and 40 mM for 24 h increased the expression of Nrf2 mRNA in primary cardiomyocytes or H9c2 cardiac cell line [15]. The up-regulation of Nrf2 and/or its down-stream antioxidant genes in response to hyperglycemia were found not only in the cultured cells, but also in the heart of diabetic mice. We have used C57BL/6 mice to make type 1 diabetes with a single dose of STZ. At two weeks after hyperglycemia, we found a significant up-regulation of Nrf2 down-stream genes NQO1 and HO-1 mRNA expression [15].

Fig. 7. SFN protected high-glucose (HG)-induced fibrotic and inflammatory response along with the activation of Nrf2 in vitro. H9c2 cells were pretreated for 1 h and then co-treated for 24 h with SFN at 10 μM in 1 g/L DMEM in the presence of HG (22 mM). The mRNA expression of CTGF and Nrf2 downstream genes (HO-1, NQO1, CAT, SOD1 and SOD2) was examined by real-time qPCR (A). The protein expression of CTGF and PAI-1 (B) and Nrf2 downstream genes (HO-1, CAT, and SOD1) (C) was detected by Western blot, respectively. Confocal observation was done for Nrf2 nuclear accumulation with immunofluorescent staining (D, 800× magnification). Data were presented as means±SD from at least three separate experiments with duplicate samples for each condition. *, pb 0.05 vs. control; #, pb 0.05 vs. HG.

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Fig. 8. Silencing Nrf2 gene abolished the prevention by SFN of HG-induced fibrosis. Efficiency of knock-downing Nrf2 at protein levels by infection with two specific Nrf2 siRNA (RSS343558; RSS343559) for 36 h were examined in H9c2 cells cultured in 4.5 g/L glucose DMEM medium without treatment, which showed that Nrf2 specific siRNA RSS343558 could efficiently knockdown Nrf2 expression at its protein level (A). H9c2 cells were transfected with siRNA RSS343558 or empty vector for 36 h, followed by 1 h pretreatment with SFN at 10 μM in 4.5 g/L glucose DMEM, and then changed with new DMEM medium containing either 5.5 mM or 22 mM glucose with or without SFN for 24 h (B), for which cellular proteins were subject to Western blot for detection of Nrf2, HO-1, CTGF, and PAI-1 protein expression. Data were presented as means ± SD from at least three separate experiments with duplicate samples for each condition. *, p b 0.05 vs. control; #, p b 0.05 vs. HG group.

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However, we also demonstrated that Nrf2 protein expression was slightly increased in the heart of mice with two month hyperglycemia, but significantly decreased in the heart of mice with 5 month hyperglycemia [20], which is further supported by the finding from the present study: increased at 3 months, but significantly decreased at 6 months of diabetes (Figs. 5,6). In fact, diabetes-down-regulated activation of Nrf2 expression was also observed in the heart of patients with chronic diabetes [20]. Tissue sections of LV were obtained from autopsy heart specimens of humans with or without diabetes (all diabetic males had histories of hypertension and cardiac dysfunction). Nrf2 expression in the nuclei was significantly down-regulated compared to control heart. In the present study, by confocal examination with immunofluorescent staining we further demonstrated that although diabetes slightly increased Nrf2 expression at 3 months as did SFN, the Nrf2 nuclear accumulation and phosphorylation as well as the expression of Nrf2 downstream antioxidants were relative lower in DM group than those in SFN group (Figs. 5,6). Based on these studies ([15,20] and the present study), we want to clarify that although the increase in Nrf2 does improve myocardial function, in particularly at the 6 months of diabetes, the diabetic changes are not due to Nrf2 decrease. Therefore, we speculate that diabetes-induced damage exists from the early stage to the late stage of the diabetes, while increased Nrf2 expression and function at the early stage of diabetes is adaptively trying to overcome diabetic damage. If there was no Nrf2 adaptive up-regulation in the diabetic heart, the cardiac damage induced by diabetes would be more evident, which has been confirmed by our early study that Nrf2-KO cardiomyocytes and heart were highly susceptible to hyperglycemia-induced damage in vitro and in vivo compared to wild-type cells and mice [15]. In further support of our speculation, here we provided the first evidence that further up-regulated cardiac Nrf2 expression and function in the diabetic heart by its activator SFN significantly prevented the development of diabetic cardiomyopathy. The most innovative finding of the present study is that treatment of diabetic mice for the first three months provided a preventive effect on the cardiomyopathy not only observed at the end of the treatment, but also observed at the 6 months of diabetes, i.e.: 3 months after the end of 3-month SFN treatment. It is known that failure of good glucose control to reverse the injury produced by prior poor glucose control is a major problem in diabetic patients. This serious clinical problem is often referred to as metabolic or hyperglycemic memory [22]. Several studies [22–26] have implied that intensive control glucose at early stage, not at late stage, often provides beneficial effect in terms of the prevention of cardiovascular diseases. However, intensive glucose control only at the late stage of diabetes often did not provide significant beneficial effect on the complication prevention. We have demonstrated that infusion of angiotensin II to normal mice only for two weeks can cause a significant cardiac dysfunction and remodeling, i.e.: cardiomyopathy, examined at the 6 months (i.e.: at 22 weeks after the end of 2-week angiotensin II infusion) [13]. We also demonstrated that treatment of diabetic mice with zinc for the first 3 months once diabetes was onset, also significantly prevents the development of diabetic cardiomyopathy, examined at the 6 months of diabetes (i.e.: 3 months after the end of first three month zinc treatment) [27]. All these data strongly suggest the importance of the early prevention for diabetic cardiovascular complications. Therefore, as long as the early diabetesinduced pathogenic changes can be efficiently prevented, the risk of development of diabetic cardiomyopathy at the late stage would be significantly reduced. It is a noticeable that as a so-called Nrf2 down-stream antioxidant gene MT gene expression in the kidney seems different from others: MT gene expression in the kidney increased from 3 to 6 months while others decreased from 3 to 6 months (Fig. 6A). This is mainly because that MT may be not completely regulated by Nrf2 although several studies have shown the association of Nrf2 expression with MT expression [28–31]. Therefore, MT may be partially Nrf2 dependent and partially independent up to conditions.

The last point worthy to be discussed here is the potential mechanism by which SFN prevents the heart from diabetes. SFN protection of cells and tissues from various oxidative stresses has been well-appreciated via up-regulation of Nrf2 expression and function [32–34]. Consistence with this, we also demonstrated the direct role of Nrf2 in the prevention of HG-induced fibrotic effect in cultured H9c2 cardiac cells (Fig. 8). However, whether the protection by SFN from diabetic cardiomyopathy in vivo is also completely mediated by the up-regulation of cardiac Nrf2 expression and function remains uncertain since we did not examine whether SFN treatment remains to protect the heart from diabetes in Nrf2-KO mouse model. However, a recent study by Zheng et al. has proven that SFN treatment significantly increased the renal Nrf2 expression in Nrf2 wild-type diabetic mice but not in Nrf2-KO diabetic mice. The SFN treatment also significantly prevented renal oxidative damage and inflammation and improved renal function only in Nrf2 wild-type diabetic mice but not in Nrf2-KO diabetic mice [35], indicating SFN renal protection from diabetes through specific activation of the Nrf2 pathway [35]. However, the protection by SFN from diabetic cardiomyopathy in vivo maybe not only mediated by the up-regulation of cardiac Nrf2 expression and function since SFN treatment also significantly prevented diabetes-increased BP at the 6 months of diabetes (Table 1). Therefore, the cardiac protection by SFN from diabetes may be not only due to cardiac direct protection from diabetes, but also, at least in part, indirectly due to the systemic improvement by cardiac and systemic up-regulation of Nrf2 expression and function. In summary, we have investigated whether SFN as one of Nrf2 activators can protect diabetic cardiomyopathy using a type 1 diabetes mouse model. We treated diabetic and age-matched control mice with SFN at 0.5 mg/kg for five days in each week for 3 months, resulting in a significant prevention of diabetes-induced development and progression of cardiac dysfunction and remodeling. The cardiac protection was even more significant at the 6 months of diabetes, i.e.: at 3 months after the end of 3-month SFN treatment. The cardiac prevention from diabetes was accompanied with a significant up-regulation of Nrf2 expression and transcription function in the heart. In cultured cardiac cell study, we defined the direct role of Nrf2 in SFN protection from HG-induced fibrotic effect because silencing Nrf2 gene could completely abolished the SFN's prevention of HG effects. These results suggest that diabetic cardiomyopathy can be prevented by SFN, which is associated with the up-regulation of Nrf2 expression and function. Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.yjmcc.2013.01.008.

Author contribution Y.B., W.C., Y.X., X.M., C.Z., Q.C., and Y.T. researched data. Y.B., Y.T. T.C. and Y.Z. reviewed the article. T.C., Y.Z. and L.C. contributed initial discussion of the project and reviewed the article. L.C. wrote and edited the article.

Conflict of interest disclosures None.

Acknowledgments This study was supported in part by the Basic Research Award from American Diabetes Association (1-11-BA-17, to LC), the Starting-Up Fund for Chinese-American Research Institute for Diabetic Complications from Wenzhou Medical College (to LC & YT), and the grant from the National Science Foundation of China (81273509 to YT).

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